title raman microscope imaging and analysis of molecular dynamics in a living cell author(s)

Title Raman microscope imaging and analysis ofmolecular dynamics in a living cell

Author(s) Okada, Masaya

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Osaka University Knowledge Archive : OUKAOsaka University Knowledge Archive : OUKA

https://ir.library.osaka-u.ac.jp/repo/ouka/all/

Osaka University

チのノる''Pλ

Raman microscope imaging and analysis of molecular ilynamics

in a lioing cell

Masaya OKADA

List of contents,

List of contents,

Introduction

Chapter 1. Raman microscope imaging of a living cell

l-1,. Raman scattering from molecular vibration

1.-2. Observation of cellular molecules by using Raman scattering: the past researdres and the

limitation in live cell imaging

1-3. Live cell imaging by slit-scanning Raman microscopy

'1,-4. Molecular identification in cell images

chapter 2. Imaging and analysis of cytochrome c dynamics in cell apoptosis

2-1. Apoptosis

2-2. The function of cytochrome c in apoptosis

2-3. Resonant Raman scattering of cytochrome c

2-4. Imaging of cytochrome c in a living cell: Experimental

2-5. Optimization of experimental condition to induce apoptosis in a living cell

2-6. Observation of cytochrome c dynamics in apoptosis

Chapter 3. Resonant CARS for cytochrome c observation

3-1. CARS

g-2. Resonant Raman effect to enhance CARS signals

3-3. Optical configuration of resonant CARS microscope

3-4. Enhancement of CARS from rytochrome c

3-5. Detection limit of concentration

Chapter 4. Raman tag imaging of cellular molecules

4-1. Raman tag: the optical and morphological features

‐

1-

List of contents,

4-2. Measurement of a spectrum from Edu, an tagged cell proliferation probe

4-3. Imaging of EdU in a living cell

4-4. Raman tag disturbs the cellular function?

Conclusions & Discussions

List of publications

Acknowledgements

Appendix

A. Derivation of nonlinear susceptibility of CARS

‐2‐

Introduction,

Introduction,

Every human body is filled with several tens to hundreds of different kinds of bones,

muscles, and organs. Each part has a precise structure, and works intricately even in a simple

motion such as a jump, a handshake, and a yawn. At the moment, it seems quite difficult to make

a human with current tec-hnologies. Until now, various humanoid robots or androids have been

invented and their appearances are similar to a human. However they still cannot mimic human

motions completely. Interestingly, a human body can evolve to adapt to its surrounding

environment. For example, African people have a lot of melanin pigment in their skins to Protect

their bodies from the strong rays of the sun. Thus, their skin color is dark. If one works out

everyday, the body becomes strong. If the one does not eat enough every day' the basal

metabolism rate decreases to save energy. Such examples show that a human body is so

complicated and has lots of ingenious mechanisms, and so I have been fascinated in them since I

was child.

A human body consists of approximately sixty trillions cells. The size of a cell is about

10-100 pm, and various kinds of biological molecules exist inside it. When a biological system

works, these molecules react to each other, which alternates their distributions or structures in

regulated fashion. Therefore, many scientists have attempted to reveal which, whery where, and

how biological molecules behave in a living cell for exploring biological systems.

For investigating biological systems at a cellular level, optical microscopy has played an

irnportant role. Since Hans and Sacharias ]anssen invented the ancestor of an optical microscope

in the 16th century, microscopic techniques have been improved significantly. Nowadays, phase

contrast, differential interference contrast (DIC), and fluorescence microscoPy are widely used to

visualize cell functions [1-a]. Phase contrast and DIC microscopy provides structural properties

of a living cell. In contrast, fluorescence microscopy visualizes structures inside a cell which is

stained with fluorophores. For example, the use of fluorescent proteins known as GFP, CFP and

YFP (gree+ cyanine and yellow fluorescent protein), avoids fixation in the staining process and

opens up visualization of molecular d;mamics in a living cell [5]' Many types of fluorophores'

which canbe applied to a live cell imaging, are now commercially available.

‐3‐

Introduction,

To expand further the capability of optical microscopy for live cell imaging I decided toexplore Raman microscopy, which is the combination of Raman spectroscopy and opticalmicroscopy, i. -y doctoral work. Raman spechoscopy monitors molecular vibrations [6]. Inbiological imaging, this feature enables us to image the distribution of cellular molecules withoutstaining. In addition to identify the molecular specieg the information about the structure andenvironments of the molecules in a sample can also be obtained because the properties of themolecular vibrations depend on these factors. Therefore, Raman microscopy gives usopportunities to analyze molecular dynamics in a new way, and this feature could contribute indiscovering new insights about biological systems. Historically, the first Raman spectrum of a

living biological specimen was reported by G. J. Puppels et al. in 1,990 [7]. The same groupperformed Raman imaging of a cell in 1993 [8]. After these reports, a lot of Raman imagingexperiments of molecules, organs, and drugs in a cell have been performed. Some scientists

reported about the investigation of cellular condition. I. Notingher et al. identified dead cellsfrom living cells in 2004191. Y. S. Huang et al. found a Raman band "signature of life,,, which isobserved only when a yeast cell is alive [10].

Although Raman spectroscopy/microscopy has been utilized in a variety of biologicalapplications, it has not been much used in live cell imaging because of the low scatteringefficiency of Raman scattering resulting to long image acquisition time. Some researchers haveattempted to overcome this issue, and K. Hamada et al. combined slit-scanning detectiontechnique with Raman microscopy and succeeded to observe cell division with 5 minutes intervalin 2008 [11].In 20L0,M. Okuno et al. implemented multi-focus Raman microscopy, and imagingof a living yeast cell for several tens of seconds was achieved t121. Recently, several physicalphenomena related to Raman scattering, such as surface enhanced Raman scattering (SERS),

coherent anti-Stokes Raman scattering (CARS) and stimulated Raman scattering (SRS), are

attracting attention for fast Raman imaging, too. In 2009, K. Fujita et al. obtained an image ofliving macrophage cells with spectral information within 2 minutes using SERS [13]. In the case

of nonlinear Raman processes (Ceng SRS), only a single Raman band is observed basically, buteven video rate imaging is possible now [1415].

Based on the background presented, the research in this dissertation was carried out toperform Raman imagrng and analysis of molecular dynamics in a living cell. First, the principlesof Raman scattering and Raman microscopy are explained in chapter 1. At the end of this chapter,Raman imaging of a living cell is demonstrated. Following this demonstratioru the dynamics

‐4‐

lntroduction,

observation of cytochrome c in cell apoptosis is described. Utitizing resonant Raman effect to

visualize cellular cytochrome c is also explained. Furthermore, the use of resonant CARS is

discussed to observe cytochrome c with higher sensitivity in the next chapter. Finally, imaging of

small molecules using a Raman tag is demonstrated in chapter 4.

References

l. 野島博′顕微鏡の使い方ノート,羊土社 (2002).

2. R Zemike′〃Phase contrast a new method for the lnicrOscopic observation of trallsParent

ObieCtS〃′Physica7%ろ 689-698(1942)。

3. Do Murphy″ Differendal interference contrast(DIC)miCrOscoPy and modulation contrast

microscopy Fundamentals oflight micrOscoPy alld dig■ alimaging″′Wiley― Liss′ New Yolヽ

153-168(1998).

4。 S.Bradbury&R Evermet″ Fluorescence microsCOpy.′ Contrast techniques in light

microscopy″ ′BIOS Sdentific Publshertt Ltd.′ Oxford′ United Kingdom(1996).

5. 0。 Shimomura■ Ho JOhnsOn&Y Saigar″ Extraction/PurifiCation and properties of

aequorれ a biOl―inescent protein from the hminous hydromedusan/Aequorea〃 ′JOurnal

of Cellular and Comparative Physiologyr 59′ 223…239(1962).

6。浜口宏夫′平川暁子′ラマン分光法,日本分光学会 (1988)。

7. G.Jo PuPpels′ R R MoMuし Co Ottoν Jo Grever Mo R― 。Nicoudν D.Jo A― 。Jovin&T MoJovれ

″Studying single l市 ing ceus and chromosomes by confocal Raman microspectroscoPy″ ′

Naturer 34乙 301‐303(1990).

8. G.J.Puppelε M.Grond′ &J.Grave′″Directimaging Raman microscope based on tunable

wavelength exdtation and narrow―band emission detection″ APPlied Spectroscopy/4ろ

1256-1267(1993)。

9. I.Notingher&L.L.Henれ″Raman microspectroscopy:a nonlnvas市 e tool for studies of

ind市iduall市 ing cens in vitr。〃′Expert Rev Med.De宙 ces′ 3′ 215-234(2006)。

‐5‐

lntroduction,

Y‐S.Huan3■ Kawashima/M.YamamotO/■ ogawar&H.Hamaguchち ″RamanSPeCtroscoPic signature of life in a living yeast cell″ ′Jouma1 0f Raman SPectЮ SCOpy 3島

525-526(2004)。

K.Hamanda Ko Fuiita/N.I.Smit M.Kobayadhし Y.Inouye&S.Kawata″ RamanmicroscOpy for dynamic molecular imaging of living cells″ ′JOumal of BiOmedica1 0ptics

13′ 044027(2008).

M.Okuno&H.Hamagucht〃 Multifocus cOnfocal Raman microscPectrOSCOPy fOr fast

mulumOde vibradonalimagmg ofl市ing cens″′35′ 244096‐4098(2010).

Fuiiセし S・ IShitObち K.Hamad～ No Io Smit A.Tagudt Y.Inouye&S.Kawat～〃Time―resolved obseⅣatiOn of surface―enhanced Raman scattering from gold nanOPartides

during transportthrough a hving cell.″ JOumal ofBiOmedica1 0PiCら 14024038(2009)。

C.L.Evanら E.O.POtma M.PuO五s:haaレ Do CO“′C.P.Lit X.S.Xie″ Chemicalinlaging of

tissue in vivO with videO_rate cOherent anti― Stokes Raman scattering microscOpy″ ′

Proceedings ofthe Nauonal Academy Of sciences USJ、 10216807(2005)。

B.Go Saar/Co W.FreudigeL J.Reichman/Jay′ C.Mo Stanleyr G.R.H01tom′ &x.S.Xiら″Video―rate molecular imaging in vivo with stimulated Raman scatterin3″

SCiencら 330′

1368-1370(2010).

12.

13.

14.

15。

‐6‐

Chapter L, Raman microscope imaging of aliving cell

Chapter 1,

Raman microscope imaging of a liaing cell

This chapter represents the principles and the importance of Raman microscopy in live cell

observation. In Raman microscopy, a Raman spectrum is obtained from each point of a cell'

Raman spectrum reflects the information about molecular species. Therefore, label-free imaging

of a living cell is possible. Also, information about the structure and its surrounding environment

of the molecules are available from the spectral shape. Raman microscopy can be applied to

observe a biological sample in water-based solution such as a living cell, because Raman signals

from water d.oes not interfere with that from the biological molecules. At the beginning in this

chapter, the principles of Raman scattering at molecular vibration are described for a detailed

understanding of Raman microscopy. After the explanation, I describe the history and challenges

in Raman obsewation of a cell with the explanation of the imaging methods. Finally, the optical

configuration of a slit-scanning Raman microscopy and imaging results of a living cell are shown'

L-1. Raman scattering from molecular vibration

When light is irradiated to a molecule, some photons that have energy of E = hvo (h: planck's

constant, vs: frequenry of the tight) head for the molecule. some of them just pass straight

through the molecule, and the others hit with the molecule and scattered. In the scattering, two

types of scattering, scattering through elastic or inelastic collision, exist. Fig. 1-1 represents the

energy variation of photons in the scattering process. In the case of elastic collisioru the scattered

photon has same energy hvs as the incident photon. This is called Rayleigh scattering and

frequenry of the incident light is preserved. In contrast, a small population of photons gives or

receives energy with the molecule through inelastic collisioru resulting in frequency shift of

incident light. This phenomenon is called (spontaneous) Raman scattering [1',2]. The amount of

the frequency shift is same as the intrinsic molecular frequency v. When incident photon gives

‐7‐

Chapter L, Raman microscope imaging of a liaing cell

intrinsic frequency: v

\^llfl/I,m#sns:v.+v

Figure 1-L Processes of Rayleiglu Stokes Raman, and anti-Stokes Raman scattering. The

frequency of incident light does not change through Rayleigh scattering. br Stokes Raman

scattering or anti-Stokes Raman scattering the frequenry of incident light becomes higheror lower, respectively.

energy to the molecule, the energy of the scattered light is v0 - v, called Stokes Raman scattering.

If incident photon receives energy from the molecule, the energy of the scattered light is v.+v,called anti-Stokes Raman scattering. The amount of the frequency shift is called Raman shift.

For analyzing Raman scattering from a sample, a Raman spectrum is measured.. A Raman

sPectrum is obtained by detecting the scattered light with a photodetector such as CCD camera

after dispersion in a spectrometer. Fig. 1-2 shows an example of a Raman spectrum of carbon

tetrachloride [11. The vertical or horizontal axis represents Raman shift or Raman scatteringintensity. Raman shift is described with wavenumber and the unit is cm-1. The unit of intensity isarbitral unit. In the spectrum, nine peaks appear. The peak at 0 cm-l derives from Rayleigh

scattering. The five peaks above 0 cm-1 or the three peaks below 0 cm-l correspond to Stokes

Raman scattering or anti-Stokes Raman scattering. Since a molecular bond shows both of Stokes

and anti-Stokes Raman scattering, the derivation of the peak around. 200, 300 or 450 cm-1

correspond to that of. - 200, - 300 or - 450 cm-1, respectively. However, the two peaks at around800 cm-l aPPear only in Stokes region. This is because the Stokes Raman scattering is stronger

than anti-Stokes Raman scattering. The reason will be explained in the quantum theory of Raman

scattering. Generally, Stokes Raman scattering is used for analyzing a Raman spectrum.

‐8‐

Chapter 1, Raman microscope imaging of a lioing cell

16∞ 800 000400~200~0-200-400-600-300-1000

Figure 1-2 Raman spectrum of carbon tetrachloride [1]. Peaks by Stokes Raman scattering,

Rayleigh scattering and anti-Stokes Raman scattering are observed at above 0crn-1, 0 cm-l

and below 0 cm-1, resPectivelY.

Classical theory

The principle of Raman scattering can be explained with classical theory. When a molecule is

irradiated with tight, electric dipole moment P is induced. When the electric field of the light is

enough weak, P is proportional to electric field E as

P=αE (1-1)

c is polarizability, which means how much electron cloud distorts. Polarization c,u:r be induced

in different direction from that of electric field, thus a is considered as a second rank tensor

which has 9 (= 3x3) components as

α =|li li l:| (1-2)

In order to make the discussion simple, let's suppose both of the polarization dipole moment P and

the electric field E are in one dimension. The electric field can be described with the maximum

amplitude of Eo and the frequency of vs as

‐9‐

Clzapter 1, Raman microscope imaging of a liaing cell

E = Eocos2fivot

By substituting Eq. 1-3, Xq. 1-1 is transformed as

P = aEocos2nvot

(1-3)

(1-4)

The polarizability o can be divided into two terms, a term co which does not change with ihe

molecular vibrational frequency and other terms which changes with molecular vibrational frequency.

3y supposing Q t=Qocos2rvt) (00 and v are the maximum vibrational displacement and the

molecular vibrational frequency, respectively) as normal coordinaie, s can be clescribed as

Q'*"' \r-u生が

１

一２‐

＋０ぬ一鐙

＋α〓α

since the terms after the third term are enough small to be

Q- Qocos2nvt in Eq. 1-5, Iq. 1-6 is obtained.

neglected. By substituting

Qo cos7rvt (1-5)

3y substituting Eq. 1-6 in Eq. 1"-4,Eq.1-Z is obtained finally as

QoE'cos2.7r(vs +v)r {1-7)

?he frequency in the first term of Eq. 1-7 is same as that of incident light. Therefore, this termrepresents Rayleigh scattering. The frequency in the second term or ihird term indicates that lightwhich has frequency of vo - v or vl+v, thus each ierm represents Stokes or anti-stokes Raman

scattering. From the equation, it is obvious that Raman scattering is induced when the second

and the third term is not zero. This condition is satisfied when {6al6e\=0 because eo and Eu

always should not zero. This means that Raman scattering generates only when polarizabilitychanges in the molecular vibration at the moment of vibrational displacement.

ぬ一η

＋α〓２ぬ一“

＋α〓α

ね一″

１

一２

＋Ｖ一ＶπＥ０ぬ一鐙

１

一２

＋γ″島α〓Ｐ

- 1U-


Theoretical intensity of Raman scattering is obtained by calculating amplitude of spherical

wave from a molecule. Here, the molecular position is supposed as the origin coordinate' The

electric field vector E, at the position R which is enough far from the origin coordinate exists

in the plane which is perpendicular to the position vector R. The polarization vector er, of the

scattered light at R is parallel to the projection of P against the plane which is perpendicular

to R. With Maxwell equations, E, can be described as

ム=洗θ〆,COS10」…ザ)

(1-8)

where ωs is angular frequency of sCattered lighちc is ve10dty of lighち E,is intensity of

icident hghし R is the absolute value of R.Ramarl mtensity is acquired by calculating ie

square of Es as

Is∝髯 (1‐9)

By substituting Eq. (1-S) in Eq. (1-9), and with Eq. (1-3), Eq (1-10) is obtained as follows'

(1-10)

νs represents dle frequency of scattered light. Fronl the equation′ it iS dear d■at Ramall

scattering intensity ls is proPo■ onal to inddent lightintensity f,Or biquadraic of the Ramarl

■equency νs.Thutt using shOrter excita■ orl wavehngtt hght iS advantageous to generate

Ralrlan scatterlng efficiently.

Ouantum theorv

ln order to understand Ramall scattering′ especially resonant Raman scattering/which is the

phenomenon used to obseⅣ e cytochrome c ln chapterつしin deta■′exPlanation with quantum

theory is requiredo ln quan― theoryr scattering of light iS considered as ttro― PhOtOn process

[1′ 21.Fig。 1-3 shows a Jab10nski diagram where interaction bebveen Photon allld molecule is

described. When a photon hits a molecule′ the energy of the molecule is excited fronl the

electronic ground state to the virtual state.Then thほ excitcd energy relaxes to lower energy state

←際 )"ゲ為

‐

11‐

Chapter 1, Raman microscope imaging of a liaing cell

electronic

excited state

electronic

ground statevibratioanal

states

Rayleigh Stokes anti-Stokes resonant pre_resonant

Figure 1'-3 ]ablonski-diagram of scattering processes. A: Rayleigh scattering. The initaland the final state are same. B: Stokes Raman scattering. The initial state is lower or higherthan the final state. C: anti-Stokes Raman scattering. The initial state is lower or higher thanthe final state. D: resonant Raman scattering. The intermediate state reaches the electronic

excited state. E: pre-resonant Raman scattering. The intermediate state reaches close to theelectronic excited state.

in the electronic ground state. At the momen! a new photon emits. The energy of the emittedphoton is described with the length of the down arrows in the figure. In the case of Rayleighscattering, the energy of a molecule is excited from the lowest state of the electronic ground state,

and the length of the uP arrow and that of the down arrow is same (Fig. L-3A), that is the energyand the frequency of the incident photon is same as those of the emitted photon. [r the case ofStokes Raman scattering the down arrow reaches excited vibrational state in electronic groundstate (Fig. 1-38). On the other hand, in the case of anti-Stokes Raman scattering, the energy of a

molecule is excited from excited vibrational state in electronic ground state, and the down arrowreaches the lowest state in electronic ground state (Fig. 2-3C).In both of Stokes and anti-Stokes

Raman scattering, the frequency of the incident photon changes through the processes. The

frequency difference is same as the vibrational frequency of the molecule which was hit by thephoton.

As mentioned before, it can be explained that why Stokes Raman scattering is stronger thanthat of anti-Stokes Raman scattering. To generate anti-Stokes Raman scattering, the originalmolecular energy state should at excited vibrational state in electronic ground state. However,

Rayleigh

‐12‐


the population at the state is few at room temperature. The population is decided by Boltzmann

distribution as Eq. (1-11) [2].

寺→やけ)

(1-11)

where vu,u isvibrational state in electronic ground state, No oI Nl is the population at v0 or

v1, respective\y, k is Boltzmann constant, T is absolute temperature. For example, under the

condition of T = 300 K and vu,6 = L440 cm-l, N6 = 1000Nr, thus the intensity of Stokes Raman

scattering is L000 times stronger than that of anti-Stokes Raman scattering'

When quantum theory of Raman scattering is explained with equations, Raman scattering

tensor that corresponds to polarizability tensor P in classical theory is calculated. Concretely, the

transition probability from the initial state Iln) to the final state (nl is acquired. Raman

scattering is considered as two-photon process where an incident photon with frequency ar, and

polarization vector e, disappears and an emitted photon with frequencY @" and polarization

vector es appears in the transition process. A component a* inRaman scattering tensor a is

expressed by Kramers-Heisenberg-Dirac (KHD) formula as

(1‐12)

where Do and, Do are components of dipole moment D. E^, E, and E, ate the energy at

initial final, and intermediate state, respectively. E; is the energy of incident light. I, is

damping constant. (mlo)e) of the first term represents that an incident light in the direction of

p disappears and ("lO"l") represents that an emitted light in the direction of o appears (Fig.

I-4A). The denominator shows the energy difference between the i state and the intermediate

state. The second terms represent that an emitted tight with the polarization of o appears and

then, an incident light with the polarization of p disappears (Fig. 1-48).

When the dispersion equation of KHD is applied to Raman scattering, the initial the final

and the intermediate state of a molecule are expressed by product of electronic state and

vibrational state with Born-Oppenheimer approximation as

%L録,″I + |

‐13‐

Chapter L, Raman microscope imaging of a liaing celt

Figure 1-4 Processes of Raman scattering expressed by Eq. 1.-12. A: Raman scattering

generates after the excitation of energy level of a molecule. B: Raman scattering generates

before the excitation of energy level of a molecule.

D=同D

０　ゅ

Ｄ　”

D=HD同=同D

(1-13)

(1‐14)

(1‐15)

where I ] * | ) is state vectors which represents electronic or vibrational statg respectively.

Here, it is assumed that the energy state does not degenerate in electronic ground state. Bysubstituting these equations in Eq.'1,-12, Eq. 1-16 is obtained as

=″

―「 ♂+島 +'鳥 lstfr (1-16)

where E*,Egr and n* istheenergystateat l"]lr), ls]l;) ""a lsilr),respectively.

Under non-resonant condition, the energy gap E*-Eri-Ei is much larger than

vibrational energy. Therefore, E* - Esi- E, can be approximated by E" - Er- E,. Alsq iI) can

be neglected because E* - Egi - Ei >>iI). Furthermore, in most cases of Raman scattering, initial

state is ground vibrational state lO) in electronic ground state ls] and final state is excitedvibrational state l1) in electronic ground state ls].With these conditions Eq. t-13 - Eq.L-L5 and

with an equation )lr)(rt= 1, Eg. t-12 is transformed as

帥耳

αρσ=(′にglΣΣ′■8 ツ

ｒｌＪＴＩＬ

ヒ

'9じ

レ1ツ)(ツレIDpヒ ]

１

‥

Ｌ

ｒ

ｉ

Ｊ

+

a* =1ilar"lf)

‐14‐

(1-171

Chapter 1, Raman micrascope imaging of a liuing cell

キigl

ヤ

一一

IDρ lg](1-18)

―E,

When the energy of incident light E0 is same (Fig. 1-3r) or close to (Fig. 1-38) the energy

gap between electronic excited state and ground state, :esonant or pre-resonant Raman scattering

generates 11,2]. 1n Eq. 1-18, the fi:stterms with denominatot of Hu,*Esi-Eo relates:o resonant

Raman scattering. Under resonant condition, Eu - Esi- Eo becomes almost zero, resulting in the

increment of Raman tensor. Since the value of r is order of 100 cm-i, the value o{ the

denominator becomes less than 11102 under resonant condition. Thus, more than 104

enhancement of Raman cross section can be expected with the conliguration of resonant Raman

scattering.

