Study of early spectral changesStudy of early spectral changesin cells transformationin cells transformation

using advanced physical and mathematical using advanced physical and mathematical models.models.

By E. Bogomolny By E. Bogomolny

Supervisors:Supervisors: Prof. S.MordechaiProf. S.Mordechai Department of Physics (BGU)Department of Physics (BGU) Dr. M.Hulihel Institute of Applied Research (BGU)Institute of Applied Research (BGU)

IR radiation IR radiation UV radiationUV radiation FTIR MicrospectroscopyFTIR Microspectroscopy Light inducedLight induced FluorescenceFluorescence. .

1) Introduction.1) Introduction.

3) Results and Discussion.3) Results and Discussion.

2) Methodology.2) Methodology.

4) Conclusions.4) Conclusions.

Experimental descriptions. Spectra analysisExperimental descriptions. Spectra analysis.. Mathematical methods.Mathematical methods.

The Electromagnetic SpectrumThe Electromagnetic Spectrum

Gamma rayGamma ray SPECT, PET Imaging Nuclear transition

X-rayX-ray Mammography ,CAT. Inner shell

UVUV Potential electromagnetic range for diagnosis Valence electrons

VisibleVisible Pathology Valence electrons

InfraredInfrared Potential electromagnetic range for diagnosis Vibrational rotational level

MicrowaveMicrowave EPR imaging Rotational levels or fine structure

RadioRadio NMR imaging Hyperfine structure

IR radiationIR radiation

• Born-Oppenheimer ApproximationsBorn-Oppenheimer Approximations ::• 1) The electronic motion and the nuclear motion in molecules 1) The electronic motion and the nuclear motion in molecules

can be separatedcan be separated • 2) nuclear motion does not induce electronic transitions.2) nuclear motion does not induce electronic transitions.

ˆ ˆ ˆ ˆ ˆ ˆel el nuc el el nuc nuc nucH T V V T V

Multi-atomMulti-atom moleculemolecule

ˆn̂ucH T U R

el nuc nucU R E R V

2

1

ˆ2

elNj

elj e

pT

m

2

1

ˆ2

elNj

nucA A

pT

M

2

1

'ˆnuc nucN N

A Bnuc nuc

A B A A B

Z Z eV

R R

2

1

'ˆel elN N

el eli j i i j

eV

r r

2

,

'ˆ Ael nuc

j A j A

Z eV

r R

IR radiationIR radiation ˆ , , , ,nuc nuc nuc nucH R R E R

2

22 2 2 2 2 2 2

, , , , , ,1 1 1 2sin , , 0

sin sinnuc nuc nuc

nuc

R R Rr E U R R

r r r r r

2

22 2 2

( )1 2 ( 1)( ) 0

2nuc

nuc

d Rd J Jr E U R R

r dr dr r

Rewriting the Hamiltonian in spherical coordinates for diatomic systemRewriting the Hamiltonian in spherical coordinates for diatomic system

Equation similar to Hydrogen atomEquation similar to Hydrogen atom

Solution depends explicitly on U(R) . By Solution depends explicitly on U(R) . By expanding U(R) in Taylor series we obtain:expanding U(R) in Taylor series we obtain:

Equation can be separated to angular and radial partEquation can be separated to angular and radial part

2

,

1 1( ) ( )

2 2J eq eq

J JE U r h n

' * ''n n nR dx

The selection rules for vibrational and rotational The selection rules for vibrational and rotational transitions: transitions: ∆n = ±1 and ∆J= ± 1∆n = ±1 and ∆J= ± 1

The transition momentThe transition moment The dipole momentThe dipole moment

Only vibrational modes that have a transition dipole moment (or component of the transition dipole moment) can be observed in infrared spectroscopy

Different Types of Vibrational modesDifferent Types of Vibrational modes

Symmetric Stretch Asymmetric Stretch

Non linear molecules 3N-6Non linear molecules 3N-6

Rocking Twisting Scissoring Wagging

(Stretching)(Stretching)

(Bending)(Bending)

Different Types of Vibrational modesDifferent Types of Vibrational modesLinear molecules 3N-5Linear molecules 3N-5

Symmetric Stretch Asymmetric StretchSymmetric Stretch Asymmetric Stretch

Bending (two types)Bending (two types)