In 1951, Albrecht iniroduced a detailed explanation about resonant Raman scattering.

According to the theory, one has to take into account vibronic interaction at the intermediate

state in Raman scattering process. Thus, molecular Hamiltonian is separated into three terms

Hr*1, Hu*o1 , and H"]o,, which relates to a coordinate about electron, that about atornic core, and

that about both of electron and atom core, resPectively. H"^, and Hu.a are ze:o-order

perturbation and Uil", is first-order perturbation. Also, three different energy stale, electronic

ground sta:e lg0], electronic excited states leu] and lso] are considered in &e theory. With these

assumptioas, the intermediate state le] in Raman scattering is described as

αρσ 〕】}{■g

置

一

　

′

牌蔚

叶丁

昭」一

，ｇ

１１∬一ユ

ｐ

一鳥

(1‐ 19)

By supposing go] as initial electronic state and final state, and le] as intermediate state, Raman

tensor about resonant Raman scattering is acquired with the first term of Eq. 1-18 as

αρσ A+β(1-20)

A=

招=P(響IQレ嗣

感平

海一畔

漂不

い，んソ

Σ

錆卜州１二Ｌ

』

葛Ｌ一プ

‐15‐

Chapter L, Raman microscope imaging of a lioing cell

A A term B Aterm

<el lg0>+0 <e1lg0>=0

<e0lg0>=1

<gl le0>=0

Figure 1-5 Enhancement mechanism of resonant Raman scattering based on A- or B-term. A -term derives from Frand<-condon overlaps in the upward and downwardtransitions. When there is a displacement between electronic excited state and ground state,

Raman cross section ( p p ) becomes not zero (A). On the other hand if there is nodisplacement, p n becomes zero and no Rarnan scattering generates (B). B-term does notdepending on Franck-condon factor. When vibronic interaction exists between two excitedelectronic states, resonant Raman scattering generates (C).

anfl,lagr represents an operator about vibronic interaction.

A-term-related enhancement derives from Franck-Condon overlaps (;lrX"lf) which isthe degree of overlap between the wave function at electric excited state and that at electronic

ΣΣ響　ΣΣ響

〓　　　　　　＋

３

or= l<110><010>1, >O

o*= l<111><110>lr>0

or= l<110><010>lr=0

o*= l<llt><t;o>12=9

<g1le1>*0

‐16‐

Chapter'1", Raman microscape imaging of a liaing cell

ground State.In order to enhance Rallnan scattering signals′the disPlacement △ between the

equilibriuln PositiOn of atont core at electronic excited state and that of electronic ground state is

required.This iS because(′ |ツ)(ツ 1/〉>O at△ >0′ while(ブ

lν )(ツ |ノ)=C at△=O as shoWn in Fig.

1-5A and B.In principle′ only when the molecular vibraEon is Symllnetric′ (メ |ツ)(ツ |ノ〉>O iS

satisfied.Thus′ Aterm―related enhancement doIIninates for syn■ lnetric vibrations.

Some nonsynlmetric vibrations also show resonal■ t Raman scattering.This derives froIIn 3

term.Fig.1‐ 5C dePiCtS the transitiOn process related to B terlln.When two different electronic

excited states exist and they interact Vibronically as the dotted line in the figure′ Raman

scattering intensity缶 omthevibrationmodewhichsatisttes lι OI(″`v/∂

οた)。 ISol・O iltcreases.

1-2. observation of cellular moleclles by using Raman scattering: the

past researches and the limitation in live cell imaging

In 1990′ G.J.Puppels et al.analyzedふ e structtre of chromosome in a tty 13].ThiS Was the first

report of Raman observation of a biological body,As mentioned before′the condition of the

sample can be estillnated by analyzing Raman spectral Shape. In 2004′ I. Notingher et al.

estilnated life and death of a human lung adenocarcinoma cell by the peak intensity or widil of

Ral■an bands whiCh derives from molecular bonds in DNA′ Phel■ylalanine and so on 14〕 .In

addition to condition of sample′ unknown lnforrYlation can sOrYletimes be obtained through

Raman obServation because a Raman spectrum contains information about all molecular

vibration in foCus spot.For exalnple′ Y.S.Huang found a Raman band″ signature of life″ which

is observed only when a yeast celliS alive 15].Recently′ L.Chiu.et al.found that the Ralnan band

derives frotrn ergosterol 16].

A Raman image contains a Raman spectrum f■ oIIn each PoSition in a sarnPle and gives us

opportunities to analyze the samPle in more detail.In general′Rarrlan ittaging is dOne with a

Raman IInicroscope.In moSt Cases′ laser is foCused on a sample to generate RaFnan SCattering and

the laser focus is scarllled two diIIlensionally(x and y)On the salrnPle.Raman scattering from

each Point is dispersed in spectrolrlleter and finally detected with 2D CCD camera.As a result′a

data set which contains three dilnensional information about sPaCe x′ y and Rarnan shift is

obtamed as shown in Fig. 1-6.Each Position in tlle sallnPle ShOWS a Raman spectru壼1■Vhich

…17‐

Chapter L, Raman microscope imaging of a liaing ceII

Figure 1-6 Raman data obtained by a Raman microscope. A Raman spectrum is obtainedfrom each point of a sample. Each Raman spectral shape reflects the information about themolecular species at the irradiated position. By reconstructing the intensity distribution of aparticular Raman shift, a Raman image of molecular vibration relating the Raman shift isobtained.

reflects the information about molecular vibration of the material there. By reconstructingintensity distribution with a peak of interes! the distribution of the molecular vibration whichrelates to the peak is obtained and the image is called Raman image.

G. J. Puppels et al reported the first Raman imaging of a biological body in 1993 [Z]. Theyobserved carotenoids in human lymphoryte. In 1996, Schaeberle et al. performedhistopathological characterization of biopsied human breast tissue containing foreign polymerinclusions using Raman microscopy [8]. In 1998, N. M. Sijtsema et al. reported three dimensionalRaman imaging of human megaloblast I9l. In 7999, Arzharttsev et al. compared a Raman image ofcellular phthalocyanine with fluorescence image [10]. Except this repor! some other researchers

also have combined Raman observation with other kinds of optical microscopy. In 2003, N.Uzunbajakava et al. analyzed nucleus distribution in an apoptotic cell using Raman andtwo-photon microscopy U1l. In 2004,H. -j. Manen et al. revealed the association of lipid bodieswith phagosomes in leukocytes [12]. V. V. Pully et al. combined Rayleigh imaging with Ramanand two-photon imaging [13]. c. Matthiius et al. combined Raman and near infrared microscopyin 2005 [14].

After 200O variety of biological samples such as protein in a cell, beta-carotene in livingcorPus luteum cells, mitochondria in a HeLa cell, and NADPH Oxidase Subunit cytochrome b55s

Raman shift

-18-

Chapter 1, Rsman microscope imaging of a liaing cell

in Single Neutrophilic Granulocytes has been observed by Raman microscopy [15-18]. A' Zoladek

et al. performed time-course imaging af a living cell and an apoptotic cell in 2010 and 2011'

respectively [19, 20]. Though visible or near infrared wavelength laser is used as an excitation

light source in Raman imaging generally, Y. Kumamoto et al' used deep ultra violei laser as an

excitation light source and observed the distribution of DNA and RNA in a fixed HeLa ceti [21]'

Raman microscopy can monitor the distributions of drugs in a celi, too. ln 2002J. Ling et al'

reported Raman imaging of cancer drug, paclitaxel, in a cell for the first time [22]. In 2004, H' -J'

Manen ei al. observed reduction of heme in a neutrophil under immune activity [23]' C' Matthiius

et al. applied Raman microscopy to follow the uptake of liposomal drug carrier systems in a cell

l1 A1l4=1.

As just described, Raman imaging of a biological body has been reported by many

researchers until now. However, in most cases, samPles were fixed with chemical such as

parafo:maldehyde before imaging, and it has been difficult to observe molecular dynamics in a

living sample in time. This is due to the low scattering efficiency of Raman scattering, resulting in

long exposure tirne (several seconds) for each point in samples is required' Raman scattering

efficienry is described with Raman scattering cross section and the vaiue is approximately 10-30

cm2. This means that 1030 incident photons are requi:ed to obtain one Raman scattering photon

under the condition that one molecule exists in the area of 1 cm2.

Recently, some feports about time-lapse Raman imaging of a living cell have been

published. In 2005, Y. Naito et al. succeeded in time-lapse imaging of &e process of dying of a

budding yeast cell using Raman microscopy with convendonal Raman microscopy (Fig' 1-7A)

[25]. They obtained an image within several minutes because imaging area was small and each

image contained 19x2i".However, in the case of larger cell such as HeLa cell, it has been difficult

to realize Raman imaging within sevetal minutes like the budding yeast cell. In 2008, Hamada et

al. implemented a Raman rnicoscope with line-focused illumination where Raman spectra from

multiple spots are detected simultaneously (Fig. 1'-m) 126, 27]' With the microscope' they

succeeded in observing cell division every 5 minutes in fime. !:r 2009, T' Takamatsu el al' applied

the slit-scanning Raman microscopy lo observe hea:t iissue [28]' M. Okuno et al' constructed a

multi-focus Raman microscope, where multiple foci distribute on a sample (Fig' 1-7C) [29]' They

succeeded in obtaining an image within 30 seconds. Like these, it is becoming possible to realize

time-lapse observation of a living cell in these years'

-19-

Chapter L, Raman microscope imaging of a liaing ceil

Figure 1-7 Scanning methods of A: conventional Raman microscopy (Single point-focus

with two dimensional scanning), B: slit-scanning Raman microscopy (Single line-focus withone dimensional scanning) C: multi-focus Raman microscopy (Multiple point-foci withtwo dirnensional scanning).

High-speed imaging technique using surface enhanced Raman scattering (SERS) has beenproposed, too. In SERS, Raman scattering from molecules at the vicinity of a metal nanoparticlein a cell is detected with high sensitivity through enhancement of Raman scattering by surfaceplasmon at the surface of the nanoparticle. In 2009, K. Fujita et al. reported the first observation ofSERS imaging of a living cell and they took one image in 2 minutes [30]. T. Itoh et al. detected

SERS from silver nanoparticles attached on cell membrane with several seconds temporalresolution [31]. Recently, J. Ando et al. observed endocytosis process by tracking a particular goldnanoparticle with detecting sERS from it in a living macrophage cell [32].

The use of nonlinear optical phenomena such as coherent anti-Stokes Raman scattering(CARS) and stimulated Raman scattering (SRS) have been studied for high-speed imaging. Inboth cases, coherent Raman signals generate from molecules. Thus, Raman signals are detected

with high sensitivity. In'l'999, A. Zumbusch et al. reported the first CARS imaging of a cell [33].The first SRS imaging was performed by C. W. Freudiger in 2008 [34]. Multi-focus CARSmiffoscopy for real-time imaging was developed using a microlens array [35]. Video rate CARS

or SRS imaging was reported, too [36,371.In addition to allowing fast imagin& the spatialresolution of non-linear Raman techniques is somewhat higher because the size of the spot inwhich the nonlinear optical excitation takes place is smaller than that of linear optical phenomena.

Recently, obtaining CARS spectra is possible using multiplex detection method [3g]. Though

‐20‐

Chapter 1, Ramsn microscope imaging of a liaing ceII

cellular components except liPids have been hard to visualize with CARS or SRS′Z.Xu et al.

reported SRS imaging of nucleic acids in living celis in 2012.

1‐3。 Live cell illllaging by slit‐ Scanning Ramartinicroscopy

Slit‐ scanning Raman llllicrosco,v

ln this research′ I constructed a slit― scanning Raman lnicroscope in order to perform Rarnan

illnaging with high telnporal resolution as shown in Fig.1-8.As mentioned in previous section′a

line―focused laser illuttlination is created on a sample and Raman spectra frorlll multiPle SPOtS are

detected silnultaneously on the line.The line is scanned in one direction to take a Raman iIItage.

3ecause of this characteristic/the temporal resolution can be improved more than 100 tiines

compared to conventional Raman lnicroscopy.For the excitation light source′532 rtlrn CW laser

derived from a frequency… dOubled Nd: YV04 1aser(Verdt cOherent lnc.or Milletrlnia― Xs′

Spectra―Physics)was uSed.The laser bea難げヽas exPanded by the beam exPander and then shaped

into a line byよ e three cylindrical lenses.After the silver coated mirror and the lens′the beam

was reflected to the galvanometer mirror by a short‐ PaSS edge filter(LP03-532RU…25,Sernrock for

532 rlm).After the galvanometer lnirror′ the laser beam went into the tticroscope through the

relay lens and the tube lens′ and in the trnicroscope it was reflected by the edge filter

((LP03-532RU… 25,Sem■ock for 532 nm)and fOCused on tlle sarnPle at■ le lnicroscope stage by a 60

X/1.2N.A.water ilrnmerslo■ objective lens(UPLSAP0 60XW′ 01ympus).The backscattered

Raman signals froln the sample were collected by the same obieCt市 e lens and renected by the

edge filter and then pass into a spectrograph(MK-300′ BunkOh Keiki)thrOugh the tube lens′ the

relay lens′ galvanolrneter rnirror′ tW0 532 nln long―pass edge filters′ the imaging lens and slit at

the entrance of the sPectrograph.The edge filters(PiXiS 400B′ Princeton lnstruments)transinit

light longer tllan 532 nm and block light shorter than 532 nlxl′ respectively.Since I Ineasured

stOkes Raman scattering in this research′ the desired signals can lvent through the filters and the

excitation light was blocked. After the spectrograPh′ the Raman signals were detected by a

cooled CCD carnera(PIxiS 400B′ Princeton lnstruments).For imaging′ the laser line is scaivled in

One direction across the sample using a single‐ axis galavnometer mirror(CSI Lumonics′ Billerica′

MA)′ the line¨ focused laser was scarlned by a single― axis galvanometer mirror(710-745825′ 000-

‐21‐


Halogen light

ilt

Edge filterWater immersionobjecive lens(x60, N.A:l.2)

Cylindrical lenses galvano mirror

Figl-8 Optical configuration of slit-scanning confocal Raman microscope. A line-shaped

laser is created by the cylindrical lenses. The laser is focused at the sample plane and isscanned in one direction by the galvano mirror.

3014076, GSI Lumonics) with a step size shown in each figure caption. The Raman spectra fromeach pixel in the line were then collected in parallel. The slit width of the spectrograph was 50 pm.A stage top incubator (INUG2-OTD-LL Tokai Hit) was equipped with the microscope to keep theenvironment around samples in a humidified atmosphere (5% COz , gS % air) at 37 "C duringexperiment. The Raman microscope was also equipped with a halogen lamp, a mercury lamp,filter cubes (G-2A, FITC Nikon), and CCD camera to acquire a bright-field or fluorescence imageof Fig. 2-24. For a bright-field image the sample is irradiated by halogen light from upper sideand the light through the sample was collected by the objective lens. Then the some of the lightthat transmits the edge filter in the microscope and is detected by a CCD camera (DS-SMC,

Nikon). For a fluorescence image, the sample was irradiated by light from a mercury lampthrough a filter in the filter cube and the objective lens. The backscattered fluorescence light wascollected by the objective lens and is detected by the CCD camera filter in the filter cube (The

edge filter was removed from the path during Raman measurement).

Slit

田囲¨

‐22‐

Chapter 1, Raman microscope imaging of a liaing ceII

focus

Optical axis

Objective lens

Figure L-9 ConJocal detection schemes. A: a pinhole or B: a slit. In the case of slit, light

from out-of-focus plane is not blocked in the direction of parallel to the slit.

The imaging characteristic of slit-scanning Raman microscopy can be considered by

modifying the theory of imaging characteristic of confocal Raman microscopy with pinhole

detection. when a pinhole is used, the signals from out-of-focus are removed as shown in Fig'

1-9A. As a result, spatial resolution increases compared to non-confocal microscopy. Especially,

improving of the resolution in z direction is the most distinct feature of confocal microscopy and

this realized three dimensional imaging. As shown in Fig. 1-98, Slit also removes the signals from

out-of-focus. However, in this case, spatial resolution in the direction of parallel to the slit does

not increase.

The spatial resolution of confocal Raman microscopy can be discussed by requiring optical

transfer function (OTF) of it [39]. For simplicity, let's consider with two dimensional model

where a sample is irradiated by u. excitation light source and the Raman scattering light is

detected through a pinhole. In the case of Raman scattering, a position in sample plane can be

considered as a point light source that is incoherent with other positions. The signal intensity at

the position is proportional to the intensity distribution of excitation light source' When

amplitude point spread function of the objective lens and the density of the sample in focal plane

arc h1(x,!) and t(x,y) respectively, the intensity distribution of Raman scattering in the plane

c is described as

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‐23‐

Cltapter 1, Raman microscope imaging of a liaing celt

t' (x",y.: x,y) * lhr(*,gl' . r(x - ,r,,y * y") (1-21)

( "" , ), ) is considered as the center of the focus spot. When Raman scattering is corrected by the

same objective, the intensity disiribution at the position of the detector tx;,1;) is acquired as

I(x",y") = J.f r'("",y"tx,t)lh27,gl'd*,d, (1-23)

* J |lhrO,yll"{* -_r",y - y")l&r( x,ySlz ax,ay

*ln,l'ln,l'at

This equation can be expanded to a three dimensional case just by replacing ft,(.r,y) withhr(x,y,z).In additiory the detector has finite size d(x,y,e) in:eal sptical system. To take intoaccount this, larl2 is replaced with larl'@d and then, Eq. 1-23 is described as

I(x",y";x,,y)- t f t,(r",y";x,y)lhr@,_ x,!i- il, a*,ay

When lhe detector size is infinitely small and (x,,t)* (0,0),

/(_r" ,y.,,r.,) * lnrl'(nr1, @ d)\ a t

d(x,Y,3) = l tr',;::r:,, = O

When a slit is used,

(1-22)

(1-24)

(1-25)

ご(χ,y,`)=δ (χ )δ (z) {1"-26)

Here, it is supposed that the slit is enough long in one direction (x) and enough small in thedirection perpendicular to x in the detector plane. By Fourier transformation of /(r",y'u,), threedi.mensional OTF is finally obtained as shown in Fig. 1-10.

In Fig. 1-10, L 1, and ( corresponds to x, y and z, respectively. When pinhole is used (Fig.

1-8A), the detectable spatial frequeacy range in the direction of y is larger than slit detection case

(Fig' 1-8 C). The resolution in y with slit detection is almost same as non-confocal case (Fig.1-gD).

On the other hand, the resolution in x and z with slit detection is comparable to pinhole case.

‐24‐

Chapter '1, Raman microscope imaging of a liaing ceII

ρ=(プ/りξ==(f/X) η=(1/り

=(ア/ガ

Figure 1-L0 Three dimensional OTF. A: pinhole, B: Slit (parallel), C: Slit (perpendicular),

D: no pinhole or slit.

Ima2in2 of a living cell

I PerfOrmed Raman imaging of a living IIeLa cell with the slit― scanning Raman inicroscope.Cells

were seeded on a quartz substrate(2.5 cm in diameter′ 0.17 mm thick,Starbar Japan)and

cultured in culturing medium in humidified am■ osPhere(5%CO″ 95%air)at 37° C.The

medium was Dulbecco′ s modified Eagle′ s medium(DMEM′ 680552′ Nissui)supplemented with

10%fetal calf serum′ 2 mM glutamine′ 100 units/ml PeniCillin′ and 100,g/ml streptomycin.

Before the Raman imaging′ the inedium was rePlaCed with a llepes― buffered Tyrode′ s solution

COmPOSed of(in mM)NaCl′ 150′ glucose′ 10,Hepes′ 10′ KCl′ 4.0,MgClν l.0,CaClν l.0,and

NaOH′ 4.0.

Fig。 1-1l shOws representative Raman spectra of living HeLa cells wid1 532 rlm excitation

wavelength.The spectrun■ was Obtained by averaging 100 Points of cytoPlasm and nucleus.h

wavenumber region of 600-1800 cm~1′ variety of Raman peaks from biological molecules such as

nucleus acids′ Phenylalanine′ allnide― I′‐II′ ―III of Protenl and CI‐ I bond ofliPid′ and this region is

called molecular fingerprint region.In high wave number region of 2800-3000 cm~1′CH bond

exists.In the Raman sPectra Of CytoPlaSm′ Raman Peaks from cytochrome c in IInicochondria

were observed in the cytoPlasm・ This is because resonant Raman effect of cytochrome c and the

detail is described in chapter 2.In Fig。 1-12A― C represents Raman images at 750′ 1687 and 2857

cnl~l which derives from pyrrole breathing mOde V15 in Cytochrome c′ AInide― I band and CH2

band′ respectively.Since cytochrome c locarizes in lnitochondria′ the image at 750 cnl‐1 lookS

mitochondrial distribution in the cells.AInide― I is contained in Protein′ and Protein distributes in

ρ

‐25‐

+ fingerprint region -+

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Chapter L, Raman microscope imaging of a liaing celt

Figure 1-11 A representative

excitation wavelength.

2800 3000Raman shift [cmi]

Raman spectrum from a living HeLa cell with 532 1m

Figure 1-L2 Raman image at A: 750 (green), B: 16g5 (blue), and C: 29g7 cm-r,which can be

assigned to cytochrorne g Amide-I and CH, stretcJring vibration mode. D is merge of A-C.

The excitation wavelength was 532 nm. The excitation laser intensity was 3.0 mW/pm2 andthe exposure time was 4 seconds per lined. The images consist of 207x29s pixels. The slit

was set to 30 pm.

-26-

Chspter 7, Rqffian microscope imaging of a liuing cell

whole part of the cells. Thus Raman scattering are observed from whole part of the cells' CH2 is

contained in lipid. Lipid localizes in lipid droplets at high concentration and also exists in cell

membranes. Since Fig. 1-12A-C were obtained simultaneously, the relative distribution of each

cellular component can be observed by merging them with color channels as shown in Fig'

1-12D.

1-4. Molecular identification in a cell image

when a Raman image is constructed, di{ferent component species are often contained in the

image. This is due to the spectral overlap of Raman bands from various molecular species'

Raman bands from different rnolecular species are characterized by different peak positions and

linewidths. Therefore, the degree of overlap of several Raman bands depending on the spectral

position. Here, I examine the image contrast at several wavenumber regions' and rely on a priori

knowledge of the Raman band assignments and characteristic spatial features of the different

molecular species in cells to determine the wavenumber regions with weak overlap as well as

strong overlap, and identify the dominant species in the Raman image [a0]' The wavenumber

regions investigated here are 122A-L510,1550-1730 and 2840-2980 cm-l, respectively' To perform

the analysis more precisely, the spectral resolution should be high' In this experiment' the

spectral resolution was approximately 3 cm-r'

1220-L5L0 cm'1

Fig. 1-13 shows a Raman image that was obtained by averaging images at 1220-1510 cm-l of

a living HeLa cell, and observed Raman spectra of in this wavenumber region' In this region,

molecular vibrations from Amide-1II, cH2 bond, ClI3 bond and cytochlome c exist' The Raman

image contains all vibrational modes shown in the spectra. Spectral shape change depending on

the position in the cell.

When a Raman image is consiructed one by one from low wavenumber in 1220-1510 cm-r,

the image contrast changes dramatically at some wavenumbers' Sometimes, molecular

vibrational mode in a image changes wiihout changing image contrast. Sased on these things, the

wavenumber region of 1220-1,510 cm-l can be distinguished into ten categories based on the

contrast differences or Raman band assignments as shown in Fig. 1-14. In 1'221'-1244 cm-1, amide-l

-27-

Chapter 1-, Rnman microscope imaging of a tiaing cell

1250 1300 1350 1400 1450 1500Raman shift [cm-l]

Figure l‐13 Raman image which was Obtained by averaging images at 1220-1510 cm‐ 1 0f a

living HeLa cell and Raman sPectra and bands which are observed in this wave number

region.The dOted line represents the shape of cells Or their nuclei.

in PrOtein beta― sheet and cH2 0SCi■atory pitching inode.TherefOre/proteln and liPid cOntributes

to the image contrast. I‐ IOwever/ since both molecular bOnds shOw weak Raman signals′

background signals from dle substrate also dOminant in the image′resulting hard t0 0bserve the

cell.h1244-1264 cnl~1′ the signals fronl the substrate become weak and dle prOtein and liPid are

observed in the image.From the prio五 knowledge ofthe Raman band assittentS′ the contrast

for prOtein is due tO anlide― I Of protein random cOil.The derivatiOn of the lipid is CI‐12 0SCillatOry

pitching mOde.h1264‐ 1285 cnl~1′ the image looks similar tO that Of 1244-1264し.11‐

1.HOWeveL in

this region/amide― I Of PЮ tein alPha― hel破 gives tte image contrast Of PЮ tein distribuuon.h

1285-1312c.11‐ 1′ the cOntrast Of the cytOplasm becOmes high due tO the existence Of Raman band

fron■ cytochrolne c in this regiOn.L■ additiOn′ nOt Only oscillatory pitching but alsO cH2 tWiSting

mode contributes tO the cOntrast Of the liPid diStributiOn.The prOtein distributiOn is given by

amide― I Of PrOtein aIPha― helix.L■ 1312-1410 Llll‐ 1′ Raman scattering froln whOle part Of the cell

becomes strOng. This is because dle existence of CH3 SyIIninetric defo...lation mOde in this

wavenumber region.This宙 bra●Onal mOdes is cOntained in both Of hPid and PЮ tein.Raman

scattering fЮm cytochЮme c in mitOchOndria makes the cytoPlasm brighter than the nucleus.h

山is regioL CH2 0Sdllatory pitching mode is includec t00.h1410-1423 cm‐ 1′ Raman scattering

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‐28‐

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Figure 1-14 Characteristic Raman images at L220-1510 cm-1. Each image was averaged

over Raman images at wavenumber region written in it. The vibrational modes contribute

to the contrast of each image. The dotted line represents the shape of cells or their nuclei.

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‐29‐

Chapter L, Rsman microscope imaging of a tiuing cell

from cytochrome c becomes weak and protein and lipid gives contrasts dominantly. As well as

1410-1423 cm-r, CH, symmetrical deformation vibration mode give conirasts of the distribution ofprotein and lipid. CH2 oscillatory pitching mode also contributes io the lipid i:nage. In 1423-145A

cm-], the Raman scattering becomes weak except lipid droplet. In this wavenumber regiory CH,scissoring mode contributes to the lipid image. In addition, CH3 symmetrical deformation modegives contrast o{ the distribution of protein and lipid. ln 1450-1480 cm-l, the whole part of the cell

gives strong Raman scattering. This is because protein is imaged by CH3 asymmetric deformation

mode. This vibrational mode and CH2 scissoring vibration gives a contrast of lipid in the image.

In 1480-1500 cm-l, the image of lipid becomes dark because the contrast is given only by ofCH, asymmetric deformation mode. Cytochrome c also contributes to the image contrast. In1500-1507 cm-r, only cytochrome c contributes to the image contrast of the image. lloweve4, since

the scattering intensity is weak, Raman signals from the substrate also appears in the image.

From above discussions, in the wavenumber region of 122A-7510 cm-1, several kinds ofmolecules consist of a Raman image in most cases, and it is difficult to observe an image whichcontains only one kind of molecules.

148$'1510 cm-1

Fig. 1-15 shows a Raman image which was obtained by averaging images at 1550-1730 cm-1

of a living XeLa cell, and observed Raman spectra of in this wavenumber region. In this region,

molecular vibrations from lipid, protein, and cytochrome c exist. Raman spectral shape can be

categorized into three. The spectrum from the cytoplasm has a Raman peak at around 15gF cm-r.

In the spec*um from the nucleus, Amide-I of protein is observed at around 1650 cm I, but nopeak from cytochrome c or C:C double bond is not observed.

In Fig' 1-16, representative Raman images are shown as well as Fig. 1-14. tn 1550-1602

cm-r, mitochondria-like structure is observed with high contrasl due to a Raman band fromcytochrome c. in addition, nucleus gives weak Raman scattering. This might be Raman bandfrom amide-I of protein alpha-helix overlaps with the cytochrome c band. In 7602-7645 cm-r,

Raman scattering from amide-I of protein alpha-helix becomes stronger and as a result, proteindistribution is ovserved' This wavenumber region also has cytochrome c distribution. ln1645-1661 cmr, lipid strongly shows Raman scattering due to cis configuration of C=C bond.

-30-

Chapter 1-, Raman microscope imaging of a liaing cell

1550 1600 1650 1700Raman shift [cmt]

Figure 1.-15 Raman image which was obtained by averaging images at 1550-1602 cm-l of a

living HeLa cell and Raman spectra and bands which are observed in this wave number

region. The dotted line represents the shape of cells or their nuclei.

protein is also observed by amide-I of protein alpha-helix exist. In 1'66L-1'668 cm-1, amide-I of

protein random coil exists and this gives image contrast of protein. In L668-1584 cm-l, lipid is

observed by cis configuration of C:C bond. Protein is also observed and the contrast is given by

amide-I of protein beta-sheet. ln 1,684-1694 crn-l, the contrast of lipid almost disappear and

amide-I of protein beta-sheet is observed selectively. Ln1694-1713 cmi, lipid droplets due to C:C

trans are observed slightly in the image in addition to protein beta-sheet. ln 1713'1726 cm-l, the

distribution of lipid appears clearly because of the existence of trans configuration of C=C bond'

In addition, protein is observed due to amide-I of protein beta-sheet.

From above discussions, only the distribution of amide-I in protein beta-sheet was imaged

selectively 11-1614-1,6g4cmi. Since the range of this region is about 10 cm-1, is necessary to obtain

Raman spectra with spectral resolution of L0 cm'l for detecting the distribution of amide-I in

protein beta-sheet selectively. In other wavenumber regions, several molecules are observed in a

Raman image simultaneouslY.

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奮ちＹＥｏＯｃ●）一あ

理Ｅく

崎００ｒ‐〇崎０

一

（Ｘ一一ｏ工‐∞ｆＱ一∞）Ｔ００事にく崎０〇一‐崎寸０一

〕

ＯＥ

ｏ」〓υｏゝ

〕

崎∞崎ｒｌ

ＯＣ一Ｃ“⊃０

０Ｃ一Ｃ００“

一卜（崎

ｒｔ

‐31‐


Figure L-16 Characteristic Raman images at 1550-L730 cm-l. Each image was averaged

over Raman images at wavenumber region written in it. The vibrational modes below thewavenumber contribute to the contrast of each image. The dotted line represents the shape

of cells or their nuclei.

‐32‐

すごす口日■■■δ妥

Chapter '1, Raman microscope imaging of a liaing cell

2840-2980 cm'1

In the wavenumber reigion of 2840-2980 cm-l, Raman band from CHz bond or CHs bond

exist as shown in Fig. L-lT,theRaman image in the figure has the distribution of protein and lipid

as well as other wavenumber regions discussed above. There are two kinds of Raman spectral

shape. Lipid droplet showed stronger Raman signals than nucleus at 2850-2890 cmr' The

difference comes from the existence of symmetric or asymmetric stretching vibration of CH2 bond

which is contained in liPid.

Fig. 1-18 represents characteristic Raman images in the wavenumber region. In 2838-2863

cm-1, the distribution of only CH, symmetric stretching vibration is observed' Thus' lipid is

imaged selectively in this region' ln 286g-2914 cm-l, CH2 asymmetric stretching mode becomes

dominant for the contrast of lipid, and CH3 symmetric vibration mode gives contrast of protein in

the image. The CH3 bond contributes to the contrast of lipid, too. In 291'4-2949 cm-1, the

contribution of CH3 symmetric stretching mode is more dominant than CH2 asymmetric

stretching mode. Thus, the distribution of protein becomes dominant for the image contrast' In

2900 2950Raman shift [cmj]

Figure 1-L7 Raman image which was obtained by averaging images at2840-2980 cm-1 of a

living HeLa cell and Raman spectra and bands which are observed in this wave number

region. The dotted line represents the shape of cells or their nuclei.

。Ｃ一〓∪一ｏ」一∽声にＣ』沐∽““

工∪

〇卜ＯＮ‐〇いＯＮ

ＯＣ一〓υ一ｏ」一∽メこＦ】ス∽ｍ

工∪

崎寸ＯＮ‐崎ｒＯＮ

ＯＣ一‘∪一０」〕∽メこＣ」沐∽“Ｎ

ＯＣ一〓〕一０」一∽´ヒＣ』沐∽Ｎ

〓∪

い０∞Ｎ‐崎寸∞Ｎ

lowJ]high

-33-

Chapter 1, Raman microscope imaging of a tiaing cett

Figure 1-18 Characteristic Raman images at 2838-2863 cm-1. Each image was averagedover Raman irnages at wavenumber region written in it. The vibrational modes below thewavenumber contribute to the contrast of each image. The dotted line represents the shape

of cells or their nuclei.

2949-2976 cm-r, only CH3 asymmetric vibration mode exists and as a result, protein and lipid are

observed in the Raman image.

ln 2840-2980 cm-l, the distribution of CH2 symmetric stretching mode or CH3 asymmetricstretching mode was observed selectively in 2838-2863 or 2949-2976 cm-r. For the selective

observation of these modes, 2s or 27 cm-l spectral resolution is required.

In this section, complementary spatial and spectral analysis of the Raman data of livingHeLa cells was performed. The analysis gave us information about the selection of wavenumberfor observing cellular molecules, and required spectral resolution to observe them. This kind ofinformation would be useful in single band detection Raman microscopy sucir as CARS and SRS.

-34-


RefeFenCeS

l. 浜口宏夫′平り|1暁子′ラマン分光法ジ日本分光学会 (1988).

2. P.R.CARItEY(伊藤紘一′尾崎幸洋訳)′ ラマン分光学基礎と生化学への応用′共立出版

ネL (1969).

3. G.J.Puppels/R R MoMuし C.OttO.′ J.Greve M.R― 。Nicoud.′ DoJ.A― .Jovin&■ MoJovin/

〃Studying single living cells and chromosomes by confocal Raman microspectroscopy″ ′

Nature′ 34ろ 301-303(1990)。

4. I.Notingher&L.L.HencL″ Raman microspectroscopy:a nonlnvas市 e tool for studies of

individual hving cells in宙 tro″′Expert Rev Med.Devices/a215-234(2006)。

5。 Y― So Huang′ ■ Kawashima/M.Yamamoto′ T Ogawa′ &H.Hamagucht″ Raman

SpectroscoPic signature 9f life in a l市 ing yeast Cell″′Jouma1 0f Raman SpectЮ scopy7 35′

525-526(2004).

6。 L.Chit F.Hunin_Matsudι T.Kobayasht Ho TOr五 &H.Hamagucht″On dle origin of the

1602 cnl‐l Raman band of yeasts,COntribution of ergosterol″ Jouma1 0f Biophotonics′ 島10′

724-728(2012).

7. Go J.Puppels′ M.Gron↓ &J.Grave′〃Direct ima〔平lg Raman microscope based on tmable

wavelength excitation and narrow=band elnission detection″ Applied SpectroscoPy/4乙

1256-1267(1993).

8。 M.D.Schaeberle′ V.Fo Kalasins町′Jo L.Luke′ Eo N.Lewis′ Io W.Levれ &P・ J・Treado′

″Raman Chenlical lmaging: I・Iistopathology of hClusions in I・I―an Breast TiSsue″

Analytical Chemistry/6町 11′ 1829‐1833(1996)。

9。 No M.Stttsem～ S・ Do Wouterら C.Jo CrauW7 C・ O仕o&J・ Grave′″Confocal Direct lma〔 Flg

Raman NIlicroscope:Design alld Applications ln Biology〃 Applied SpectroscoPy/52′ 348-355

(1998)。

loo S.Y.Arzhantsev/ A.Y Chikishev/ N.I.KoЮ teev/ J.Grave′ C.Otto & N.M.

StttSema ″Loca■zation study of CO―Phthalocyanmes in cells by Raman

micro(spectrO)scOpy″ ′Jouma1 0f Raman Spectroscopy/3Q205-208(1999)。

11. N.UzunbaiakaVa A.Lenfertt Y Kraan/E.Volokhina G.Vrensen/J・ Greve&C。

OttQ 〃Nollresonant Confocal Raman lmaging of DNA and Protein Distribution in

APoptotiC Cens″ ′BiophysicalJoum札 84(2003)。

‐35‐

Chapter 1, Raman microscope imaging of a liaing cell

L2' H-. K. Manen, Y. M. Kraary D. Roos & C. Otto, "Intercellular chemical imaging ofheme-containing enzymes involved in innate immunity using resonant Raman

microscopy", Joumal of Physical chemistry B, L0& 19762-tgr7r,, (2004).

13. V. V. Pully, A. Lenferink & C. Ofto, 'Hybrid Rayleigtu Raman and two-photon excitedfluorescence spectral confocal microscopy of living cells" |ournal of Raman spectroscopy,

41,6, (2009).

L4. C. Matthiius, S. Boydston-white, M. Miljkovic, M. Romeo, & M. Diem, ,,Raman and infraredmicrospectral imaging of mitotic cells", Applied spectroscopy, 60,'1,,1-g (2006).

15. N. uzunbajakava, A. Lenferink, w. B. Kraan, G. vrenser; I. Grevg & C. ofto, ,,Nonresonant

Raman Imaging of Protein Distribution in Single Human Cells", Biopolymer s,71, 1. (2003).

1'6' S' Arikan, H. S. Sands, R. G. Rodway, & D. N. Batchelder, "Raman spectroscopy andimaging of beta-carotene in live corpus luteum cells", Animal Reproduction Science 7'!,, g-4,

249-66 (2002).

17. C. Matthtius, T. Chernenko, J.A. Newmark, C. M. Wamer & M. Diem, ,,Label-free detection

of mitochondrial distribution in cells by nonresonant Raman microspectroscopy,,,

Biophysical fournal, 99,2, 668-6Tg (2007).

18. H-. J. van Manen, N. uzunbajakava, R. van Bruggen, D. Roos, & c. otto, ,,Resonance

Raman imaging of the NADPH oxidase subunit cytochrome b558 in single neutrophilicgranulorytes", Joumal of American Chemical society, 12s,40,121,12-9 (2003).

19' A. Zoldek- F. Pascut, P. Patel & I. Notingher, "Development of Raman Imaging System fortime-course imaging of single living cells" spectro scT|y,24, L-2,131,-196 (2010).

20' A. Zoldeh F. Pascut, P. Patel & I. Notingher, "Non-invasive tirne-course irnaging ofapoptotic cells by confocal Raman micro-spectroscopy" Joumal of Raman Spectroscpy, 42, 3,

251.-2s8 QOtt\.

21'. K. Kumamoto, A. Taguchi, N. I. Smith & S. Kawata, "Deep ultraviolet resonant Raman

imaging of a cell" Journal of Biomedical Optics, 17,7,07600.1, (20t2).

22. J.Ling, S. D. Weitrnan, M. A. Millea R. V. Moore & A. C. Bovik, "Direct Raman imagingtechniques for study of the subcellular distribution of a drug", Applied Optics, 41

6006-5017 Q002).

23. H-. K. Manen, Y. M. Kraan, D. Roos & C. Otto, "Intracellular chemical imaging ofheme-containing enzymes involved in innate immunity using resonant Raman

microscopy", Journal of Physical chemistry B, r.0g, 19762-1g17l, (2004).

‐36‐

27。

Chapter 1", Raman miuoscope imaging of a liaing cell

c. Matthiius, A. Kale, T. chernenko, V. Torchilin & M. Diem, "New ways of Imaging

Uptake and Intracellular Fate of Liposomal Drug Carrier Systems inside Individual Cells,

Based on Raman Microscopy", Molecular Pharmaceutics,5, 2,287-293 (2008)'

Y. Naito, A. Toh-e & H. Hamaguchi, "In vivo time-resolved Raman imaging of a

spontaneous death Plocess of a single budding yeast cell", Journal of Raman spectroscopy'

36,837-839 (2005).

K. Hamada, K. Fujit4 N. I. Smith, M. Kobayashi, Y. Inouye & s. Kawata, "Raman

microscopy for dynamic molecular imaging of living cells", journal of Biomedical Optics,

L3,04/i027 (2008).

K. Fujita & N. I. Smith, "Label-free molecular imaging of living cells", Molecules and Cells'

26,530-535 (2008).

M. Ogawa, Y. Harad+ T. Yamaoka K. FujitA H. Yaku, T. Takamatsu, "Labe-free

biochemical imaging of heart tissue with high-speed spontaneous Raman microscopy"'

Biochemical and Biophysical research communications, 382,2,370-374 (2009)'

M. Okuno & H. Hamaguchu "Multifocus confocal Raman microscpectroscopy for fast

multimode vibrational imaging of living cells", 35,24, 4096-4098 (2010).

K. Fujita s. Ishitobi, K. Hamada, N. I. Smith, A. Taguchi, Y. Inouye & s. Kawata,

,,Time-resolved observation of surface-enhanced Raman scattering from gold nanoparticles

during transport through a living cell." Journal of Biomedical Optics, 'l'4,024038 (2009)'

A. Sujith, T. Itotu H. Abe K. Yoshida M. s. Kiran, v. Biju & M.Ishikawa, "Imaing the cell

wall of living single yeast cells using Surface-enhanced Raman sPectroscoPy", Analytical

and Bioanalytical Chemistry, 394, L803-1809, (2009)'

I. Ando, K. Fujita N. I. Smith & S. Kawata, "Dynamics SERS imaging of cellular transport

pathways with endocytosed gold nanoparticles", Nano Letters, 1'!',L2,53M-5348 (2011)'

A. Zumbusch, G. R. Holtom, & x. s. Xie, "Three-dimensional vibrational imaging by

coherent anti-Stokes Raman scattering", Physical Review letters, 82,41'42-4L45 (1999)'

c. w. Freudiger, w. Min, B.G. Saar, S. Lu, G. R. Holtom, C. He, J. C. Tsai, J. X. Kang, & X. S.

Xie, "Label-free biological imaging with high sensitive by stimulated Raman scattering

microscopy", Science, 322, 1857 -L861 (2008)'

29。

30。

●０

32.

34.

‐37‐


35. CoLo Evane E.O.POtma7 M.Puoris'haaレ Do CO歓毎Co Po L士しX.So Xi%″ Chelnicalimaging of

ussue in v市。 wi■ video―rate cOherent and― StOkes Raman scattelng miCrOscOPy″ ′Proceedings of■ e Na■onal Academy Of sciences Usへ 10216807(2005)。

36. Go Saaし Y.Zeng′ Co W.Freudiger/Y.―S.Lit M.E.Hinlmeし X.So Xie/&s._Y.Ding/

"Label― freら real‐time inOnitoring of biomass PrOCessing with stimulated Raman Scattering

MicroscOPy"Angewandte Chemie lntemauonal Edi■ On/4%5476‐5479(2010).

37. Bo Go Saat Co w.FreudigeちJo Reichmatt Jay′ C.Mo Stanley2 G.R.H01tom/&Xo s.xie

″Video‐rate molecular imaging in v市 O wid■ sumulated Raman scatterL℃′

″SCience′ 33Q

1368-1370(2olo).

38。 H.Kano&H.Hamaguchち ″Vibra■ onally resOnant imaging of a single l市 ing cell by

supercontinuum‐ based multiplex cOherent and_stOkes Raman scattering

microspectroscOPy″ ′OptiCS Expres,13′ t1322-1327(2005)。

39.河田聡′超解像の光学,日本分光学会 (1999).

40。 A.T.k,Raman spectroscOpy in biology:PrinciPleS and APPlicatiOns′JOhn wiley&sOns/

hc.(1982)。

‐38‐

chapter 2,Imagbzg and analysis of cytochrome c dynamics in cell apoptosis

Chapter 2,

Imaging nnd analysis of cytochrotne c dynamics in cell

apoptosis

As mentioned in chapter 1, the slit-scanning Raman microscopy enabled us to observe a living

cell with several to several tens of minutes. In this chapter, I apply it to observe and analyze

molecular dynamics in a cell. As the target phenomenon, I chose apoptosis that is one o{ the most

important cell functions to keep biological body in propel condition' In past days, C' Otto et al'

and J. s. Maier et al. repo:ted Raman imaging of fixed apoptotic cells [1]. Here, I aim to observe

apoptotic cells without fixing and. especialiy focus on cytochrome c dynamics, which has an

impotant role to drive apoptotic process. This chapier starts with overview of apoptosis and

how researchers have revealed the mechanism. After the description, I explain how to visualize

cellular rytochrome c :rsing the Raman microscopy. Then, I show the observation results of

cytochrome c in apoptosis. Furthermore, I discuss about the redox state of cytochrorne c in

apoptosis which has never been analyzed in aiao'

2-1. Apoptosis

Apoptosis is known as active and regulated cell death in multi-cellu1ar organism such as human

[2-g]. Because of this feature, apoptosis is sometimes called programmed cell death. Not only

cells which have error for some reasons but also cells during development undergo apoptosis' By

inducing apoptosis properly in a body, cell homeostasis and tissue atrophy such as

disappearance of tadpole's tail on the way of metamorphosis are controlled, and serious

disorders such as cancer are protected. The molecular mechanism of apoptosis is different from

that of necrosis, which is passive cel1 death caused by damage from the outside' Revealing the

molecular mechanism enables us to understand the origin of disorders such as cancer, and

-39-

Chapter 2,Imaging and analysis of cytochrome c dynamics in cell apoptosis

＼凩ン .B

Figure 2-1 Processes of A: apoptosis and B: necrosis. In apoptosis, a cell forms blebs on itsmembrane (I). The cell breaks into pieces without breaking up cell membrane and

membranes of cellular components (II). Finally, the cell is eaten by a phagocyte. ln necrosis,

cell membrane breaks up and the cellular components are scattered.

autoimmune immune disease, and it leads to establishment of effective medical treatment or newdrug discovery. Therefore, many researchers have tried to reveal the molecular mechanism andvariety of different genes and proteins which are related to apoptosis have been discovered one

after another in these 20 vears.

The existence of attractive cell death was pointed out by C. Vogt n1,B42and apoptotic cells

were observed by W. Flemming in 1885 for the first time. They found cells whose chromatin iscondensed and split in follicle of lat, and called it chromatic cell death. However, the importance

of this phenomenon was not noticed and research about apoptosis has not been progressed

significantly in those days. In 1972,1. F. R. Kerr, A. Wylie, and A. R. Currie, who were medicalpathologists, defined the word "apoptosis" as cell death with chromatic condensatiory shrinkageand segmentation of nucleus or cytoplasm.Fig.2-1, represents how a cell is removed frorn a bodythrough apoptosis. At first, chromosome is condensed, cytoplasm shrinks, and blebs are

appeared on the surface. Subsequently, small apoptotic bodies form and they are endocytosed byanother cells such as macrophage cell. Lr addition, phospholipids appear on the surface of cell

＼＼鰊瑶 |≫ ′咆ヽ~

‐40‐

chapter 2,Imaging and analysis of cytochrome c dynamics in cell apoptosis

DNA ladderchromatin condensation

Figwe 2-2 Molecular Process of apoptosis. Apoptosis starts with DNA stress' ER stress or

FAS. Most processes proceed via mitochondria. From the mitochondria, various proteins

such as cytochrome c are released and, an apoptotic cell shows apoptotic morphological

dnanges at the last stage-

membrane on the way of morphological changes and it is considered that this has a role as maker

to draw attention of macrophages. since cell membrane does not break uP in the processes of

apoptosis, the contents do not scatter in the body and the apoptotic cell does not influence its

surrounding cells. On the other hand, in the case of necrosis, cell membrane is broken and

contents in the cell are spread in the body.

Between the stage I and II in Fig. 2-1, cellsignaling process is activated as shown nFig' 2'2'

The signaling process starts with three kinds of stimuli as follows.

. Activation of p53 gene through the damage of DNA by stimulus such as irradiation of UV

light or X-Ray, reactive oxygen species (ROS), or drug

. stimulus to endoplasmic reticulum (synthesis of abnormal protein etc.)