Introduction FTIR-MSPIntroduction FTIR-MSP((Fourier Transform Infrared Micro-Spectroscopy)Fourier Transform Infrared Micro-Spectroscopy)

Introduction FTIR-MSPIntroduction FTIR-MSP

MichelsonMichelsonInterferometerInterferometer

IR Source (Globars)

Sample

Microscope

Lens

Detector MCT

Michelson InterferometerMichelson Interferometer

IR spectra of bio-moleculesIR spectra of bio-molecules

Cell parameters Values

Radius [µm] 100

Volume [µm3] >10000

Generation time [h] 20-24

proteins % [w/w] 60

RNA % [w/w] 3-4

DNA % [w/w] ~1

Lipids % [w/w] 15-20

Polysaccharides %[w/w]

6-84000 3600 3200 2800 2400 2000 1600 1200 800

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

carb

oh

ydra

tes

nu

cle

ic a

cid

s

proteins

2958 C

H 3 str

asym

m

Lipids vib modesWater vib modes

2364

(C

O 2)

2917

CH 2, C

H3 s

tr. a

sym

m

3219

OH

str

. ban

d o

f w

ater

2849

CH 2, C

H3 s

tr. s

ymm

1740

CO

str

. (li

pid

s)

1652

am

ide

I

1542

am

ide

II14

52 C

H 3 asy

mm

. ben

din

g

1080

PO 2 s

ymm

. str

.

A

bso

rpti

on (

A.U

.)

Wavenumber (cm-1)

S0

S1

T1

transition involving emission/absorption of photons

radiationless transition

fl

uo

resc

ence

flu

ore

scen

ce101

0-6-6 t

o 1

0 t

o 1

0-9-9 s

ec s

ec

--hνhν

in

tern

al

inte

rnal

co

nve

rsio

nco

nve

rsio

n

inte

rsys

tem

inte

rsys

tem

cr

oss

ing

cro

ssin

g

FluorescenceFluorescenceThe Jablonski diagramThe Jablonski diagram

Phos

phor

esce

nc

Phos

phor

esce

nc

ee1010

-4-4 to 1

0 se

c

to 1

0 se

c+h+h

Ab

sorp

tio

nA

bso

rpti

on

1010 -1

5-1

5 s

ec

se

c

Fluorescence properties Fluorescence properties

(Quantum yield )

Number of fluorescent (emitted ) photonsNumber of absorbed photons

Q

Compounds with multiple conjugated double bonds, Compounds with multiple conjugated double bonds, i.e. extended i.e. extended electronic systems, has high quantum electronic systems, has high quantum yield and therefore detectable fluorescenceyield and therefore detectable fluorescence

1) Scattering of light due to particulates in the sample. 2) Phosphorescence of the sample . 3) Shifts in chemical equilibrium as a function of concentration. 4) Non-monochromatic radiation, 5) Stray light.

)(max nm )(max nm

Endogenous fluoropores

Excitationmax

Emissionmax

Q (Quantum yield)

Tryptophan 279.8 348 0.20

Tyrosine 274.6 303 0.14

Phenylalanine 257.4 282 0.04

NADH 340 440 0.019

Intrinsic fluorophoresIntrinsic fluorophores

Schematic diagram of Schematic diagram of spectrometerspectrometer

Emission spectra Excitation spectraEmission spectra Excitation spectra

250 260 270 280 290 3000

2

4

6

8

10

12

14

16

Emission monochromator set on 340

Aromatic amino acids fluorescence

Inte

nsi

ty

wavelength nm300 320 340 360 380 4000

2

4

6

8

10

12

14

16

Excitation monohromator set on 285 nm

Aromatic amino acids fluorescence

Inte

ns

ity

wavelength nm

• Main goals: Main goals: Study cancerous transformation using FTIR and LIF . Study cancerous transformation using FTIR and LIF .

Special emphasis was made to identify spectral changes before cell cultures Special emphasis was made to identify spectral changes before cell cultures

passed complete morphological transformation.passed complete morphological transformation.

• Experimental stepsExperimental steps

• Growing the cells Growing the cells

• Infection of the cell cultures by murine sarcoma virus (MuSV) (known to induce Infection of the cell cultures by murine sarcoma virus (MuSV) (known to induce

cancerous transformation)cancerous transformation)

• Measuring samples using FTIR and LIF as function of post infection time.Measuring samples using FTIR and LIF as function of post infection time.