-4L-


Figure 2-3 TEM image of mitochondria t9]. A mitochondrion has inner and outermembrane. some proteins such as cytochrome c existbetween the membranes.

' Conjugation of molecules sudr as tumor necrosis factor-or, lymphotoxin, Fas ligand withaccepter at the surface of the cell

Though apoptosis proceeds by different molecular mechanism depending on the type ofstimulus, many types of pathways are related to mitochondria. A mitochondria is subcellularorganism where ATP (Adenosine Triphosphate) is synthesized [9]. Fig.2-l shows a TEM imageof mitochondria and it has outer and inner membranes. Between the membranes, several kinds ofproteins (cytochrome g Smac/Diablo, and HtrA2/Omi) that are related. to apoptosis exist. Theseproteins translocates from mitochondria into rytoplasm after apoptotic signal reaches

mitochondria due to MOMP (mitochondria outer membrane perrreabilization). The mechanismof MoMP is explained in two ways [10]. ln the first explanation, permeability transition pore(PTP) which locates at contact point of outer and inner mitochondrial membrane is related toMoMP' In a healthy cell, PTP closes. When RoS or Ca2* is increasesd. by apoptotic stimulug pTp

oPens after depolarization of mitoc-hondrial membrane potential. Therg proton or smallmolecules whose molecular weight is below 11000 Da flows into mitochondria. Finally, a

mitochondria swelles and MoMP occurs by destroying mitochondrial membrane. In anotherexplanatiory Bcl-2 family protein is related to MOMP. Bcl-2 family proteins are categorized. as

three Bax subfamily (Bar Bak), Bcl-2 subfamily (Bcl-2, Bcl-xl, Mcl-l) and BH3-only protein (BidBik)' Bax subfamily promotes MOMP while Bcl-2 subfamily suppresses MoMp. BH3-onlyprotein controls the function of Bax subfamily or Bcl-2 subfamily by conjugating with them.

‐42‐

chapter 2,Imaging and analysis of cytochrome c dynamics in ceII apoptosis

When apoptotic stimulus reaches mitochondria, pores form at mitochondrial outer membrane via

activation of Bax subfamilY'

After proteins are released from mitochondria into cytoplasm, caspase cascade is activated'

Caspase is a kind oI protease and this is synthesized as inactive Precursor, Procaspase in a cell'

When caspase is activated once, the activated caspase activates other caspases and this escalates

protein degradation into a chain reaction. Procaspase has cysteine which is active site, and is

activated via disconnection of asparagine acid by other caspases' In the past, 14 kinds of caspases/

caspase-1 to -74, have been discovered. Especially, it is known that caspase-3 or -7 cut other

proteins and induces apoptotic morphological changes such as cell shrinkage and memb:ane

blebbing occurs in the last stage of apoptosis'

In past days, to identify proteins and genes that are related to apoptosis and to investigate

their roles in apoptosis, researchers often isolated cellular components such as cell exttact'

nucleus and mitochondria from cells, and then they investigated what kinds o{ proteins or genes

is related to apoptosis. For example, in 1996, X. Wang et al' found that the Apaf-L' rytochrome c

and caspase-9 are related to apoptotic mechanism using cell extract' In 1997, M' Enari et al

succeeded in purification and cloning of CAD which induces DNA ladder using extracted

nucleus. The discovery of MOMP was also done by using extracted mitochondria' Except the

methods, many kinds of biological assays such western blotting, two-hybrid system'

immunostaining method, electrophoresis, and knockout mouse have used to analyze apoptotic

mechanism, too. However, it has been still di{ficult to observe how the proteins or genes behave

in an apoptotic cell. optical microscopy is one of the possible methods to observe the dynamics

and some researchers have reported observation of molecular dynamics in apoptosis by

combining optical microscopy with GFP, fluorescence lesonance energy transfer or plasmon ruler

[11-13].

2-2.The function of cytochrome c in apoptosis

In apoptotic process, cytochrome c is one of the most important proteins because its release from

mitochondria triggers caspase cascade. Fig.2-4 depicts the schematic images of cytochrome c [14]'

Cytochrome c has a porphyrin ring including heme protein. Depending on the charge number of

-43-

Chapter 2,lmaging and analysis of cytochrome c dynamics in cell apoptosis

Figute 2-4 Schematic image of A: cytochrome c [1a]. B: Heme protein at the center ofcytochrome c.

ferric iron in heme protein +II or +IIL cytochrome c shows reduced. or oxidized form. Generally,

rytochrome c exists in reduced form in a living cell [14].In 2000, D. Green et al. has reported theobservation of the release Process of cytochrome c in apoptosis as shown in Fig. 2-5 [11]. Theyprepared living HeLa cells in which rytochrome-GFP (green fluorescent protein) is expressed.Apoptosis was induced to the cells by irradiating UV light and then the distribution change ofcytochrome c-GFP was observed.

After cytochrome c is released, it forms complex apoptosome by connecting withProcasPase -9 together with Apaf-l that activates caspase -3 or -7. At this moment, the necessityof oxidation of cytochrome c is suggested by some researchers through the experimental resultsbut it is still under controversial t141. br order to investigate the red.ox state of cytochrome c inaddition to its release Process in a cell, Raman microscopy is promising because it is possible todistinguish rytochrome c redox state from the shape of Raman spectra.

2-3. Resonant Raman scattering of cytochrome c

In order to observe cytochrome c dynamics in apoptosis, it is necessary to detect the distributionof cytochrome c in a living cell with enough contrast to investigate the dynamics. Flowever,distinguishing particular molecular species is difficult in Raman image because Raman peaks

NNFe

NN

‐44‐

chapter 2,lmaging and annlysis of cytochrome c dynamics in cell apoptosis

Figure 2-5 Time-lapse imagng of Cytodピ OIFle C―GFP in apoptosis 1111.CytOChrOme c― GFP

in■にcell surrounded by white line was released in tirle.

from target molecules often overlaps with Raman peaks from other mole(■■es ln the cell as

mentioned in chapter l.Here′ I attempted to Obtain cytochrome c distribution selectively in a cell

by enhallcing only Raman scattering from cytochrome c by utilizing resonant Raman scatterng

effect.

Resonance RaIIlall sCattenng froln cytochrome c was diSCOvered by T.SPiro et al.in the

1970s115′16].As mentioned abovら heme is composed of a ferric iron and a porphyrin ring′ and a

porphyrin ring contams four modified Pyrr01e subu面 ts intercollnected at thett alPha carbon

atoms宙a methine bttdges.Heme strongly absorbs light at PartiCular wavelengi and resonance

Raman scattem■ g from cytoふrome c denves from vibra■ on mode in POrphyrin ring.Fig.2-6

shows absorption spectra of reduced and oxidiZed cytochrolrle c[17].Absorption peaks aPpear at

arOund 410 rwL and 500-550 nm/whiCh are called soret banC alpha arld beta band respecavely.

Resonant Raman scattering that is related tO Soret band derives from A te...l of Eq. 1-20.

Resonant Ralrlan sCatering that is related to alPha and beta band der市es from B term ofthe

equationo When cytochrome c is reduced′ lllltensity of beta band increases.

When excitaion wavelength dlanges as Fig.2-乙 Raman peak intensity of cytoChrome c

alsOふ anges due to resonant Rallnan effect.The figure represents Raman spectra Of OXidized

cytochrome c with 532 and 488 11m exdtaion wavelength[181.Bo■Spectra have several Ralnan

Peaks.For 1374 and 1502 cn■‐1′ resonant Rallnan scattering is induced by resonance with soret

45‐

Chapter 2,lmaging and analysis of cytochrome c dynamics in ceil apoptosis

Figure 2-6 Absorption spectra of reduced and oxidized rytochrome c [171. Both types ofcytochrome c have absorption bands ar around 410 nm and 500 - 550 nm.

band' Therefore, these peaks aPPear more strongly with 488 nm excitation wavelength. Otherpeaks are related to alpha or beta band at 500-550 nm. Therefore, their intensity is higher with 532nm excitation.

The Raman spectral shape alters depending on redox state of rytochrome c. Fig.2-g showsreported Raman sPecfta of reduced and oxidized cytochrome c solution at 530.9 nm excitation

[19]' one of the remarkable differences between the samples was Raman peak at around 1635

cm-l that aPPears only when cytochrome c was oxidized. Aside from the difference of peakpositions, the Raman scattering intensity of reduced rytochrome c shows several times strongerpeaks than that of oxidized cytochrome c.

Fig' 2-9 rePresents the comparison of Raman spectra of cellular cytoplasm with that of purereduced cytochrome c. Since various Raman peaks from cytochrome c appears in the cellularspechum, I confirmed that Raman scattering from cytochrome c are able to be detected in a livingcell by using resonant Raman scattering.

-46-

Chapter 2,lmaging and analysis of cytochrome c dynamics in cell apoptosis

Figwe2-7 Raman spectra of cytochrome c solution with 488 and 532 nm excitation [18]'

+ll+hskumcnt" R.roblion

Figure 2-8 Raman spectra of reduced and oxidized cytochrome c U'9]'

cytochrome c shows stronger Raman peaks than oxidized cytochrome c.

DAr(cm-t)

Reduced

Ｅ

ＥＰＳ　　″

上¨Ｔ

800 1000 1200 1400 1600

l.r'55o'9tm

‐47‐


1200 1400 1600 1800Raman shift[Cm■ ]

Figure 2-9 COmparison of Raman spectra of cellular cytoPlasm(upper)wiふthat Of pure

reduced cytochrome c(bottonl).Ramall peaks(75Q l12乙1175′ 1314136■ 1400 and 1585

Lニユ1‐

1)frOm dle cellular spectmm cOrresPOnd tO Ramall peaks of cytochrome c.

2-4.lmaging of cytochrome c in a riving cell Experimental

I exallnined the suitable Raman peak tO construct the distributiOn of cellular cytOchrOme c。

Fig。 2-10 represents Raman images of a l市 ing HeLa cell at several Raman peaks that are assigned

to the vibrational mode Of cytOchrome c.The inset shOws a RalrLan Spectrunl of cytoplasnl of施

cell.750cニュ1-l shOwed dle strongestimage contrast and Peak intensityJh contrast Raman images

at odler peab′ background signals from the subsLat liPiC and PrOtein appeared in■e images.

TherefOre′ I chOse tO this peak to obseⅣ e cytOchrome c distributiOn in this research.

To llnake sure dlat the inage represents cytOchrOme c distributioL I cOmpared Raman

ilnage at 750し..1‐l widl an hnmunofluorescence unage of cytochrOme c as shOwn in Fig。

2-11.The

iminunofluOrescence llnage was Obtained at first and d■ en/Raman image was obtained after

bleaching the fluOrescence.The images showed similar dist五 butiOn and this result suggested that

cytochrome c cOntributed tO the contrast Of ■ e Ranlall imageo The prOtOcol fOr the

immunOstaining is shown be110w.

ｍＯ“一

守Ｆｍ”

〇〇寸

Ｆ‐

［．コ．ユゝ一∽ｃｇＥＣＣＥ●∝

‐48‐

Chapter 2, lmaging and analysis of cytochrome c dynamics in cell apoptosis

Figure 2-10 Raman images of cytochrome c in living HeLa cells at various wavenumbers

that are related to molecular vibrational modes of cytochrome c' Raman image at 750 cm-i

showed the strongest image contrast. Other images contained background from the

substrate, or other proteins aside from cytochrome c. The contrast of the images was tuned

in the same signal range so that the signal intensities can be compared between the images'

び〓

鮫軍崎卜ｒｒ‐‐‐‐‐‐‐

-49'

Chapter 2,lmaging and analysis of cytochrome c dynamics in ceII apoptosis

Figure 2-1l A:Raman image of cytochrome c(750 LI.l‐ 1)in HeLa cells.B:Inlmunostaining

image of cytochrome cln same cen.

I. Fix the sample with 4%Parafo...laldehyde fOr lo minutes

Ⅱ. Membrane permeatiOn by O.05%Triton_x for 5 minutes

IⅡ.B10cking cells by l%BSA

IV.Incubate the salnPle Wiふ 1%Primary and‐bOdy(and―cytochrome c mAb′ InvitrOgen)fOr l

hours

V.hcubate the samPle with l%Secondary and― bOdy(Alexa Fluor@532)for l hours

※ The sample was rinsed in PBs_after eaこ h step.

Fur■ermOre′ I invesugated the pOssibility that Other types of cytOchromes cOntribute to the

image contrast Of Raman image at 750 cm・ because they alsO absOrb visible light and have

Raman peak at around 750 cm‐ 1.Table 2-l shOws variety tyPes of cytochrOmes.The type of

cytochromes is categorized into fOur groups′ cytochrOIIne a/b′ c and d depending on which type

of heme′ heme ar b′ c and d′ is cOntained 1201.Each type of cytOchrOme is categorlzed in detail

depending on its functiOno Since a HeLa cell was used as a samPle′Cytochrome a b′ and b5 Can be

candidates Of a molecule that cOntributes tO the Raman image in additiOn tO cytochrome c.Fig.

2-12 shOws dle structure of heme a in cytochrome a′ heme b in cytochrOme b Or heme b5 in

cytodhrome b5。 Heme a′ b and c has different side chain and this makes dle characteristic Of

absorbance Of each cytochrOme different as shOwn ln Fig.2-13.In the absOrption spec―Of

cytochrOme a7 no beta band exists while alPha(60511m)and soret band(443 1un)appear[21‐23].

Hencer itiS cOnsiderable that resOnant Raman scattering of cytochrOme a dOes not Occur wid■532

すごす口回□■■δ〓

‐50‐

chapter 2,lmnging and analysis of cytochrome c dynamics in cell apoptosis

Tabte 2-L Various types of cytochormes.

type ofheme

type of Peak Positioncytochorome of o band [nm] derivation

mitochondria of almostall kinds of eucaryotesome kinds of bacteriasome kinds of aerobic bacteria

mitochondriabacteria, mitochond ria

yeastmicrosome, mitochondriagreen plantbacteriamitochondriamitochondriapurple non-sulfur bacteria

sulfate reducing bacteria

algaech roloplast, cya nobacteriapurple photosynthetic bacteria

denitrifi cation bacteriabacteria

aa3

ba3

baa3

alcl

b

bl

b2

b5

b-559

C

595-607

-610

598

587

560-563

557-560

557

554-556

549-558

550-554550-552

552-553

553-554

553-554

625

635

Ｃｌ

Ｃ２

Ｃ３

Ｃ６

ｆ

ご

Ｃｄ‐

ｂｄ

Figare2-l2 Structure of A: heme a and B heme b'

‐51‐

chnpter 2,Imaging and analysis of cytochrome c dynamics in cell apoptosis

.ro0 450 500 sso eoo--Wovelonglh - nm

Figure 2-13 Absorption spectra of A: cytochrome a, B: cytochrome b C: cytochrome b5

121-231.

nm excitation, and the Raman image at 750 cm-l does not contain cytochrome a. Resonance

Raman effect from cytochrome b5 can be generated with 532 nm excitation because it hasabsorption peak of beta band. ln additibru cytochrome b5 has a Raman peak at around 250 cm-l(Fig' 2-L$. Thus, cytochrome b5 might contribute to the image contrast of Raman image at 750

cm{ [L9]. However, since cytochrome b5 exist not only in mitochondria but also in endoplasmicreticulum which distributes differently from mitochondria as shown in the immunostainingimage (Fig. 2-15). Therefore, I concluded that cytochrome b5 did not contribute to the imagecontrast of the Raman image. CytocJrrome b also has an absorption peak of beta band around 550nm and has a Raman peak at around 750 cm-1 (Fig. za"a) t191. It exists in mitochondria like

rytochrome c. Though resonance Raman scattering does not generate effectively unlikecytochrome c because the absorption peak is a bit far from 532 nm, it is still difficult to judge ifcytochrome b contributes to the Raman image at750cm-1only from those information. However,with the result of Raman observation of an apoptotic cell, it is concluded that cytochrome bwould not be contained in the Raman image (the detail of this discussion is mentioned in section2-6).

…uCED+co、

しへ ″ REOuCE0

400

‐52‐

Chapter 2,Imaging and analysis of cytochrome c dynamics in cell apoptosis

wavenumber lcmj]

Figure2j],4 Raman spectra of A: cytochrome b, B: rytochrome bs []'9]'

Figure 2-15 Immunostaining image of cytochrome b5. since cytochrome b5 exists in rytochrome c

and E& the fluorescence signals aPpear at whole part of the cytoplasm'

-DO'

Chapter 2,Imaging and analysis of cytochrome c dynamics in ceil apoptosis

2-5. Optimization of experimental condition to induce apoptosis in aliving cell

l investigated the experilnental conditiOn to induce apoptosis in a living cells.Generally′some

medicine or Uヽ″irradiatiOn is utilized.In this research′ l used Actinomycill D tO induce apoptosis

in a living lieLa cell.Actinomycin D is kinds Of cancer drug and it suppresses RNA synthesis by

connecting with guanhe in DNA[24].As a resulし mitOchondria is stilntllated tO release

cytochrome c and Other II101ecules into cytOPlasm/resulting in activating casPaSe cascade and

occurring aPoptotiC Inorpho10gical changes such as lnembrane blebbing and cell shrinkage.In

order to check apoptosis is induced in HeLa cells by Actinomycin D properly′I observed lteLa

cells incubated 、vith i llM Actinomycil■ D by phase― cOntrast nlicroscOpy in tillne. In this

experilnent a stage tOp incubator was set On the lllicroscOpe stage and kept tlle temperature and

the hulnidit1/37° C and 4%around the sample inside tl■ e incubator.when HeLa cells were

incubated witl■ Actinomycin D′ a cell shO、 ved apOptotic cl■ aracteristic lnorpho10gical cllanges

such as cell shrinkage and membrane blebbing as shOwn in Fig.2-16.After 6 hours′sOme cells

shrunk by apoptosis and no cell showed cell divisiOn(Fig.2-18A).On the Otller handノin COntrol

cell′ cell divislon was observed as shOwn in Fig.2-1ろ but apoptotic ll10rphological cl■anges did

not occur even after 12 hours Observation as shown in Fig.2-18B.Froln the results′I cOnfirined

that cell apoptosis is induced witl■ the additiOn of i llンl Actinomycin D.

‐54‐

chapter 2, lmaging and analysis of cytochrome c dynamics in cell apoptosis

Figure 2-16 Time-lapse phase contrast observation of cell apoptosis.The time in each

lmage showS incubation time of HeLa cells with Actinomycin D. The intewal was l

mmutes.At around 244 mmutes′ 血 Cell started sh血は′and membrane blebbing was

obseⅣed.

‐55‐

chapter 2,lmaging and analysis of cytochrome c dynamics in cell apoptosis

Figure 2-17 Time-lapse phase contrast observation of living HeLa cells. The time in each

image shows elapsed time of the observation from the starting time. The interval was 5minutes. Unlike apoptotic cells, a cell was divided into two cells after shrinkage.

-56-

chapter 2, Imaging and analysis of cytochrome c dynamics in cell apoptosis

Figure 2-18 Phase contrast images of A: HeLa cells after 5 hours Actinomycin D

incubation. The black arrows indicate apoptotic cells. B: Living HeLa cells attet L2 hours

time-lapse observation.

2-5. obsewation of cytochrome c dynamics in aPoPtosis

I performed time-Iapse Raman observation of an apoptotic HeLa cell' Fig' 2'19A shows the

resulting bright-field and time-lapse Raman images of apoptotic cells, reconstructed from the

distribution of the Raman scattering intensity at 750 cm-1. The cells were seeded on a CaF2

substrate (To compensate for the difference of refractive index between glass and CaF2, the

correction collar of the objective lens was set to 2.03) and cultured with 1 pM Actinomycin D for L

hour to trigger apoptosis. The solution was replaced with DMEM and incubated for 5 hours to

remove Actinomycin D from the cells so that the interaction between light and Actinomycin D

will not alter the cellular condition during the time-lapse Raman imagrng. The time-lapse Raman

imaging was performed at 5 minutes intervals and the medium was replaced with a

Hepes-buffered Tyrode's solution. After 30 minutes observation, the Raman signal distribution

was seen to diffuse into the cell body. This diffusion of the signal continued for approximately 5 -

L0 minutes, and no further significant change in the distribution was observed. A living HeLa cell

showed no distribution changes in Raman peak intenslty at 750 cm-1 without Actinomycin D

incubation as shown in Fig. z-LgB.From these results, it can be guessed that Raman image at750

cm-t does not contains the distribution of cytochrome b because rytochrome b still remains in

mitochondria, but no mitochondrial distribution is observed in Raman images of apoptotic cells

at 750 cm-1.

-57-

chapter 2,lmaging and analysis of cytochrome c dynamics in ceII apoptosis

5 10 15 45 50 55

す

0す

]O〔

Figure 2‐ 19A Time-lapse Raman lFnageS of apoptotic Hela cell.The area indicated with

the dOtted line in the bright― ■eld image was ObseⅣ ed.Raman images of cytOchrome c(750

Cm‐1)′ prOtein(2968 cm‐ 1)and Overlay of them are shown.The images were obtained at 5

mlnutes intervals with a frallne rate of 80 seconds/image.At 30 minutes′cytOchrOme c

started to be released frOnl nlitOchOndria.PrOteln shOws the shape Of the cell and the

distribution did nOt change through the obsewatiOn.The excitation laser intensity was l.5

mW/11Π12 and the exPOSure time was l.5 seconds per line.The images consist of 360×20

pixels.The slit was sett0 50 11m.

ｅｔｉｍ

３。

ｎａｔｉ。

２５

Ⅳｂ

Ｓｅ

２０

０ｎ‐

４０

Ｉｍｉ

３５

ＴＥυｏ崎卜じＯＥｏ』〓ｏｏス∪

ＴｒＣυ

∞０いＮ

Ｃ一０一〇」」

Ｃ一０一〇』α

一）ＯＥ

Ｏヽ二）ｏ一ｘ∪

‐58‐

Bright-leld

10μ m

10

chapter 2,lmaging and analysis of cytochrome c dynamics in cell apoptosis

Observa■on time[min]15 20 25 30 35 ４５

ＴＥ）ｏ崎＾じｏＥｏ』〓∪ｏゝ∪

ＴｃＣυ∞００Ｎ

Ｃ一ｏ一０」」

10μ m

すごす［国■■■び〓

Figure 2-19B Time-lapse Raman llnages of living Hela cells. Both cytocrhome c and

PrOtein did not change in their distributtons through the observauon.The excita■on laser

intensity was l.5mW/11m2alld the exPosure dme was l.5 seconds per line.The images

Ｃ一０一〇」α

一）。Ｅ

Ｏ」ｆ∪ｏ〕、∪

‐59‐

Chapter 2, Imaging and analysis of cytochrome c dynamics in cerl apoptosis

Fig.2-20A represents the tilne series of Raman spectra Obtained fronn the cellular cytoPlaslln

of an apoptotic cell.Each spectrurTl was averaged froln 180 points.The decrease of Raman signals

assigned tO cytochrOme c was Observed in the cellincubated with Actinomycm D′corresponding

to the release of cytOchrOrne c and subsequent dil縫siOn tl■ rOugh the cell.The intensity of other

Raman bands remained at siIFlilar intensities during the observatiOn.As lnentioned before′the

shape Of the Ralnan spectruln alsO reflects the redOx state of cytochrOrne c which has been

reported tO change during apoptOsis.In Fig.2-20A′ the POsitions ofthe Raman peaks corresPond

to those of cytOchrOme c in the reduced state′ and the relative peak intensity is allnost silnilar tO

that in the cOntr01 result(cells iIIlaged without Actinomycil■ D)shOwn in Fig.2-20B.This implies

that the redOx state of mOst cytochrOme c was maintained during the release from the

FnitOChOndria in tl■is ObservatiOn.

To further investigate■ e redOx state of cytOchrOme c′ I have Obtained the Raman spectra

of a cytochrOIne c solution in reduced and oxidized form with 532 nrll excitatiOn as shown in Fig.

2-21A.The cOncentration of each s01utiOn was lmg/ml,Since oxidized cytOchrOIne c sOlution can

be obtained by just diss01ving cytochrOme c PO、 νder in DDW because it is easily oxidized by

oxygen in the air.The reduced cytochrome c s01ution was prepared by adding lmヽ4 ascorbic acid

to oxidized cytochrol■ e c s01ution tO ntake the final cOncentration l llng/1nl.Aside frOIII the

change Of peak PositiOns′ the Rarnan spectra for reduced and Oxidized fOrlns shOw a significant

difference in tlle Rallnan signal intensity as、 vell as the repOrted spectra in Fig.2-9.Fron■ this

result and the fact tl■ at tlle peak intensity of the Raman signals in early aPOptotic cells、vas

lllaintained at a silnilar level to cOntrol cells′ Our ObservatiOn indicates that the cytOchrOme c does

not change the redOx state as it is released from lllitochOndria.TO cOnfirTn that the Raman signal

acttally decreases during oxidizatiOn of cytochrome c in a cell′I obtailled Ralrl■ an illnages of live′

and then paraformaldehyde fixed]ieLa cells′ which were then treated、 vitl■ lI120″ tO ensure the

oxidizatiOn of cytOchrOme c.I then cOmpared the images ofthe salne cells:live′after fixation/and

then after additional I1202 treatllent aS Shown in fig.2-21B′ C and D.The cOmparison of the

images clearly shOws that the Oxidized cytOchrOme c cannot ProduCe a Raman signal high

enough to be detected in the cells,Fig.2-21 shOl、ァs the Ralllan spectra averaged in the region

indicated by tlle white line in each illnage.TIne Raman peaks assi8med to cytOchrOme c(indicated

with the arrOws)decreased in the fixed cell and tOtally disappeared with H202~OXidatiOn.These

results imply that the resPiratOry functiOn in lnitochOndria Fnay nOt be degraded significantly

‐60‐

Chapter 2, Imaging nud analysis of cytochrome c dynamics in cell apoptosis

during stimulation by Act D, which is supporled by the fact that Actinomycin D actually induces

apoptosis by interfering with RNA synthesis'

0 min(

10

t5

20

25

30

35

4A

45

50

55

14b0 1800 2200 3000

Raman shift lcm1]

0 min

l015

25

30

35

40

45

50

55

Figure 2-20 Time-lapse observation of averaged Raman spectra obtained from cytoplasm

(180 pixels) of A: an apoptotic cell and B: a living celf in the case of apoptosis' Raman

intensitv from cvtochrome c decreased at around 30 minutes' Raman peaks at75A' L1'27'

『●．●］メ〓∽Ｃ〇一Ｃ一Ｃ∞経ｏ∝

一．３

．●］

沐

〓

いＣ

〇一Ｃ

一Ｃ

●

Ｅ

●∝

‐61‐

1400 1800 22AA

Raman shift [cm']


lXed+H2°2

Figute 2-21, A: Raman spectra of a cytochrome c solution und.er the reduced and oxidizedstate. B: Raman images of living HeLa cells, and same cells were observed after C:

paraformaldehyde fixation and D: the H2O2 treatment following the fixation. For each

image, Raman intensities at 7s0 (green), 1dg6 (blue), artd, 2sg7 (red) cm-r. After H2o2treatmenf the distribution and the peaks of rytochrome c disappeared.. The contrast of theimages was tuned in the same signal range so that the signal intensities can be compared

between the images. The excitation laser intensity was 3.5 mW/um2 and the exposure timewas 4 seconds per line. The images consist of 329x178 pixels. The slit was set to 50 pm.

Raman shift [cmi]

‐62‐

chapter 2, Imaging and analysis of cytochrame c dynamics in cell apoptosis

In order to confirIIll the distribution of the Raman signalsヽVith high sPatial resolution′ I

fixed HeLa cells after being incubated、 vith Actinornycin D for 2 1■ours and observed by the

slit― scanning Raman lllicroscope.Raman images constructed by tlsing the peak intensities at 750′

1684′ al■d2857 cn■‐l are shown in Fig.2-22A― C′ and the color ll■ erged image is shoヽへ′n in Fig.

2-22D.Ralllan images of IIeLa cells、 vithout ActinoIInycin D treatlnent are also obtained and

sho、vn in Fig. 2-221E一 H. The comparison of the figures sho、 vs the Clear difference in the

distribution of cytochrolllle c in the cells、 vith and without tl■ e Actinomycin D treatntent.In Fig.

2-22A′ cytochrome c is diStributed in the、 vhole part of the extranuclear region′ but not in Fig.

2-22E.We also recognized the、 veak Raman signal of 750 cn■‐l in the intranuclear region in Fig.

2-22A′ 、vhich is presumably fron■ cytoChrotrne c above or belo、v the nuclei′ and is observed in the

irnage due to the lo、 v axial resolutiol■ inherent llll slit― confocal detection.These results stlpport the

cOnclusions dra、 vlt fronl the tilne～laPSe observation results sho、 vn in Fig.2-20A.

The distributions of proteins shOヽ v■o significant differences in eitl■er samPle′ and PrOVide

further evidence that the cytochrome c distribution changed independently of tl■e cell

mOrPhological change.Colrl■ Pared tO the control result′the apoptotic cells exl■ ibit a larger number

Of liPid Vesicles′ which inight correspond to the accurllulation of intracellular vesicles in the stage

of rrlorphological change in apoptosiS 125′ 26].HoweVerノ theSe lipid vesicles are often also

observed in non―aPoptotiC Cells(See Fig.2-21B for example)′ alld frorrl the rnorpllology′ the cell

observed in Fig.2-22 was presullnably imaged before forF、 ing the apoptotic bOdy discussed in

Refs.25 and 26.The distribution of lnitochondria vvas alS0 0bserved tlSing nlitotracker red of

apoptottC Cells and perforrTling Raman illllagil■ g of the cytochrome c distribtttion as shoWn in Fig.

2-23.To lnake this sample′ l incubated HeLa cells、げitl■ lFnitotracker red in a C02 inctlbator for 10

111intttes after i pM Actinomycin D treatment for 10 hours.Finally′the cells were fixed by 4%

ParafOrmaldehyde.This restlit supports the conclusion that the Rarllal■signal distribution in Fig.

2-20A and Fig.2-22A is the result of release of cytocltroll■ e c frolrn Htitochondria by aPoPtoSiS′ and

corresPondS tO a previotts report of fluorescence observation of cytochrome c―GFP dynarnics in

apoptosis llll.

…63‐

（０

一υくヽ

Chapter 2, Imaging and analysis of cytochrome c dynamics in ceil apoptosis

750 cm-t: Cyochrome c 1684 cm-r: protein 2857 cm{: Lipid Cyochrome c Protein Lipid

Figwe 2-22 Raman images of apoptotic cells. Raman images of apoptotic or non-apoptoticHeLa cells at A, E: 750,8,F:7684, C, G:2gs7 cm'1. D and H were constructed by mergingimages A through C or E through G with color channels. In apoptosis, cytochrome c

distributed in wole part of the cytoplasms where cytochrome c localized in mitochondria inthe non-apoptotic cells' Protein and lipid did not show remarkable differences in theirdistributions between two samples. The excitation laser intensity was 3.3 mw/pm2 and theexposure time was 5 seconds per line. The images consist of 154xgg pixels. The slit was set

to 30 um.

（０一∪く０ヽ〓）

すごすロ

ー・ヽ・―

出■■δ〓

-64-


Figure 2-23 A: Fluorescence and RaIIlan mages of a fixed HeLa ce■ treated wi■

Actinomycin D.The release of cytochrome c can be confirmed from the comparison of the

images.The excitadOn laser intensity was 2.7 mW/pm2 and the exPoSure tlme was 2

seconds per line.The images consist Of 291× 67 PixelS・ The slit was setto 50 11m。

Mitochondrial inembrane potential is known to change during apoptoSis and lnay in fact

be responsible for the release of the cytochrome c as mentioned ln section 2-1.In order to

investigate the link be缶″een tt mtochondrial membrarle potential and cytochrome c′ we

monitored mitochondrial membrane depolarization and lnvestigated dle distribution of

cytochrome c in apoPtotiC Cellso L■ order to rnonitor initochondrial rnembrane depolarizatiott we

introduced JC‐ l dye′ which has a■ uorescence peak that changes with mitochondrial membraFle

potential Polarization from 529 rlm t0 590 rlm due to forlnation of J―aggregate ln the

mitochondrial membraner tO Lve HeLa cens in order to monitor dle change ofthe potential[271.

The JC-l dye was PurChased frolrl CaylrLan Chelrucal and followed the protocol reconllrlended by

the mallufacturer.The original JC-l reagentin tt kit was diluted with DMEM by l10 times and

used to incubate the cells for 15 minutes in order to load dle cens widl Jc-l dye.Then the cells

were incubated for 7 hours after replacing tt mediuln wi■ l DMEM containing l llM

Actinomycin D.The solution was replaced with a HepesLbuffered Tyrode′s solution7 and■ e

mitochondrial lnembrane potential was lnonitored by fluorescence rnicroscopy.After lnonitoring

すごす［国■■■σ〓

‐65‐

Chapter 2,Imaging and analysis of cytochrome c dynamics in ceII apoptosis

Mitochondrial Membrane c:750 cn丁 1

Figute 2-24 Relationship between the distribution of rytochrome c and mitochondrialmembrane potential. A, C: The mitochondrial membrane potential images of HeLa cells

incubated with or without Actinomycin D were obtained by lC-1 dye. Fluorescence

intensities of the emission at around 529 and 590 nm are shown in green and red,respectively. B, D: The Raman images of HeLa cells incubated with or without Actinomycinwere reconstructed using the distribution of Raman signals at7S0 cm-1. The excitation laser

intensity was 2.67 mw/um2 and the exposure time was 4 seconds per line. The imagesconsist of 235x111 pixels. The slit was set to 30 pm.

the mitochondrial membrane potential, I observed the cells by slit-scanning Raman microscopy.For monitoring mitochondrial membrane potential, JC-1 dye was excited by a mercury lampfocused on the sample by the same objective lens used for Raman imaging. For observing red orgreen fluorescence, G-2A (Excitation filter: 510 - 560 nm, Emission filter: 5g0 nm, Dichromaticmirror: 575 nm) or FITC (Excitation hlter:465-495 nm, Emission filter: 515-555 nm, Dichromaticmirror: 505 nm) filter cube was used, respectively. Fig. 2-24A and B show fluorescence andRaman images of a HeLa cell in which membrane potential depolarization was confirmed from

もく

露ヽ）∽

もくｏヽ３）“

すごす同国目■一δ〓

-66-

Chapter2,lmagingandanalysisofcytochromecdynnmicsincellapoptosis

the fluorescence emission only at 52g nm. After confirming the membrane potential

depolarization from the fluorescence image, Raman observation was performed' From Fig'2-248'

we can confirm the cytochrome c signal is distributed in the extranuclear region of the cell' Fig'

2-24C and, D shows a Raman image aI 750 cm-1 and a fluorescence image from a HeLa cell

without membrane depolarization, which was confirmed by the fluorescence emission at 590 nm

from the aggregated JC-1 as shown with the red color in the image' In Fig' 2-24D' we confirmed

the localization of the Raman signal in the microstructure which aPPears to be mitochondria in

the cell. Taken together, these observations support the above conclusion that the significant

change in the distribution of the cytochrome c signal at750 cm-l was induced by apoptosis'

In addition to the experiments above, I performed Raman imaging of uV-induced

apoptotic cells as shown in Fig. 2-25. When cells were irradiated by uV light, the medium in the

sample was replaced to tyrode to avoid absorbing uv light by the medium' After the irradiation'

the tyrode was replaced to medium and incubated in an incubator. Apparently, the cytochrome c

spread out in whole part of the cytoplasm as well as Fig. 2-22A while other molecular

distribution was similar to Fig. 2-228 or C. This result implies that Raman microscopy can

monitor apoptosis with different stimulus. The comparison among apoptotic cells with different

stimulus might reveal the differences of molecular dynamics among them'

Figure 2-25 Raman images of uv-induced apoptotic cells at A: 750, L684,2857 cm'l

D: merge of A, B and C with color channels. Raman signals from rytochrome c

detected from whole part of the cell. The images consist of 154x88 pixels'

and

was

‐67‐

Chapter 2, Imaging and analysis of cytochrome c dynamics in ceil apoptosis

In this chapter/1 studied the dynallnics Of cytochrOme c during apOptosis withOut using

labelingo CytOchrOIne c release from lnitOchOndria in an apOptosis was observed in tilne lvith the

slit―scanning Raman ttlicroscope. The label― free Observation has advantages especially in

observation Of dynanlics Of bio10gical mOlecules because labeling markers may disttrb the

motion of target m01ecules with a size and、～reight greater than or cOmparable to the target.For

exarnPle′ cytOChrOlrne c cOnsists Of lo4 amino acids and weighs abOut 12.4 kDa′whereas a typical

GFP cOllsists Of 238 atrninO acids and weighs abOut 27 kDa[28′ 291.The released cytOchrOme c

maintained its redOx state befOre and after the release in a cell.Observation Of cytOchrOme c

redox state in a cell has never dOne by Other lnethOds.In apoptotic study′bi010gists are interested

in the relationshiP betWeen cytOcl■ rome c redox state and casPase-9 because tl■ ey cOnsider tl■ at

only oxidized cytOchrome c activates casPase-9・ TherefOre/ siinultaneous Observation Of

cytochrome c redox state with capase-9 will be important tO reveal apoptotic process ill detail.

AlsO′ tl■e result implies the pOssibility of redOx state analysis of cytochrOme c in metab01ism Of

nlitochOndria because cytochrome c carries electrol■ s by alternating its redOx state tO produce

ATP.

Aside fron■ the apoptOsis PrOceSS I Observed tllis time′ there is anOther apoptosis process

that dOes nOt involve lnitOchondria.In this case′ apoptotic mOrph010gical change such as cell

shrinkage occurs witl■ Out cytochrOme c release.This can be used to clarify if aPOptOtiC stimuli

relate to rnitOchondria Or not.

References

J.L.Panza′ &J.S.Maie与〃Ralnan SpectrOscOpy and RallYlan Chemical lmaging of APoptotiC

Cells″Society of PhOtO_optical lnstrumentatiOn(2007)

大山ハルミ′山II武′細胞の自殺―アポトーシスタ丸善株式会社 (1995).

中西義信ノ細胞の運命Ⅲ―細胞の生死づ株式会社サイエンス社 (2005).

田沼靖一′細胞の運命 III―糸露胞の生死～株式会社サイエンス社 (2005).

辻本賀英′細胞死・アポトーシス集中マスタータ羊土社 (2006).

三浦武志′曲圏武志′アポトーシス′株式会社羊土堂 (2000).

２．

３．

４．

５．

６．

‐68‐

7.


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(2000).

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生物学第4版″Newton Press(2004)。

J.D.Ly7 Do R.Grubb′ &A.Lawen/″ The mitochondrial membrane potentia1 0ψ m)in

aPOptOSiS'an update″ APoptoSiS 8′ 115-128(2003)。

J.C.Goldstein/NoJ.WaterhOuse′ R Jui■ G.Io Evan.&Do R.GreeL″ The coordinate release

of cytodIOme c during apoptosis is rapic cOmplete and kineically invariant″Nature Cen

Biology 2 156-162(2000)

F.Wang′ To Chen7 D.Xing′ J′Wang&Yo W↓ ″

Measuring dynamics of CasPase~3 activity in

l市ing cens using FRET technique during apoptOSiS induced by high■uence low― POWer

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Y JiL So Sheikholeslamち Do R.HostetteL C.TaiOn/c.S.Crait&A.R Al市isatos′

″Continuous IIrlaglng of Plasmon rulers in live cells reveals early―

stage casPase~3 activatiOn

at■e single― molecule level〃 ′Proceedings of the Na■ onal Academy of SCience島 106′ 42

17735-17740(2009)。

Go C.Brown dtt V.Borutaite/″ Regulation of apoptosis by the redox state of cytochrome c″

BiOchimica et BiOphysica Actみ 177乙 877-881(2008).

■ G.SPir%&TC・ Streka助″Resonance Ralrlan spectra of herrLOg10bin and cytochrolne c:

Inverse polarization and Vibronic sCattering″ ′Proceedings of d■e National Academy of

Sciences USへ 69′ 9′ 2622-2626(1972)

T.Go SPiЮ′&Strekas T C.′〃Resonance RAman spectra Of heme proteins.Effects of

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so Hし I.Ko Morris′ J.Po Sin」しKo M.S,ith&To Go SPir07″ COmplete assig― ent of

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浜田啓作′ラマン散乱による細胞内分子分析イメージングラ博士学位論文 (2008).

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levels of the iron ion〃′The Journal of Physical Chemistry′ 8Z2230-234(1978).

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８

　

　

９

10。

12.

13.

16.

17.

14.

15.

８

　

　

９

１

　

　

１

‐69‐

う４

chapter 2,Imaging and analysis of cytochrome c dynamics in ceil apoptosis

R V Argade/Yc.ching/&Do Lo Rousseaし ″Resonance Raman spectralis01ation Of the a

and a3ChЮ mOphOresin cytochrOme oxidase″ ′BiOphysicalJOurnaL 5Q t 613-620(1986)。

Ko Lo POff&ⅥL L.Bulteし ″spectral characterization of the photOreducible b― type

甲 tottmne of dictyos縫 lmm discOideum″ Plant Physi010gy 5島 42Z429(1975)。

B.Hagihara&T Kaiiaharar〃 AbsorpuOn spectra Of cytochrome b5 in Crysta■ine and

diss6●ed states″ Proceedings Ofthe JapanAcademy 44′ 1,35-39(1968).

J.Kleet M.Korrunam/H.Sawhneル M.Korc〃Actinomycin D induces apoptosi6 and

inhibits growd1 0f Pancreaic caner cells〃 ′IntJ Cancer 86 399‐ 407(2000).

Zhang L Reedy Mc HarlllurL聯ヽ Obeid LM(1999)mibition Of casPases inhibits dle

release of apOptotic bOdies:Bcl-2 inhibits thtt initiation of fbェ..latiOn Of apOptotic bOdies in

chelnOtherapeutic agent― induced apoptosis.J Cell Blol 145:99-108。

N.UzunbaiakaVヽ A.Lenfermt Y Kraan/E.V010khina7 G.VrenseL J.Greve&C.

Otto′ ″Nonresonant COnfocal RaⅡLan lmaging Of DNA and PЮ tein Distribution in

APoptotiC Cells″ ′BiOphysicalJOumaし 84(2003).

S。 ■Smileyr Mo Reers/Co MOtt01a‐HartshOrn/M.Lれ A.Chenr T w smit Go DoSteeleJr&

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23.

24.

25。

26.

27.

29.

・70‐

cたα′たγ3′ Rθsθπαtt CARSルγッわ磁ЮZι C θbSι″α′わ

“

Chapter 3,

Resonant CARS for cytochrome c obseraation

The dynamics of cytochrome c in a apoptodc Cells was successfully visualized using resonant

Raman scatteringoln this chapter′ the possibility of using resonallt CARS to observe cytochrome c

with higher sensitivity is diSCussedo As mentioned in chapter l′CARS g市es strong Raman

scattering bccause it is coherent process 11-5].Some researchers have reported real―time imaging

ofmolecular dynamicsin a living cell has alreadybeen achieved using CARS 16-7].HoweVeち量is

difficult to observe cellular rnolecules such as cytochrome c that have Raman bands in fingerprint

region.I expected that enhancing CARS by resonant RalrLan effect might OVercome■e issue.

Here′ l examirled the sensitivity of resonant CARS using cytochrome c.This chapter starts wi■

dY〕 exPlanatiOn of princiPleS Of CARS.Then/dI〕 resonant CARS microscOpy widl the optical

cOnfiguraion is described.Finalし I ShOW the resu■ s of measurement Of Cytochrome c and

compare the sensidvity ofresonant CARS wi■ resonant Raman scattering.

3‐ 1。 CARS

CARS is a nonlinear oPiCal PhenOmenon.It iS a four wave mixing process including d■ree laser

excita■on pulses which are called pumP beam El(ω l)′StOkes beam E2(ω 2)and PrObe beam E3(

ω 3)・In mOSt Cases′ the■ght ield of pump beam El(ω l)iS Often also used as a probe beaⅡ し tOo.

CARS was discovered by Maker and Terhune when they tried to lneasure■ird order nonlinear

susceptibilities using ruby lasers 18].The first high resolution CARS imaging of a cell was

reported by A.Zumbusch et al.in 1999 and after that a lot Of(EARS imaging of biological

specimen has been reported 19-181.

Fig。 3-l shows he Jab10nski diagram of CARS process.CARS is as a four―wave m破ing can

be resonately enhallced by molecular vibrations.When a paricular molecular vibration

‐71‐

Chapter 3, Resonant CARS microscopy for real-time imaging

electronicexcited state

a

pump beam (o,)

Stokes beam (orr)

Vibrational state

Ground state

CARS (ul.o.r)

Figure 3-L process of CARS.

frequency corresponds to the difference between the frequenry of pump beam and that of Stokes

beam, a)1- Q)2r the molecular vibration is resonantly and coherently excited. Then, by probing theexcited molecular vibration with probe beam, a resonantly enhanced CARS signal generates.

In general, linear and nonlinear responses are observed when a specimen is irradiated withstrong light source. The polarization in the irradiated sample as Eq. 3-1.

p - yoB +y@EE+y<ttg"u *...(3-1)

where P denotes polarization, fr) denotes optical susceptibility and especially y@) 1n=2, 3, 4...) represents nonlinear susceptibility and E is electric field of incident light [19,20]. Thepolarization P produces a new electric field leading to a variety of optical phenomena. The firstterm denotes linear interaction such as transmissior; refraction, reflection and one-photonabsorption. The second term relates to second harmonic generation (SHG), sum frequencygeneration (SFG) and difference frequency generation (DFG). The third terms relates to manykinds of optical phenomena such as third harmonic generation (THG), optical Kerr effect,two-photon absorptiorg saturable absorption and four wave mixing (FWM). In fact, optical

phenomena related to yG) generates from any kinds of sample while X@ _related opticalphenomena can generates only in a sample which d.oes not possess inversion symmetry.

‐72‐

Chapter 3, Resonant CARS for cytochrome c obseroation

When optical phenomena related to the■ lird term are exP10red′ a specilnen is irradiated

with the light ield E(r,′)WhiCh COntains three different monochromaic electric ields as Eq.

3-2.

E(F,′)=El(F,′ )+E2(r,′ )+E3(r,の +ε・ε・

=ElexpF01″ ―れ・r)]+E2eXp[J02′~ち・r)]+E3eXp[′03′ ~ル

3・r)]+ε・ε。(3-2)

where E,′ ω,′ λ: and r are amplitude7 frequency′ wave vector and PoSitiOnal vector of

electric fiel↓ reSPectiVely.6。 co iS the complex c6niugate・ By Substituting Eq。 3-2in Eq.3-1′ a lot

ofterms(6× 6×6=216)are obtained and they can be categorized by the frequency included in each

term.Among thenし the terms which includes ωl― ω2+ω3← ωCA3)g市eS the CARS Polarization

according to Eq.3-3.

=tミ(r,″)=縫濃El(r,′)E'(r,″)E3(r,′ )

=XCARSEIE]E3(eXp[JOtts′ ―にctts・ →]+C.ε 。} (3-3)

where tctts=れ ―た2+・3・The latter equation if known aS Phase matching conditiono Classical

electrodynalrlics dictates that the intensity of the CARS signal is proportional to the square of

absolute value of induced Polarization/ふ us itis also proportional to the square 6f absolute value

of nonlinear susceptib■ ity for ktt aSShOwnin Eq.3-4.

Icrns nlP.*rlt

= lx, * rl'p rEi n rl= lx c ensl' t r' rt, (3‐4)

The■ird order susceptibility for CARS′ xcARS Can be der市 ed by dassical or quantum

physics.In order to include the effect of coherence lifeime in the discussion t0 1ifetime quanhm

■eory ls required.Concretely′ lt can be exPresSed with resonant and nolvesonant contributions

as Eq.3-5 based on Liouv■ le equation with density matriX(the deta■ is described in AppendiX

A).

‐73‐

Chapter 3, Resonant CARS miuoscopy for real-time imaging

ωl´´´´

ω2

Figure 3-2 Process of nonresonant background. A: Nonresonant CARS from an electroniccontribution where the dotted lines indicate virtual states. B: Electronic contributionenhanced by a two-photon resonance of the pump beam associated with an excited

electronic state.

A

χαRs=XNR+χR

=獅 +#σ乳″μめμ"μ“ωぼq_″現

(3-5)

ω姥―(■ ―ω2)~J=な ω偲-01-ω2+ω3)~′L

where ハr is number of molecules per unit v01ume ' is imaginary number/ 力 is Planck′ s

const耐′μ〃 (χ orッ is α′ b′ θ Or g in Fig。 3‐1)is transition dipole moment from states χ

tOツ′ω,iS the energy difference beれ veen χ andッ′b is the hOmogeneOus linewidd1 0f theassociated(electrOnic Or vibrational)transition beれ veen χ alld ッ′and dle permutation and

sunllnation runs over all dle vibrOnic states of the mOlecule.Since χR reSOnantes with the

molecular vibratiOn/a CARS spectrum can be obtained by Plotting the Observed CARS intensity

versus n―ω2・ χNR COntains■ vO kinds Of cOntributions as shOwn in Fig。 3-2.Fig。 3-2A is

nolllresonant contribution iat is independent Of Raman shift and Fig。3‐2B depicts enhanced

nonresOnant cOntribution due tO● ″0‐phOtOnさ lectrOmc resOnance.In facし these nOIlresonant

contributiorts cause a frequё ncy‐invariant signal which is detected together with target CARS

signal as background signal. χR lll Eq。 3-5 also can be described as

‐74‐

‰=#孝

α姥(ωl)〓茅枇α場(OCARSトフ

Chapter 3, Resonant CARS for cytochrome c obsensation

(3-6)

(3-7)

(3‐8)

When both of ωl alld ωcARS are far from ω

“ and ω%′

that is the transition is far from

resonallce/the fractions u■ Eq.3-7 and Eq.3-8 can be considered as frequency― invariant constants。

By using this′ Eq。 3‐5 is silnPlifies to

Rk黙=χNR+δ

″…,■

δr=Ω ―(ωl― ω2)

Q is the vibrational frequency of particular molecular bond In CARS, the background signals

which derives from 6yp distort the shape of a CARS sPectmm and the peak position is always

blue-shifted 13,21.-23l.This can be interpreted by Eq. 3-10 which is obtained by substituting Eq'

3-9 in Eq. 3-4.

t =lXl' = XX* = Pr+Pr+P, (3‐10)

P3=轟

where Pl is the square of the norlresonant background susceptibility which is a real constant

when the transition is far from resonance.P2denOtes a dispersive cross term which is zero at

δr=0′ POSitiVe for δ″>0′ and negative for δ″く0′ and whose magnitude depending on the

product Of χtt and χR.P3iS Lorentzian of width■ Centered at δ″=0.In Fig。 3-3′ the

spectral shape of each terin is shOWn and a CARS spectruln can be obtained by superposing them

(3-9)

‐75‐


400

′(ω )

200

-200-5 0

J′

Figure 3-3 calculated spectral shape of term p1, p, and.p, in Eq. 5-r.0 and cARs undernonresonant condition. The CARS spectrum is superposition of the spectra of the three terms.

Xwn=IS, R =10 and F":1 were used to obtain these spectr a [21,].

[21]' Since P, has a peak at 4 t 0, the peak of the CARS spectrum appears at 6, > 0. Therefore,

the peak shifts to lower frequenry.

3-2. Resonant Raman effect to enhance cARs signals

With resonant Raman scattering effect, CARS signal intensities can be enhanced as well as

resonance (spontaneous) Raman scattering. From Eq. 3-5, it is obvious that yr*, increases wheno\ ot acARs matches with the electronic transition frequency of specimen, that is when ar, orcrr"*, is absorbed by specimen. As a result, the CARS intensity also increases because it isproportional to the square of Xcens as shown in Eq. 34. With this method, it should thus bepossible to measure CARS signals of dilute solutions. However, when the resonant effect isinduced by matchinl acans with an electronic transition frequency, it is necessary to considerthe cJrange in the electronic resonant condition due to the change of the CARS signal wavelengthwhen ar, - a, isplotted in order to obtain a GARS spectrum of the specimen.

-76-

Chapter 3, Resonant CARS for cytochrome c obsentation

In 197C I. Chabay′ B. Hudson alld their group members reported h〃O papers about

resonant CARS measurement Was published 123′ 24]for the■rst time.They measured dilute

solution of diphenyloctatetraene in berlzene.The irst biological samples measured were

cytochrome c and cyanocobalamm 125].In 197ろ resonant CARS experlments of the highly

fluorescent biological chromophores/flavil、 and its binding to the active site of a■ avoprotein7

glucose oxidase were reported[261.After dlese reports′a variety of samples have been measured

[27-37].In many Cases′ the energy of pump beam is matched Jtt electronic transi■on energy of a

sample in order to detect resonant CARS signalsr and tuning either of pump or stokes beam was

used to obtain a CARS spectrum.In 2003′ rutric oxide was ineasured by tuning pr6be bean■ tO

match the CARS signal With■F electroniC transition energy[38-401.Recently/W.M.So Lu et al。

performed a triple resonant CARS measuremenし Where bo■ of pumP and PrObe beam are ttmed

fOr■e resonant effect(anOther resonance is CARS resonance)′ and measured chemical

compound′ llleocyarune[411.In ie Paper′ the sensitiVity was demonstrated to approach 102

neocyaninein DDW(distilled delonized water)widl Raman spectralideniicaiott thus boosting

the sensit市 ity ofthe reported CARS detection by 103t。 105f。ld.

Under resonant condition/the fractions in Eq。 3-7 allld Eq。 3-8 should consist of ilnaginary

parts in addition to real PartS because light absorptton occurs.In order to take■is into account R

in Eq.3-9 is replaced Wid■ A+沼 00■ A and B are real constantS numbers)。 Similarly′

introduction of an ilnaginary part is required due to the existence of some nonresonant

absorptions.Alsc if χNR alters sigttficantly with frequencyr it has to be included in the

resonance part of χ.Here′ only resonant CARS situations are treated whiCh can be described as

χ=臨 +端 )+br+尋

獅 =為 +臨

where χ爆′χんR are real const,ntS numbers.By subsituting Eq.3-10 in

which describes the spectral shape in resonant CARS situation as

I =lxl' = xi =S, +S, +S3 +Sa

(3‐11)

Eq. 3-4, an equation

SI=lχNR「

‐77‐

(3-12)


S2=繹

S3=

S4=

0‐ike Eq.3-11′ dle equatton cOnsists Of fOur terms.The first three te...ls Of ISl′ s2and S3represent the abs01ute square of the nOrlreSonallt background susceptibility7 a dispers市

e crOss

term/a Lorentzian cuⅣ e as well as Pl′ P2and P3・ The difference is the fOuri term and its

spectral shape is a negOtive Lorentzian curve as shOwn in Fig. 3-4 [21]. As a result Of

superposition of dFse cOntributions the peak Of the resOnant(]ARS spectmm shifts.The amOung

of the shift alters depending on the experlmental cOnditiOns such as h specific vibration under

observatiOn and the concentratiOn Of the sample.

400

′{ω )

200

Figure 3-4 Calculated spectral shape of term 51, 52, S, and So

CARS under resonant condition. Xkn = lS, Xka: S, A = _ 10, Bwere used to obtain these spectra[ZL].

in Eq。 3-12 and

=5 alld■ =1

０

み

・78‐

Ｓ　免

し:ダ耳

Chapter 3, Resonant CARS for cytochrome c obseraation

electronicalexcited states

CARS (or.o.r)

pump beam (ur,)

Stokes beam (olr)

Vibrational state

Ground state

Figure 3-5 Process of resonant CARS. The signal (908 - 915.5 nm), L054 nm, and 532 nm

picoseconds pulse lasers were used for pump, stokes, and probe beam respectively' The

generated CARS signal was 489.9 - 492Jnnr'

In this research, I performed resonant CARS measurement by matching the GARS

frequency with the electronic transition frequenry of a sample' For the sample, cytoclrrome c was

used. Fig. 3-5 shows the energy transition Process driven for resonant CARS' For pump and

Stokes beam, picoseconds pulsed lasers with wavelength of -910 nm (908 - 915'5 nm) and L064

nm, respectively, were used to resonate with one of the strongest Raman band at 1585 cm-l from

cytochrome c. In order to obtain a resonant Raman effect, a 532 nm wavelength picoseconds

pulse laser was used. for probe beam. When the foci o{ the three different lasers colors are

overlapped spatially and temporally at the sample plane, CARS signals are generated at around

4g0 nm (4sg.g - 4g2.1nm). This wavelength should be absorbed by rytochrome c for resonant

Raman enhancement.

3-3. optical configuration of resonant CARS microscope

Fig. 3-6 represents a schematic image of the optical configuration for the resonant CARS

microscopy. In the setup, L054 nm picoseconds pulse laser is emitted from Nd:Yag laser and 532

nm picoseconds pulse laser is also emitted by doubling the 1064 nm laser in same laser system'

'79-

Chapter 3, Resonant CARS microscopy for realiime imaging

The OPo in■e Optics mainly consists of a nonlinear crystal within a cavity.In the crystat

■e inddent 532 11111 PulSe laser was cOnverに d into ttO different frequencies called signal and

idler beam.The cavity was chOsen tO be resonant wi■ the signal and cOherent signal and idler

output was generated.I used the signal beam for the exPeriment and b10cked the idler beam at

the exit Of tte OPo.The Output frequency was determined by phase matching condidOn as

shOwn in Eq.3-13.

ち吻 =れ"J+λ

″α (3-13)

where kp*p, kris*1, drrd k,o,n ate wavenumber of pump laser, signal beam, and idler beam,

respectively. The advantage of using an OPO is that it is possible to change the wavelength ofoutput beam continuously over a large wavelength range, by changing the temperature of thenonlinear optical crystal and by adjusting the cavity wavelength. Since the OpO laser pulsesderive from the same initial pulse, problems with timing jitter and the repetition rate differencesare circumvented. The adjustrnent of angle or temperature of the nonlinear crystal is adjusted tosatisfy the phase matcJring condition. Since the origin of the OpO lasers are the same, the pulsewidth and the repetition rate are completely the same, and they are approximately 4 picosecondsand 78 MIlz, respectively. This feature made it easier to overlap every pulse temporally.

The three different color pulse beams were overlapped spatially before the microscopebody. To achieve this, I overlapped 1054 nm beam with the signal beam from the opO andoverlap of 1064 nm pulse laser with the 532 nm pulse laser were done separately. For the spatialoverlap of 1054 nm pulse laser with the signal beam from the OpO, a dichroic filter, whichtransmits 1064 nm and reflects signal beam from the OPO, was used. The spatial overlap of l0G4nm pulse laser with the 532 nm pulse laser was done by using an edge filter which transmits L064

nm and reflects 532 nm (the signal beam also transmits the edge filter).

The beams of the three pulsed lasers overlaps not only spatially but also temporally at thesample plane to generate CARS, by overlapping 1064 nm pulse laser with the signal beam fromthe OPO and overlap ping L054 nm pulse laser with the 532 nm pulse laser separately. Thetemporal overlap of 1064 nm pulse laser with the signal beam was achieved by adjusting theoptical path length of the signal beam using delay stage 1 and by monitoring autocorrelationusing a commercially available auto-correlator. 532 nm pulse laser was overlapped with 1064 nm

‐80‐

C力α′`″

3′ &sθ 4′πι CARS/o/c1/10C力 γο“ι θ θbs`″α′われ

filters for blocking

Ａ＝Ｖ

畢

ＢＢ。

Ａ＝Ｖ

ＡＶ

口Ｈ国ＰＤ

,‐‐……CrOSS co‖erator‐ Ⅲ……

:

auto correlatol

excitation light‐|ヽ「¬PMT

condenser lens

piezo stage

objective lens

/1鬼耽:l‖鷲=∵

;1壁Fn

Edge filter

Dichroic filter

Figure 3-6 Optical configuration of resonant CARS microscope'

- 1064 nm or signal (-910 nm)

- 532 nm

Figure 3-7 Telescope for chromatic abberation comPensation. A: Before compensation.

Two beams does not overlap completely at the focus. B: After compensation' By moving

one of the lens in the telescope, focus position of.1064run or the signal laser also moves and

overlaps with 532 nm beam.

lde!ay 2

‐81‐

1064 nm 泄

OPO(908-916 nm)

3諭」鈴:電sg～4 psec′ 78MHz


pulse laser temporally by using a home-built cross-correlator. Both beams are reflected by a

silver-coated mirror (this mirror was removed when CARS was measured) after the edge filter,and then sent to the cross-correlator. In the cross-correlator, both beams were focused on a BBO

crystal by a plano-convex lens. When the both pulse lasers temporally overlaps each other bychanging the optical path length of 532 nm pulse laser using the delay stage 2, SFG generated

from the BBO crystal and was detected by a photo-diode through two lenses which were mad.e ofCaF"' In order to satisfy phase matching condition to generate the SFG, the polarization directionof each beam was set same by rotating a half-wave plate in the optical path of 532 nm pulse laserand the angle of the BBO crystal was adjusted. In the setup, the telescopes were also installed inthe optical path of 1064fuxm and the signal beam to overlap their foci at the sample plane bycompensating chromatic aberration as shown in Fig.3-7.

After all pulsed laser beams overlapped spatially and temporally, they were introducedinto microscope body and focused on the sample by an objective lens. Forward generated CARS

signals, which including other signals based on nonlinear optical phenomena and excitationlasers, were collected by a condenser lens and detected by a PMT (Photomultiplier Tube) throughfilters which passed only required CARS signals. Imaging was done by scanning the microscope

stage in two dimensions.

In order to verify that CARS signals were generated with the setup constructed by taking

Figure 3-8 CARS images of 10 11m Polystyrene beads Obtained by the resOnant CARS setup.

Only when a11 lasers(The signaし 1064-/and 532 nm picosecond PulSer lasers)were

tunled on/■e distribu■ on Of POlystyrene beads was Observed.A:All lasers ON.B:Signal′

OFF′ 1064 rlm′ ON′ 53211m,ON.C:Signal,ON′ 1064叫 OFL 532 nm,ON.D:Signal′ ON′

1064 rlm,ON′ 532 nmメ OFF.Excitauon intensity:OPO′ ～139.3mW/1Шl年 1064 nm′ ～38.5

mW/pmL 532 rlm′ ～21.9mW/pm2.Image acquisitiOn dme 20 secondso ResolutiOn:512x

512 pixels.

すごす同国■■■δ〓

‐82‐

C力α′たγ3′ R`sO“α“

′CARS/oγ りめεた70“ι ε θbS`″α`:θ“

images of POlystyrene beads as shOWn in Fig.3-8 were recordedo When the sample wasirradiated

with a■ ■e pulse lasers′ the distribuaon of lo μm polystyrene beads was obseⅣ ed(Fig。 3‐8A).

on d■e other hand′ when one of dle lasers was blocked′ no image contrast was Obtained(Fig.

3-8B‐D).From these results′ it is PosSible to conclude■ at the signals in Fig.3‐ 8A were derived

frOm CARS of Polystyrene beads(the cARS signal was not resonant CARS because polystyrene

beads does not absorb any ofthe excitation hght).

3-4. Enhancement of cARs signals from cytochrome c

I measured cytochrome c with the setup.To prepare the sample′ I first dissolved cytochrome c

POWderin DDW.The concentration was 30 mM.The cyto山ome c solution was dropped on a

cover glass sliP.TheL I Put anOther cover glass shP on h S01ution to lnake the samPle thiCkness

hin(aS ShOWn in Fig.3-6)becauSe it was not possible to detect CARS signals in dlick samples

due to the absorption of dle CARS signals by the samPle.The waveleng」■ofthe signal beam was

set around 912 nmo As PolyStyrene beads′ only when dle sample was irradiated with allthe lasers′

signals were detected from whole area as shown m Fig.3-9.Thus′ I confirmed that nonlinear

reSPOnse was obseⅣ ed from the cytottome c solution.

Figure 3‐9 CARS images of 30 mM cytottome c solution obtained by the resonant CARS

setuP(512× 512 PixelS)・ Only When alHasers(The signaL 1064ふ,and 532 nm picosecond

PulSer lasers)were turned oL■ e distribu■ on of Polystyrene beads was observed.A:AⅡ

lasers ON.B:Signal,OFF′ 106411mジ ON′ 532 nm,ON.C:Signal,ON′ 106411m,OFF′ 532 nm′

ON.D:SignaL ON′ 1064m′ ON′ 532 rlln,OFF.Excitation inteFISity:OPO,～ 139.3

mw/.m2′ 1064 nm′ ～38.5mW/pm2′ 532 nm′ ～21.9mW/Fm2.Image acquisidon time:20

seconds.Resolution:512× 512p破 els.

すごす口四■■■び〓

‐83‐

Chapter 3, Resonant CARS microscopy for resl-time imaging

In Fig' 3-9A, not only CARS signals but also nonreonant background signals shoutd becontained. ln addition, fluorescence signals which were generated by the processes shown in Fig.3-10 also might be contained because the energy state of cytochrome c was excited to the real(electronic excited) state [42]. In order to investigate the existence of the fluorescence signals, Iput a band-pass filter, which opened for 500 - 540 nm, in front of the pMT. Since CARS and thenonlinear background signals should be narrow and the wavelength of them were around 490

nm, all of them should be blocked with the filter. In contrast, the fluorescence signals should bebroad and it is not blocked by the filter as shown in Fig. 3-114. When the filier was put, almostall signals disappeared as shown in Fig. 3-118. From this result, it can be insisted that almost allthe signals in Fig' 3-9A was derived from CARS and nonresonant background signals. (Since thefilter also may block a part of the fluorescence signals in this experiment, the signals might be

stronger' Also, since the fluorescence signals reflects the information of target molecularvibration as well as the CARS signal, generation of this signal is not matter for the purpose ofobserving cytochrome c. However, to consider the enhancement of the CARS signals by resonantRaman effect as shown in the next section, investigating the existence of the fluorescence is

important.)

electronicexcited states

pump beam itl,)

Stokes beam (atr)

Vibrational state

Ground state

Figure 3-10 Process of fluorescence generated with three pulse lasers. Wilh the pumpand Stokes beams, the population at the excited vibrational state in ground state is

increased' Then, the population is excited to electronic excited state and relaxed to the

lowest level in the electronic excited state. Finally, fluorescence signals which reflectsthe vibrational information of the molecular bond (ao - a) is generated.

probe beam (o,)*__..-

'84-

A 490nt, CARS, nonresonant background (narrow)

Chnpter 3, Resonant CARS for cytochrome c obseraation

blocked

wavelength [nm]

Figure 3-11 A: Schematic image of relationship the wavelength of CARS nonresonant

background signal and the fluorescence generated by the three pulse lasers, and

band-pass filter put to block CARS and nonresonant background' B: when the

band-pass is put, almost all signal disappeared. This implies that almost no fluorescence

was generated in cytochrome c measurement by the resonant CARS setup.

To make sure that the signals in Fig. 3-9A contains CARS and nonresonant background

signals from cytochrome c and to know the wavenumber of the peak position of signals from

cytochrome c by resonant CARS measurement, I compared CARS spectra of cytochrome c

solution with that of solvent, DDW. The spectra were obtained with the following protocol' The

concentration of the solution was 25 mM.

Take four CARS images (256 x 256 pixels) of the substrate. At that time, the focal spot

positionwas approximately 5 pm below the upper surface of the bottom substrate' Then

calculate the average intensity of every pixel in the four images'

Take four CARS images (2SO x 256 pixels) of cytochrome c solution. Then calculate the

average intensity of every pixel in the four images'

Tune the wavelength of the signal pulse laser and rePeat 1 and 2. The signal pulse laser was

tuned from 908 nm to 9L5.5 nm with 0.5 nm step'

Plot the average intensity as horizontal and vertical axes are Raman shift o and intensity,

respectively for the measurement of substrate (I) and cytochrome c solution (II)' Raman

shift was calculated by l/A =llap,no-11u5,o1,",)'

Calculate (the spectra of the cytochrome c / the spectra of the substrate)

すごす目

――圏■一δ三

IV.

V.

-85-


1

寵青.1

盤弯・1

く1

滝1

°0.

1560 1580Raman shift[cm」

]

1600 1540 1560 1580Raman shift[Cm・

]

1600

Figure 3-12 CARS spectra of A: 25 rl■ M cytOcllrOrne c s01utiOn and tlle substrate

measured by resonant(]ARS settP′ B:after caiculating ratiO of cytochrome c s01ution tO

that Of substrate′ C:CARS spectra Of DDW and the substrate measured by the typical

CARS setuP′ Di after calculating rati0 0f DDW 10 that Of the substrate. Excitation

intensity:OPO′ ～139,3mW/pmt 1064細町～38.5mW/μ m2′ 532 nm′ ～21.9mW/藤 lln2.

The purPOse Of Obtaining cARS spectra Of the substrate and the calculatiOn atthe prOcess V

was removing intensity difference Of the excitatiOn lasers among measurements at different

、vavelength of the signal beam.In fact the signal bean■ from oP⊂ )often fluctuated and alsO the

transmittance of the optical components such as dichrOic filter/edge filter alld obiectiVe lens

between the OPo and the saIYIPle′ fOr different wavelength Of the signal laser was different.

These ll■ ade the signal bealll intensity at the fOcus different all10ng different wavelengths.As a

result the spectrttm Of cytochrome c(Fig. 3-12A, solid line)sl10uld reflect the signal beam

intensity dttrerenceノ t00・ In Order tO remOve this factOr from the spectrunl as trlluch as possible′I

calculated ratio Of the spectra tO that of the substrate as described in the prOtOc01.This is because

the a spectrum from the substrate reflects the signal laser intensity difference as well as

cytochrOme c spectra(Fig.3-12A,dotted line)and it should be flat if there is nO signal beam

intensity difference. Fig. 3-12B shOws tl■ e spectra Of cytOchrOme c s01utiOn

ｎψ　　Ｏ｝●」一い０３∽ヽ）０ゼハ）

D

1560 1580Raman shifticrn■

]

1540 1560 1580 1600Raman shift icm¬

]

‐86…

Chapter 3, Resonant CARS for cytochrome c obseraation

pump beam (o:,)

Stokes beam (urr)

Vibrational state

Ground state

CARS (ul*rr)

Figure 3-13 Process of typical CARS. The signal (908 - 915.5 nm) laser was used for pump

and probe beams, 1064 nm picosecond lasers was used for stokes beam' The generated

CARS signal was792- 803 nm.

after the calculation and a strong peak was observed. The spectrum of DDW in Fig' 3-11D was

also obtained by the method (Raw spectra was as shown in Fig. 3-LZC). Unlike cytochrome c, the

spectrum of DDW was almost flat. Therefore, it can be considered that the peak derived from

cytochrome c in cytochrome c solution. The peak position was 1572 cm-l and this was shorter

than the peak position of the target wavenumber 1585 cm-l in this experiment. This is due to the

result of interference of the CARS signals with nonresonant background signals'

In order to investigate if enhancement of cytochrome c CARS signal was observed with the

setup, I compared the CARS signal intensity of cytochrome c measured by the resonant CARS

setup with that by a typical CARS setuP. In the case of the typical CARS, I used the signal beam

of the OpO as pump and probe beams, and 1.064 run as Stokes beam as shown in Fig. 3-L3' The

CARS signal wavelength was 792 - 803 nm (oPo was tuned from 908 - 915.5 nm). Fig' 3-t4B

shows the obtained spectrum obtained in a manner similar to that resonant CARS measurement

(Fig. 3-144 shows the raw spectrum of rytochrome c and the substrate). Unlike resonant CARS

measurement, some peaks appeared and these derived from cytochrome c because the spectrum

of DDW was almost flat as shown in Fig. 3-'1,4D (Fig. 3-14C shows the raw spectrum of DDW and

the substrate).

To know the enhancement of cytochrome c CARS signal by resonant Raman effecL it was

also necessary to take into account the sensitivity difference of the PMT for CARS signals with

the resonant CARS setup and the typical CARS setup in addition. In order to calibrate the

‐87‐

Chapter 3, Resonant CARS mi*oscopy for real-time imaging

Figure 3-14 CARS spectra of A:25 1nM cytochrOme c solutiOn and the substrate measured

by typical cARS setup′ B:after calculating rauo of cytOchrome c sOlu■ On tO that Of

substrate′ C:CARS spectra of DDW and dle substrate measured by dF typical CARS setuP′

D:after calculating rato of DDW to■ at Of dle substrateo Excitation intensity:OPO′ ～139.3

mW/pm:′ 1064■■ ～38.5mW/Pmち 532 nm′ ～21.9mW/pm2.

wavelength [nm]

Figure 3-15 Absorption spectrum of pure benzonitrile.

Ｄ）　

ｏ一２

一い０コ∽ヽ

Ｓヽ●

６

１

１

１

１

１

０

０一四場０ついヽυｏゝ

υ

1560 1580Raman shift[Cm-1]

‐88‐

C力α′′ιr 3′ Rθsθ 4α″ CARS/oγ ッ"arOttι

ε οbsιγυα′jOη

sensitivity of the detector, I compared the peak intensity of benzonitrile measured by the

resonant CARS and the typical CARS. Since CARS signal intensity of benzonitrile should not be

enhanced by the resonant CARS setup because all of PumP, Stokes and CARS signal were not

absorbed by it as shown in Fig. 3-15. Fig. 3-15 shows the CARS spectra of benzonitrile measured

by the resonant CARS setup and the typical CARS setup. They were obtained in the same

miurner as that of cytochrome c. The peak intensity measured by the typical CARS was almost

2.g4 times stronger than that measured by resonant CARS. In principle, th"y should be the same

or the CARS intensity measured with resonant CARS setup is stronger because more excitation

laser is irradiated to the sample. By taking into account this informatiory I compared the

spectrum of cytochrome c by resonant CARS setup with that by typical CARS setup as shown in

Fig.3-LT.Apparently, the peak intensity of GARS signal from rytochrome c increased as a result

of combining the resonant Raman effect with CARS'

- resonancecARS

- - - typicalCARs

1540 1560 1580

Raman shift [cm-]]

Figure 3-16 CARS sPectra of pure benzonitrile. The CARS signal intensity measured

by the typical CARS setup was approximately twice higher than that by the resonant

CARS setup. Excitationintensity: OPO; -139.3 mW/lrm2, 1064nm; -38.5 mW/pm2'

′

′‐

‐

‐

‐

‐

‐

‐

か

「

、‐

、‐

、

『コ．‘］ゝ〓∽Ｃ〇一Ｃ一

‐89‐


Raman shift[Cm‐ 1]

Fi〔晏re 3-17 COmparison of CARS spectra from cytoふ rome c Obtained by the resOnant

CARS setuP and dlat by■e typical CARS setup.Excita饉 on intensity:OPO′ ～139。3

mW/pm%106411m′ ～38.5mW/1m:′ 532 run′ ～21.9mW/pm2.

3-5. Detection limit of concentration

I investigated the detectiOn limit Of resOnant CARS signals of cytochrome c.Resonant CARS

spectra of 3Q 2島 20′ 15 and 10 mM cytochrome c s01ution were lneasured as shOwn in Fig 3-18。

However′ no peak appeared be10w 15 1nM.

The cytOchrOme c s01utiOn l have measured existed in oxidized fOrm because it is easily

oxidized in the air cOnditiOno HOweveL since cytochrOme c exists in reduced state in a living cell

generaltt inVesugating of dle detecuon limit of reduced cytochrOme c is necessary tooo h order

to reduce cytochrOme cr ascOrbic acid was added to lhe Oxidized cytochrOme c solution to lnake

the final cOncentration 20 mM.As shOwn in Fig。3‐1%abSorbance around 490 11m Of reduced

cytochrome c was approximately half Of that Of the oxidized state.Fig。 3-20 shOws cARS spectra

of oxidized and reduced cytochrome c.when cytochrome c was reduced′ the spectral shape was

distorted.ThOugh the absOrbance of reduced form islower than oxidized fOrm′signal intensity Of

reduced fo...l seems to be a bit stronger than that Of Oxidized fOrm but they are still comparable。

The difference can be attributed to a higher intrinsic Ramalll intensity of d℃ reduced fOm

COmPared tO the oxidized fO...l as mentioned in chapter 2. I‐ IOweveL the concentration Of

cytochrorme c m cells is approxllnately 10 11M.TherefOre/1 cOncluded dlat detecting cytochrOme

‐90‐

Chapter 3, Resonant CARS for cytochrome c obserz:ation

c in cells with the resonant CARS microscopy was difficult [43]. on the other hand, resonant

Raman scattering can detect even when the concentration of rytochrome c solution is at several

tens of pM order. For example, Raman peaks from cellular cytochrome c is detected clearly with

532 nm excitation as shown in Fig. 2-L0. In order to comPare the sensitivity of resonant Raman

scattering with that of resonant CARS here, Raman peak intensity at 1585 cm-1 with -490 nm

excitation should be used. In Fig. 2-8, the peak intensity at 1585 cm-1 with -490 nm excitation was

comparable to 532 nm excitation. These results implies that resonant Raman scattering is more

suitable to observe cellular cytocfuome c than resonant CARS.

一一-30 mM

一―-25 mM

20 mM

一一-15 mM

一一-10 mM

――― DDW

Figure 3-18 Resonant CARS spectra of cytochrome c solution at different concentration'

When the concentration was below l'5 mM, CARS signals from cytocJrrome c was hard to

detect.

490 nm

|

460 480 500 520 540wavelength[nm]

580 600

Figure 3-19 Absorption spectra of reduced and oxidized cytochrome c '(20mM)'

［。っ。●］沐〓∽Ｃｏ一Ｃ一

［．コ．∞］ｏ∪Ｃ∞０」０いＯｏ

440420400 560

Raman shift [cm-']

‐91‐


Figure 3…20 Resonant CARS spectra Ofreduced and oxidized cytOchrOme c.

In tllis cllapter′ I PerfOrmed resonant CARS measurement Of cytOchrOme c. When the

concentratiOl1 0f cytochrome c sOlutiOn was above 15 mM′ resOnant CARS signals were detected.

In order to illlPrOVe the sensitivity of the llleasurerllent fOr example′ using triPle reSOnant

conditiOns would be better:411.Also′ rerrloving nonresonant background sigl■ als is important tO

realize higher sensitivity detectiOn.For suppression Of the nonresOnant background signals′a

variety of techniques such as epi― detected CARS′ polarizatiOn CARS′ time―res01ved CARS′ andheterOdyne CARS have been proPosed i44-47].

In additiOn/when resOnant CARS technique is used for imaging of cellular inolecules′the

concentration of the trn01ecules is quite important fOr the measurellylent sensitivity.1離is is because

CARS signal intensity has quadratic dependence of lie number Of vibratiOnal oscillatOrs in

princiPle lll. When sample concentration is higL resonant CARS sensitivity increases

draIItatically.In cOntrast When satrnPle cOncentratiOn is 10w′ the sensitivity is lower than resOnant

Raman scattering because resOnant Raman scattering intensity is prOportiOnal tO salylPle

concentratiOn.TO cOmpare the sensitivlty of resOnant(=ARS with resOnant Raman scattering by

theOretically′ both real and ilYlagil■ ary part of third― Order nOlllinear optical susceptibility χ(3)。

f

cytocllrome c are required because resonant CARS intensity is proPortiOnai to square Of χ(3)and

resonant Raman scattering intel■ sity is proportiOnai tO imaginary part of χ(3).

Raman shift [6p"t1

‐92‐

c力甲たγ 3′ Rθsθ

“

α″ CARSル γッわε力rOzι ε θbS`惚α

`jθ“

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Raman scattering(E‐ CAItS)miCrOscope with high spectral resolution and high sensitiVity7〃

The JOumal of Physical Chemistry B.105′ 1277-1280(2001).

J.―X・ Cheng/L.D.Boot&X.S・ Xie′″Polarization coherent anti― Stokes Ralnan scattering

microscOPy′″Optics Letters。 26′ 1乙 1341-1343(2011).

A.Volkmer/L.D.Boot&Xo S・ Xie′″Time― reso市ed coherent anti― Stokes Raman scattering

microscopy:haging based On Raman free induction decay′″APPlied Physics Letters.80′ 9′

1505′ (2002)。

E.Q PotOmar Co Lo Evan勁 &X.S.Xie7″ Heterodyne coherent anti― StOkes Raman scattering

(CARS)imaginレ″Optics Letters.31′ 2241-243(2006).

47.

‐97‐

Chapter 4, Raman tag imaging of cellutar molecules

Chapter 4,

Raman tag imaging of cellular molecules

Nowadays the capability ofimaging and analyzing molecular dynamics wi■oPical micrOscOPy

are exPanding.One ofふ e mOst crucial challёnges in biological imaging■ eld is visualizauOn of

small cellular molecule,which has Only several tens tO hundred6 of m01ecular weights.In Past

days capturing the dynamics in a■ ving ceⅡ has been impossible even with well established

imaging tednique such as■ uorescence micrOscOpy.This is because the size and molecular

weight Of■ uOrescent probes are comparable or even larger than■ e small molecules arld■ e

funcuOns Of the small mOlecules are disturbed.TherefOrQ a tiny tag is desirable for imaging of

small molecules.h this chapteち Iintroduce an ima〔興lg technique using a tiny tag/called Raman

tag.Atthe beginning′ the optical and inorpho10gical features of Raman tag is exPlained.Thett I

shOw an application using Raman tag and discuss whetter Raman tag disturbs the functiOns Of

tagged molecules.

4-L. Raman tag: the optical and morphological features

As Raman ta,lM70 important properties are required.One is that dle tag should be tiny as

mentioned abOve.Even a tag ls tiny/RamarL microscOpy can detectit because Ralrlan microscOPy

observes molecular vibration.The Other required PrOperty is that a Raman tag has an unique

peak to avoid overlapping with Raman peaks■ Om original bi010gical mOlecules in a cell.

Attne′ nitrilQ azide Or deuterium can be candidated fOr a Raman tag.Table。4-l shOws

each structure its m01ecular weight and Peak POSiuon ll′ 2].Al1 0fthem cOnsist only a few atOms

and has small molecular welght u」ike cOnvendollal■ uOrescent molecules.In addidOnr each

sttucture has a disinct Raman peak in silent reglott where nO Raman peak from Orlglnal

bio10gical molecules from cen exist as shOw in table 4‐ 1(Depending on the kind Of atOm

COniugated wi■ Raman tag/its peak PosidOn shi■s but still in silent regiOn)。 Thutt the

‐98‐

Chapter 4, Raman tag imnging of cellular molecules

Table 4-1 Candidates of Raman tagl1.,2l

combination of Raman tag with Raman microscopy lealizes minimum disturbance by labeling in

live cell observation. lndeed, Raman imaging of fully deuterated fatty acids has already been

reported [2].

4-2. Meas:lrement of a spectrum of Edu, an tagged cell proliferation

probe

ln this research′ I attempted to prove the concept of Ralllan tag by using alkyne as a RaIItan

tag. For the delllonstration of Raman ilnaging using alkyne tag′ I used EdU aS a model

compound(Fig.4-lA).EdU(5-ethynyl-2′ ―deoxyuridine)iS alkyne tagged dU(2′―deoxythymidine′

Fig.4-13).dU haS quite similar structure with dT(2′―deoxyuridilte′ Fig.4-lC)whiCh iS

incorporated into cellular DNA durillng DNA replication i3].ThuS′EdU is also incorPorated by a

cell via same pathゃ vay 、vith dT and acCullnulates in nuCleus finally. The fact tl■ at EdU is

incorporated into cell nucleus can be confi■ lmed by click chemistry as shOヽ νn in Fig.4-2.In click

chetrnistry′ EdU is incorporated and accullllulates in nucleus of a living cell.Then/the cell is fixed

peak position [cm-']

盗:…⊂≡C― 胸2alkyne

N―三N十_N

2220～ 2250

ね、m2-⊂一D

/胸t

deuterium

‐99‐

Chapter 4, Raman tag imaging of cellular molecules

B 。

OH

C

HO

Figure 4-l Structures Of EdU′ du and dT.EdU has all alkvne(highlighted in red).

and fluorescence probe/which cOnnects with EdU selectively′ is intrOduced into the cell.Then/

the distributiOn Of dle EdU is Obtained by observing the fluorescence probe with a■uOrescence

microsc6pe.By comparing Fig 4-2 A and B′ EdU is acculnulated in cell nucleus apparently.In

contrast to click imaging′ EdU with Raman■ licroscOPy dOes nOt require fixing′ and this lnakes it

possible to Observe the dynanlics Of EdU in a Lving cell.

Fig.4… 3 shows Raman sPectra OfbOth sample and EdU shOwed Raman peak at 2122 cn■ ~l in

silent region while dU did not shOw any Peak in ie region.This result suggests that the Raman

peak at 2122 cm‐ l was derived from■e alkynein EdU.

Figure 4‐2 Fluorescence images of a HeLa cellcultured with EdU.A:stained with nucleus

marker7 HOechst 33342.B:stained with nucleus lnarkeL EdU― Alexa647.C:merge of A and

B.D:rnerge of A′ B and a bright― ■eld image.

‐100‐


alkyne5du

<- silent region representativecellular signal

2000

Raman shift lcmll

Figure 4-3 Raman spectra of EdU, dU, and a living HeLa cell' EdU shows a Rar:ran peak

at 2123 cm'l and dU does not have any peak in silent region' The Raman peak in silent

region in the spectrum of the cell is derived from nitrogen'

4-3. Imaging of EdU in a living cell

In order to investigate whether the signal and/or the distribution of alkyne in EdU can be

detected in a living cell, I incubated Edu (loading concentration: 20 pM) with living HeLa cells

for 6 hours and obtained Raman scattering images of the sample by a slit-scanning Raman

microscope. The solution was replaced to a HEPES-buffered tyrode's solution before Raman

imaging. The intensity of incident laser at the sample plane was 3'52 mW/pm2 and image

acquisition time was 49 minutes. Fig. 4-4A shows intensity distribution of Raman peaks at 749

cm-tfrom pyrrole breathing mode in cytochrome c,2123 cm-1 from alkyne in EdU, 2849 cm-1 CHt

bond in lipid and merge of them, respectively and Fig. 4-48 are those of living HeLa cells without

treatment of EdU were also obtained as a control. Apparently, Raman peaks at2123 cm-1 locate in

the nucleus o{ the living HeLa cell with EdU treatment and there is no contrast of the peak in the

control while disiribution of cytochrome c and lipid did not show particular dif{erences between

the samples. I also obtained the averaged Raman spectra from 10 x 10 pixels in he cytoplasm and

nucleus in each sample as shown in Fig. 4-5. Only in the spectra of nucleus in the living HeLa cell

with EdU treatment, Raman peak at 2123 cml from alkyne bond of EdU was observed and no

peak at 2123 cm'1 was observed in other Raman spectra. These results suggested that the

［．っ

．●］

沐

〓

∽Ｌ

Ｃ一Ｃ

一Ｃ

●

Ｅ

ｏ∝

-101-


Figure 4-4 Raman imaging of EdU in living HeLa cells. A: Raman image obtained fromHeLa cells treated with EdU. B: Raman image obtained from control HeLa cells. Blue: 249

cmi, Red: 2'!'23 cm'l, Green: 2849 cm-l. The light intensity at the sample plane was 3.52

mW/Fm2. The exposure time of each line was 20 second.s. The total number of lines ofexposure was 127 for image A and 1'4'l' for image B resulting to an image acquisition time of42 minutes and 47 minutes, respectively.

すごす口日□■■δ〓

‐102‐


2123 crn‐1

|

8oo 1200 1600 2000 24002600

Raman shift [cm-t]

Figure 4-5 Average Raman spectra obtained from cytoplasm or nucleus (L0 x 10 pixels) of

HeLa cells. A: HeLa cells treated with EdU. B: HeLa cells treated without EdU. The nucleus

of Edu treated cells showed a Raman peak from alkyne in Edu at 2123 cm-l'

Raman signals and the distribution of alkyne of EdU in a living cell was imaged successfully and

it is possible to observe distribution of molecules in a cell using alkyne tag selectively.

In order to investigate the potential utility of our Raman imagrng technique using alkyne

tag, I investigated the detection limit for the loading concentration of EdU. As results the minimal

concentration was approximately 5 pM (Fig. 4-6). In additiorU I found that the fastest image

acquisition time with the slit-scanning Raman microscope was aPProximately 14 or l'L minutes

for one image with 20 pM of loading concentration (Fig. 4-7A artd B). These results show that the

Raman imagrng technique with alkyne tag has sufficient sensitivity and imaging speed to make it

useful for real time detection of small molecules in a living cell.

［．●．二

、一一∽ｃｏ一Ｅ

ＣｏＥ

“∝

［．コ

。“］

ゝ一一いＣ

ｏ

一Ｃ

一Ｃ

”

Ｅ

Ｃ∝

‐103‐


2250 2300

Figure 4‐ 6 DetectiOn lilnit of EdU in living HeLa cells by Raman nlicroscopy.Loading

concentration was 5 11M with incubation time Of 21 hours. The ilnages shOw the

distribuuons of cytOchrOme c(749 cm~1)′ EdU(2124 cm‐ 1)′ liPid(2849 cm~1)and an overlay

ofthem.scale bar is lo llmo Raman sPectruln is averaged over 30× 30 PixelS inset dle image

of lipid.The excitadon laser intensity was 4.2mW/pm2 and the exPOSure dme was 20

seconds Per line.The slit was setto 50 11m.

Figure 4-7 Raman images of living HeLa cells cultured with 20 pM EdU with shorterimage acquisition time. The images show the distributions of cytochrome c (249 cm-l), EdU(2124 cm-l),lipid (2849 cm-l) and an overlay of them. Scale bar is 10 pm. Raman spectrum isaveraged over 30x30 pixels inset the image of lipid. The excitation laser intensity was 3.4

mW/pm2 and the exPosure time was 5 seconds per line. Image acquisition tirne was 11

minutes. The slit was set to 50 gm.

８

６

４

２

０

［．コ．Ｃ］沐〓∽Ｃ〇一Ｃ一

alkyne

2100 2150 2200Raman shit[cm・

]

10W― lhigh

2050 2100 2150 2200 2250 2300

10Wtthigh

‐104‐


4-4. Raman tag disturbs the cellular function?

In order to investigaie EdU is incorporated into living HeLa cells ai proper timing, I also

performed time-course Raman observation of EdU incorporation into living HeLa cells' For this

experimen! a 40 x11.15 N.A. objective lens (olympus) was used to observe many cells

simultaneously. The intensity of incident laser at the sample plane was 6 mW/um2 and image

acquisition time was 26 minutes for each Raman image' Fig' 4-8A shows a Raman image of living

HeLa cells without Edu treatment and Fig. 4-9B-F show Raman images of living HeLa cells

incubated with Edu (loading concentration: 20 pM) fot 3,9,12,14,21 hours respectively' without

EdU treatment, no cell showed a Raman signal at2123 cm-t as shown in Fig' 4-8A' Depending on

cell proliferation, the number of Edu-positive cells grew up with time as shown in Fig' 4-88-E

and almost all of the cells showed Raman signals from EdU in nucleus after 2l hours as shown in

Fig. 4-gF. These results agreed with the reported doubling time of HeLa cells [4]. Therefore, l

concluded that alkyne tag did not disturb the molecular dynamics in living HeLa cells.

As shown in this chapter, Raman imaging technique with alkyne tag was applied to

observe small molecular distribution in a living cel1. This time, EdU, which is alkyne tagged dU,

was imaged. The result implies that Raman tags including alkyne tag can be applied to other

small molecules such as DNA bases and drugs. However, several tens of minutes are required for

imaging. The imaging speed would increases by using structures that are linkage of some

alkynes such as diynes, triynes and so on as Raman tags' In this case, molecular weight of a tag

increases, too. Thus, investigating how many alkynes can be connected is importanl' In addition'

faster imaging using Raman tag might be promising with SRS or CARS' By combining other

types of Raman tag, multi-color imaging also would be possible. Furthermore, I expect to use

Raman tag in tissue imaging. In2007, C. R. Bertozzi has succeeded in copper-free click chemistry

with tissues [5]. Thus, small Raman tags including alkyne are able to be introcuced in a tissue' To

detect signals {rom Raman tags in a tissue, using longer wavelength light such as near-infraed

light might be necessary to avoid autofluorescence from a tissue and low invasion depth'

‐105‐


Incubation time [hr]912

Figure 4-8 Time-course imaging of EdU incorporation in living HeLa cells. Loadingconcentration of EdU was 20 pM. The images at each time show the distribution ofcytochrome, alkyne, lipid and an overlay of them. 40x, 1.15NA objective lens was used. The

total number of lines of exposure was 120 for each image. Exposure time was 10 sec/linelaser intensity :6 mw/pm2 and image acquisition time was 26 minutes.

ＴｒＣ）〇崎卜

υ

ＯＥ

Ｏ

〒″こυ

寸倒ｒ一

⊃ＯШ

一卜に）０寸∞Ｎ

０一Ｑ一コ

一Ｑコ

⊃ＯШ

∪ すごす［固■■■び〓

‐106‐

１

　

　

２


References

浜口宏夫′平川暁子′ラマン分光法,日本分光学会 (1988).

H―.J.Van Manen/A.Lenferink&C.OttQ"Noninvasive imaging of prOtein metabolic

labeling in single human cells using Stable isotopes and Ralllllan microscoPy"Analyical

Chemistry′ 80′ 249576-9582(2008).

Ao Sahc&T.Jo MitChison7″A chemical method for fast and sensit市 e detection of DNA

synthesis in v市 0″′Proceedings of■ e National Academy of Sciences USA′ 105′ 2415(2008).

Zo M.Mu′ X.Fo Le′ S.Valhan/A.B.Glasslnan&Ko S.Chang′″Stable overeXPreSSiOn of

IDML alters regulation of Cell cycle progression lll HeLa cells′′′ Carcinogenesis′ 18′ 11′

2063-2069(1997).

J.M・ Basktt J.A.Prescheち S.T.Laughlitt N.J.Agar↓ P,V.Chan3 1.A.M■ leL A.LQ Jo A.

Codellち &Co R.Bertozzち〃COpper‐ free dick(ielnistry for dy,amic J“ υjυθ imaging〃′

Proceedings of dle National Academy of SCiences′ 10416793-16797(2007).

4.

‐107‐

Conclusians,

Cθπε″“

sづο″s(レ D′sczssづθπみ

h■isrseartt IPerfOmedimagingandanalysisOfm01eculardynamicsinaliving∝Ⅱ by ushg

Raman microscOpyo Raman micr。,COpy pro宙des us plenty of informatton such as molecular

distributiOns′ the structuresr and its surrOunding envirOrments in a cell withOut labelingo This

feature would be promising to address deep understanding of bi010gical funcuOIIso Though the

imaging of l市e cell has been difficult due tO the 10w Ralnan scattering effidencyr combining

slit―scaming tettque with Ralrlan lnicrOscOpy achieved Raman imaging wid■several tO several

tens of lninutes tempOral res01ution.

With the Raman microscOp%I Performed the observatiOn of cytochrome c dynamics in cell

apoptosis.Cenular cytochrome c was imaged with high sensidvity by enhancing its Raman

scattering by resOnant Raman effect.Through Raman inaging of the apoptouc cellin ime the

release of cellular cytochrome c hm mitOchOnd五 a tO cytoplasm was Observed.I fbund that the

redox state of cellular cytottOme c was maintained during its release process by udhzing the

intensity difference between reduced and Oxidized cytOchrOme c.

For detecting cytochrome c with higher sensiti宙 ty/1 attempted to use resOnant CAItSo with

resonant Raman effect CAtt intensity Of cytOchrOme c sOlu■ on increased.HOweveし dle

detection limit with the resOnant CARS was several tO several tens Of mM.Since concentration of

cellular cytochrOme c is several tens Of llM I cOnchded that resOnant CARS is not suitable to

observe cellular cytochrome c。

I examined dle imaging capability of small molecules widl mOlecular weights Of several

tens to hundreds by using Raman tag.Among some candidates for Raman ta&I used alkyne as a

tiny tag.Akyne has an unique Raman Pet and it is detectable in a cell wid10ut interference

fЮm odler Raman peaks Of cellular biomoleculeso As a demOnstratiOL 1 10aded EdU′an

alkyne―tagged du cOmmonly used as a cellular probe for DNA synttsis in living HeLa cells and

observed it with Raman■ licroscOPy・ After 21 hrs cOrresPOnding to the HeLa cell cyde time

almOst an cells had EdU in d■ eir nudei.This result implies that alkyne did nOt disturb the

functiOns of du in■ e cells。

‐108‐

Conclusions,

As lnentioned above′ Raman miCroscopy is becolning a powerful technique in bi010gical

imagngo One Ofthe important issues is he low sensiivity.Even widl slit―scarning techniquら it

sun takes several mmutes at least.Therefore′ obseⅣing high speed molecular dynamicS Such as

inllnune reaCtiOn.Besides high excitation laser power is required in Ramall obServation.Thuシ

cells sometimes show Photo damages.In order to increase the sensiti宙ty7 increasing the

山 oughput of Raman miCroscope is requhedo The use of high sensitive Raman me■OdS such as

SERS′ CAItS and SRS might be promising′ toooln dT case of CARS and SRS′ single Raman peak

is observed.Another issue in RalrLan miCroSCOpy is loW molecular selecivity in imaging.ThiS is

due tO the overlaPS Of Raman barlds in fingerPrlnt region.Resonant Raman sCatterng and Raman

tag are possible lne■ods to overcome thiS issue.Resonant Raman scattering can be aPPlied tO

molecubs′ whiCh absorbs light such aS Cytochromes′ carotenoiC retinOL DNA bases and drugs.

Raman tag would exPand the molecular species which can be visualized in a cell by Raman

micЮscOpy EsPecia■y small molecules such as DNA and drugs also Can be visualized′too。

HoweveL analysis of rnolecular structure is not possible with Raman tag imaging teChnique.In

additott increasing spectral resoluion or sPaial resohion in Ramalll lIIlaging would helps to

increase d■e molecular species.

‐109‐

Lお′げ′“

bι J“サわ“

s′

List of publications,

Original papers

1' Masaya Okada, Nicholas I Smith, Almar F Palonpon, Hiromi Endo, Satoshi Kawata, MikikoSodeoka and Katsumasa Fujit4 "Label-free Raman observation of cytochrome c dynamicsduring apoptosis," Proceeding of the National Academy of Sciences Vol. 109, No.l, pp.28-32 (20L2).

3. Masaya Okada, Almar F Palonpon, Jun Ando, Katsumasa Fujita Nicholas I. Smith, andSatoshi Kawata, "Analysis of hyperspectral Raman data for label-free imaging of molecularcomponents" (in preparation).

Review paper

HiЮyuki Yamakoslt Kosuke DodQ Masaya Okad～ Jun AndQ Ahnar FKatsumasa Fuiite SatOshi Kawata and MikikO sOdeokら ″Imaging Of Edl鴫

tagged cell Prohferaion ProbQ by Raman microscOpル ″Joumal of Ame五 can

Sodety 133 6102‐ 6105(2011)。

山越博幸′間閥孝介′Almar F PalonPOL岡田昌也′安藤潤′藤田克昌′河田聡′

ラマン顕微鏡を用いたDNA合成プローブEdUの生細胞イメージング (L市e ceⅡ

Edu a DNA syndlesis pЮ be/by Raman microscOpy)′ ″ケミカルバイオロジー

Blology)第8号 vol t No2(2011)。

1. 平成24年度日本分光学会年次講演会若手ポスター賞

Palonpon,

an alkyne

Chemical

袖岡幹子〃

imaging of

(Chemical

Award

‐110‐


Intemational conferences

lo Masaya Okada′ Nicholas I SmiぜしAlmar F PalonPon/SatOShi Kawata7ゝ江ikiko Sodeokar and

Katsumasa Fui■ %″ Label―free Raman observatOn of Cytoぬrome c dynamicS in aPoptOuc

ce■s′″Jont Meeting ofISDB 451h&JSCB 64■ しWS4b-9&P3-115′ (Kobe′ 2012).

2. Masaya Okada7 Nicholas I.Smi」し Almar Fo PalonPoL SatOShi Kawat%Mikiko Sodeok～

and KatsumaSa Fuiita″ Label―free Raman imaging of CytoChrome c in Cell apoptosis′〃14th

intema■onal COngress of histochemistry alld Cytochemistry′ P2-15′ (Kyoto′ 2012).

3. Katsumasa Fuiit～ Masaya Okad～ Hiroyuki YamakoshL Ahar F Palonpon7 Mikik0

Sodeok～ Satoshi Kawatみ〃Ralnan miCroscoPy fOr molecular imaging ofl市 ing cells′

″芽折学

術領域シンポジウム′(TaiWan/2012).

4.Katsumasa Fuiita/HirOyuki Yamakoshち Kosuke Dodo′ Almar F Palonpon/MaSaya Okada/

Jun Ando′ and NIlikiko Sodeokar″ Raman imaging of alkyne as a small tag for biological

molecules′〃SPIE BIO勁 8225-41′ (San Francisco′ 2012).

Masaya Okad～ KatsumaSa Fuiita Nicholas I.Smidし and Satoshi Kawatar〃 Raman

microscope imaging of cen aPoptosis′″ Focus On MicrosCOPy 2011′ TU―AF―PAR―E′

(Konstarz′ 2011).

Masaya Okada7 Katsumasa Fuiita7 Nicholas I.SmiJ、 and Satoshi Kawata7〃 Dynamic

Raman observation of cytochFOmeS in apoptotic cells′〃SPIE BIOS′ 7902-3′ (Sarl Frallcisco′

2011).

Hiroyuki Yamakosht Kosuke Dodo′ Ahnar F PalonPon7 MaSaya Okad%Jun Ando′

Katsumasa Fuiitar SatOshi Kawata and Mikiko Sodeokar ″Click―free imaging of

alkyne‐ tagged molecule by Raman microscopy′″

11■ iCeMS interna■ Onal Symposium′

Chemical control of Cens′ (Kyoto′ 2011)。

Ahar F PalonPon7 MaSaya 9kad～ Jun Andor Hiroyuki YamakoshL Kosuke Dodo/Mikiko

Sodeoka/ SatoShi Kawata/ arld Katsumasa Fui■ ar ″Slit― scarlrung confocal Raman

microscoPy:practical applications in l市 e cen imagng/″ IQEC/CLEO Pacific Rim 2011′

2250-CT―C(Sydneル 2011)。

5。

7.

8.

‐

111‐

List ofpublications,

Masaya Okad4 Jun Ando, Hiroshi Kasai, Katsumasa FujitaKawat4 "Raman microscopy for label-free observation

Microscopy 2070, A27, (Shanghai, 2010).

Nich01as l smiふ ′and satOshi

of living cells/″ Focus On

9.

10. Katsumasa Fujita, Masaya okad4 ]un Ando, Nicholas I smith, and satoshi Kawata,"D)mamic Raman/sERS imagrng of living cells by slit-scanning microscop y,,, xrrICoR,ThP 054 (Boston,2010).

]un Ando, Hiroshi' Kasai, Masaya Okada Nicholas I Smith, Katsumasa Fujita and SatoshiKawata, "Metallic nanoparticles for enhanced Raman imaging of living biological cells,,,sth PARC symposium, 72, (Mie,2010).

Domestic meetings

1. 岡田昌也′Almar F PalonPOL Nicholas I.smi■ し河田聡′袖岡幹子′藤田克昌′″Raman

microscOpe imaging of cytOttrOme c dynaIIlics in APoptoSt″平成24年度日本分光学会年

次講演会′(東京′2012).

岡田昌也′Nicholas l smit Ahnar F PalonpOL河田聡 ′袖岡幹子′藤田克昌′″Raman

micrOscOpe imaging of non-labeled apoptouc cell助 ″OsA―JSAP JOint Symposiar(愛媛 ′

2012).

Ahar F PalonPOn7岡田昌也′河田聡′袖岡幹子′藤田克昌′″Complementary sPatal and

specttal analysis Of the hyperspecttal Raman data of ce■勁″OSA―JSAP Joint symposia(愛媛′

2012)。

山越博幸′間間孝介′Almar F PalonPOn/岡田昌也′安藤潤′藤田克昌′河田聡′袖岡幹子′〃低分子化合物のアルキンタグラマンイメージング′

″バィォ・ラマン201乙 (埼玉′2012)。

邸亮逹′岡田昌也′河田聡′藤田克昌′″生体分子イメージングヘの新たな挑戦～ラマン分光、

プローブレスイメージングによる分子機能解析とがん診断への応用～′″

第35回日本分子生

物学会年会′2W4Ⅲ2(福岡′2012)。

邸亮逹′岡田昌也′河田聡′藤田克昌′″Label― free imaging of cytOchrOme cin l市 ing cells by

resonance enhanced Raman spectroscOpル ″第85回日本生化学会′2P-355′ (埼玉′2012)。

4.

‐112‐

7。


山越博幸′間間孝介′岡田昌也′安藤潤′Almar F PalonPon/藤田克昌′河田聡′袖岡幹子′

″アルキン修飾化合物のClick―■eeラマンイメージング′″ 日本薬学会第132回年会′

31Ell―Pm05′ (本L幌′2012).

Almar F Palonpon/山越博幸′間間孝介′岡田昌也′安藤潤′河田聡′藤田克昌′袖岡幹子′

″Raman imaging of alkyne― tagged biomolecules in l市 ing cens′〃opiCs&PhOtonics Japan

2011′ 29aD5′ (大阪′2011).

安藤潤′山越博幸′閥間孝介′Almar F PalonPon7岡田昌也′藤田克昌′河田聡′袖岡幹子′

〃炭素二重結合を利用した細胞増殖プローブEdUのラマン分光イメージング′

″第37回レー

ザー顕微鏡研究会 (東京′2011).

山越博幸′間間孝介′岡田昌也′安藤潤′AlmaF F PalonPo島藤田克昌′河田聡′袖岡幹子′

〃ラマン顕微鏡を用いたアルキンタグの Click―freeイメージング′″ 日本薬学会第131回年

会′29G‐ amll′ (静岡′2011)。

山越博幸′間間孝介′Almar F PalonPon/岡田昌也′安藤潤′藤田克昌′河田聡′袖岡幹子′

″ラマン顕微鏡を用いたDNA合成プローブEdUの生細胞イメージング′

″ 日本ケミカル・バ

ィォロジー学会第6回年′P-115(東京′2011).

岡田昌也′藤田克昌′Nicholas I Smi■ ′河田聡′″DynamiC ObSeⅣ ation of apoptouc cens

using Raman miCroscopy7〃平成22年度日本分光学会年次講演会′P-31′ (京都′2010).

岡田昌也′藤田克昌′Niふolas I Smit and河田聡′〃Labe卜free observatton of apoPtoic

cens using Raman miCroscopル〃日本分光学会分光ナノ分光部会第2回シンポジウム′POt

(男護病ミ′2010)。

岡田昌也′浜田啓作′藤田克昌′Nicholas I Smit河田聡〃ラマン分光法による細胞内分子

の無標識イメージング′″電気学会光・量子デバイス研究会′OQD-10-22′ (大阪′2010)。

藤田克昌′安藤潤′岡田昌也′″ラマン散乱を利用した細胞/生体組織の無標識イメージング′

″

第31回日本レーザー医学会′(愛知′2010).

安藤潤′葛西洋志′岡田昌也′Nicholas Io Smit藤田克昌′河田聡′″金属ナノ粒子を

用いた生細胞の増強ラマンナノイメージング′〃レーザー顕微鏡研究会36回′(埼玉′2010)。

9.

10.

11.

12.

13.

14.

15.

16.

‐113‐


17.岡田昌也′浜田啓仁安藤潤′藤田克昌′Nich01as l smit河田聡′〃共鳴ラマン散乱による

細胞内cytochromeの無標識イメージング′″2009年秋季第 70回応用物理学会学術講演会′

8P―ZM―ι(富山′2009)。

18.岡田昌也′浜田啓仁安藤潤′藤田克昌′Nicholas l smi■し河田聡′″ラマン散乱顕微鏡によ

る細胞内分子の無標識観察′″Opics&PhOtOnics Japan 200%25PB乙

(新潟′2009).

‐114‐

Acknowledgements,

Acた“θωJθ4ζθπθ

“tS/

1 wish to exPress my deepeSt appreciatiOn to Professor Satoshi Kawata Of OSaka University/

Deparb■ent of APPlied Physics′ for super宙sing my s破 years′ WOr■ of studies in his laboratory I

have learnt a 10t from his attitude toward research and it is a pleasure worldng under hiS

direction.I also appreciate d■ at he gave lne a chance to go abroad for oversea education for 10

mOnths.This experience is a great treasure for me′ arld Will also be in the future.

Suggestions and conl■ lents for this dissertation from Professor Yasushi lnouye of OSaka

University7 Department of APPlied Physics′ Professor Tetsuro Takamatsu of Kyoto Prefectural

University of NIledicine′ lDeparment of Pa■ ology and Ce■ Regulation/ Professor Masanori

Hangyo of OSaka Un市 ersity/Department of Applied Physictt alld Associate P■ofessor

Katsumasa Fujita of OSaka Umversityr Deparhent ofApplied Physics are deeply acknoWledged.

My deepest appredatiOn gOes to Associate Professor Katsumasa Fuiita Of OSaka

Universityr Deparm■ent Of APplied Physic`′ for his great technical support and ad宙 ce.HiS

corrments alWays shoWed me a right direcion for my researches.I also leamed the importance

and fascinating aspect of SCience●rOugh scienti■c and daily discussion with him.

Associate Professor Nicholas Io Smit OSaka University7 1mmuno10gy Frontier Research

Cente■ gave me a lot ofinsightful comments and sugges■ons for ths research.Itmly would like

tO eXPress lny gratitude to him。

I would like to dlank Associate PFOfeSSor Yuika Saito′OSaka University/Deparm■ent Of

Applied Physics′ and Assistant PrOfessor Satoru Shojt Osaka University/Deparm■ent Of

Applied Physics for their precious colnlnents for iny research and creating adequate situation fOr

iti

l would like to extend lny deep gratitude to PFOfeSSor Andreas Zumbusぬ ′for accepting

my studies in hiS laboratoryr and his kindness for my ten monthS Stay in Konstanz/Ge....any.I

also want to thank his laboratory members′ especially7 Mattin Winterhalder arLd D■ Romedi

Selm for ieir kind supports in my exPeriment.I really enioyed everything in the beautiful Place

‐115‐

Acknowledgements,

and will never forget the experiences.

chief Scientist Mikiko Sodeoka, RIKEN, and Dr. Kosuke Dodo, RIKEN, indicated me thenew way of Raman microscope imaging technique through the collaboration work. I deeplyexpress my gratitude to them.

I would like to exPress my deep appreciation to dear Dr. fun Ando, Dr. MasahitoYamanaka, Dr. Almar F. Palonpon, Dr. Hiroyuki Yamakoshi and Dr. Keisaku Hamada for theirtechnical supports and advices. Their attitude on research had much effect on my attitude on it.

I would like to deeply thank to Dr. Thro lchimura, Dr. Shin-ichi Tanaka and Dr. Thka-akiYano for their precious advices in -y laboratory life.

special thanks to Mitsuhiro Hond4 Hiroyuki Morimura and yoshito okuno, whoassisted me throughout my college life in osaka university. Their positive attitudes to researchesalways encouraged me in the life.

I also would like to thank Dr. Liangda-Chiu, Dr. Kazumasa Uetsuki, Dr. Kyoko Masui, Dr.Takeo Minamikawa in Kyoto Prefectural University of Medicine Jun yu, yasuo yonemaru,

Kentaro Mochiduki, Kozue Watanabe, Shota Ushiba, Toshihiro Mino, Kazuki Bando, yutaKunihisa for their supports. I also express my deep appreciation to all other lasie members.

Finally, I wish to express my sincere appreciation and thanks to my dear family for theirlong-term watching over my growth with gentle patience.

‐116‐

Appendix A, Deriaation of nonlinear susceptibility of CARS

Appendix A,

Derivation of nonlinear susceptibility of cARs

Derivation of the nonlinear susceptibility of third-order nonlinear optical resPonse including

CARS by considering how density matrix evolves as time goes on' The density matrix p

describes a quantum system in a mixed state, and is described as

where tt is the probabihty of i― th sta“ レj)。 The time evoluuon Of density matr破 obeys the

Liou↓ille equatiott Which derives from the density matr破 and the Shrё dinger equation.

ρ=Σ′jlψj)0:|

ry=!lns1,p1t1ldt ih'

fI(r) is the Hamiltonian of the total system. Eq. A-2 can be transformed as

(A-1)

(A-2)

p r,> = j I'--ln g 1, p 1t' llat (A-3)

This equation is solved with successive approximation sc-heme with assumption of P= Po' that is

all molecules in the system exist in the ground state, at t = -q. Then, the integration can be

expanded for the third-order term as,

がkの =静 I∞ル3几務2ム 41〃Q),I″り,I″a),ρ°11

(A-4)

The Hmiltonian composes hVO terms Hamiltonian of molecular system(″ 。)and Hamiltonian

of interaction between the incident light and dle molecule(″ h(″))。 ″im(の iS equ市 alent to

―μE(4r)where μ is the dipole moment ofthe system and E(4r)iS the incident electric ield

‐117‐

Appendix A, Deriztation of nonlinear susceptibility of CARS

in the current system. Therefore, Hamiltonian of the total system is described as

H(t)= Ho+H^r(t)- Ho- ttE(t;r)

′(3)=Mレ (ρ(3レ

)=ΣΣρ9μ″″

This suggests that the p(:l corresponds to third order of electric field and thus, this term derivesnonlinear Raman processes. The third order components p@ of polarization density p isdescribed as

(A-5)

は-6)

N is the number of molecules in a unit volume. To obtain Eq. 4-6 the equatior, (o)= Tr(po),where (O) is ensemble average of physical quantity O, was used.

By expanding the commutators of.Eq,. A-4, eight terms are obtained as

lr rA,l, <,,1, [rtr, l, po]]] - H e ) H (t r) H (t) p0 - H e ) H (t ) po n e,)

- H(4)H(t)p0H(tr)+ H(h)poH(t)H(t) (A_s)- H (t) H (t) po H (tr) + H (t) p0 H (tt) H (h)+ ne)po H(tr)H(tr)_ po ngrlngr)H(tz)

The former four and the latter four has same physical sense, and each term in the former four (orthe latter four) terms relates to variety types of third order nonlinear optical processes includingCARS.

Among the eight terms in Eq.A-& the first (or fifth) term contributes to cARs process. Bysubstituting the first term of Eq. A-8 to Eq. A-4 and Eq. A-6, the third order polarization p,(r) isobtained as

メつ=#。ン″・

°C~ω 8ンI∞名μめθ・°あ―ωれ

(A-9)XIL″

2μ加~'°・つ。)ち ILttμ

“ι~'°

8つα)4(ρo)ggE(ち)E(ら )E(4)

where α′b and θ represents eigenstates Of tt molecules and ω′(p=α ′b′ c andg)is

‐118‐

Appendix A, Derittation of nonlinear susceptibility of CARS

eigen-frequency for each state. In the Eq. A-9, for example, the integral over tr is acquired as Eq'

A-10 and this corresponds to the first order perturbation term of the density matrixp0' Also' this

equation suggest that the system interacts with the incident electric field at t = \ arld the state

changes from Po to Pt.

(A-10)(が )“

″チム4μttι・°8-ω♪4ΦOLЦゆ

In Eq. A-& the Hamiltonia n H(tr) operated to the density matrix p0 from the left side and as a

result, the subscript g change s a after p0 c-hanges pt i" fq. A-10' This change can be

considered as a kind of dipole transition. As well as integral ov€f {r the integrals over t, and

13 represents that states of the system transits. H(tr) opetates to (Pt)o, uttd th"tt' (p\ur

generates. Subsequently, H(tr) operates to (Pl)r, and theO (P')", g"""rates' Finally' p'tr)

generates after operatin g ps" from the left. By summarizing the discussions in this paragraptr' it

is understood that the density matrix transits as

{po)r, - (P\ o, - (P\ u, - 1Pt)"r[- @)rr] (A-11)

In earher discussions′ 血 Phase relaxation was beerL COIllSidered fOr simphcity.HoweveL in

real systemS′ phase relaxa■ on/dbr市 es from■ermal relaxauOn/has to be considered.For this

purpoSe′ Eq.A-2 is modified as

響♀=務 [″(め ,ρ (」―瑞脇∽≠の (A-12)

The second term of Eq. A-12 represents that p^, decays exponentially to zero' L' is phase

relaxation time between the state lre) and In). Here, relaxation of diagonal elements of the

density matrix is neglected. The formal solution of Eq. A-12 is described as

P^, = jr-'^' I'-*lnG'),P{t'1\^,er^'' dt'

‐119‐

(A-13)

Appendix A, Derhtation of nonlinear susceptibility of CARS

This solution c€m be expanded to the third-order polarization based on theapproximation as

ガつ=#。ン″C~ωp+「cgJrI∞名μメ・°ら~け%―りち

X几ち μ腸`・

I°α―ωル %―「 ag】ちム名 μ

“

ι{・ 8~ωα》ragン1(ρ。

)“E(ち )E(ら )E(1)

successive

Nonliear susceptibly of cARS can be obtained using Eqo A‐14.h generaし a sample is

irradiated with h″ 01■OnochrOmatic electric fields with different frequencies which is described

as

(A-14)

(A-14)

(A-16)

E(r,の =El(「 ,の十E2(F,の +ε .ε .

=El exp[ノol′ ―ム・r)卜 E2eXp[J〔 D2′ ~ち・r)]+θ .6.

By substituting this to Eq. A-'l'4,64 terms are obtained. Among them, only the terms with thefrequency of 2otr-ar, contributes to CARS. By discarding other extra terms, Eq. A-15 isobtained.

メ幼=# \rl), & scQ coF uP ae --- ---.^-

ω“―fDl― JE場ω姥―(q― ω2)~JLο,ら ,α

~101~ω2)く 2tl―た2ンLE2E3

(A‐ 15)

ω彎-2o―ω2)… ′L

From dI〕 equation/it can be understoOd that the third―Order p01arization creates electric field

which has frequency of ωl=2ωl― ω2and Wave vectOr of 24-た2・

SinCe no electric ield with

ω“

is contained in■e incident fielc the electric ield becomes血0五gin of new electric ield.TO

generate dle elect五 c ■eld in mate‖ als Phase matching cOnditiOn shOuld be satisfied. The

wavenumber Of dle electric field wi■ ω“

is exPreSSed as

た“=

ω“

C

When tt matches with■ e wave lllumber Of the new ehctric fiel↓ ふat is塩 =pれ _ち♭the new

‐120‐

A′′ιη′jχ ス′Dιriυα′jθ4 0/πθπ:'4θαγ sツSCグ′bjJ:υ げCARS

wave can generates and the wave is CARS'

ThenonlinearsusceptiblyofCARSl^isdefinedbytherelationshipbetweenthenew

electric field ant the third power of the incident field as ,{z) =,tt)ggB' thus Eq' A-15 is obtained

finally as

χR=:許`翌

a gο

μCbμ ttμ

“山 (A‐ 17)

1 1

, rJr, -rtD - iT r, ttt "r

- (2ar - a 2) - il ",

In chapter 3′ lhe incident electric fidd iS Supposed to cOntain three different frequencies but the

χR Can be obtaned Simply by replacing one of ωl of Eq.A-17 with

ω3・

‐121‐

title raman microscope imaging and analysis of molecular dynamics in a living cell author(s)

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