• Spectra analysis by software algorithms and obtaining spectral biomarkersSpectra analysis by software algorithms and obtaining spectral biomarkers

• Results of analysis by mathematical methodsResults of analysis by mathematical methods

MethodologyMethodology

MethodologyMethodology

Murine fibroblast cell lines (NIH/3T3)Murine fibroblast cell lines (NIH/3T3)normal normal transformedtransformed

Mouse embryonic fibroblast (MEFMouse embryonic fibroblast (MEF)normal normal

PropertiesProperties NIH/3T3NIH/3T3 MEFMEF

Source long term cycle in vitro

stem cells from embryos during mouse pregnancy

Susceptible to murine leukemia virus, murine leukemia virus, murine sarcoma virus murine sarcoma virus ..

Cell cultures use advantages

High utilization, less sensitive to environment condition than MEF cells

Primary cells

High gene expression

MethodologyMethodology

MuSVMuSV

ControlControl

14 d14 d

1 d1 d3 h3 h

1 d1 d

7 d7 d

7 d7 d

1)1) The cell cultures were grown in RPMI medium supplemented with 10% or The cell cultures were grown in RPMI medium supplemented with 10% or 2% (or mixture of them depends on cell condition) new born calf serum 2% (or mixture of them depends on cell condition) new born calf serum (NBCS) and the antibiotics such as penicillin, streptomycin and neomycin.(NBCS) and the antibiotics such as penicillin, streptomycin and neomycin.

2) MuSV was obtained from centrifuged highly concentrated transformed NIH ) MuSV was obtained from centrifuged highly concentrated transformed NIH cell lines. cell lines.

3) Measurements as a function of time were taken3) Measurements as a function of time were taken

a)a) FTIR measurements. FTIR measurements. Drop which contain 2 Drop which contain 2 l of PBS with concentration of l of PBS with concentration of 1 million cells/ml seeded directly on 2x2 cm2 ZnSe crystals slide. After ensuring 1 million cells/ml seeded directly on 2x2 cm2 ZnSe crystals slide. After ensuring complete dryness, the FTIR measurements were made.complete dryness, the FTIR measurements were made.

b)b) Fluorescence measurementsFluorescence measurements were taken in cuvette or microplate into PBS were taken in cuvette or microplate into PBS

3 h3 h

FTIR spectra analysisFTIR spectra analysis

100015002000250030003500

Wavenumber cm-1

0.0

50.1

00.1

50.2

00.2

5

Sin

gle

cha

nne

l

Background spectraBackground spectra

Baseline correctionBaseline correction NormalizationNormalization

Fluorescence Spectra analysisFluorescence Spectra analysis

Mathematical methodsMathematical methods Cluster analysisCluster analysis

Cluster Analysis ( Ward`s method )

0 2 4 6 8 10

Heterogenieity

5 days

3 days

1 day

4 hours

NIH/3T3

13 days

7 days

11 days

9 days

NIH/MuSV

Normal state classification

Cancerous state classification

2

1

Ward method ( minimum variance)kn

kk iki

E z z

Euclidean distance between two points p and qEuclidean distance between two points p and q 21

N

i ii

p q

2958 2852

1120 1015

1059 1099

1028 1085

2

A /A

A /A

A /A

A /A

symPO Shift

Mathematical methodsMathematical methodsDiscriminant classification functionDiscriminant classification function

D = w0+w1Z1 + w2Z2+ w3Z3 + .... wiZi

 

discriminant score

weighting coefficient

variable

MusvMusv NormalNormal 4h4h 1d1d 3d3d 5d5d 7d7d 9d9d 11d11d 13d13d

Z1 4.2 1.6 1,7 1,8 2,2 2,9 3,7 3,9 4,04 4,29

D 100 0 4.5 6.5 24.2 50.7 80.4 83.4 93.8 102.3

D = w0+w1Z1

W0=-61.7 W1=38.5

FTIR data analysisFTIR data analysis

I arealipids

II areaNucleic acids

FTIRFTIR data analysis data analysis

AA29582958/A/A28522852

FTIR spectral indicators FTIR spectral indicators (lipids absorbance)(lipids absorbance)

A2958/A2852 NIH/3T3 NIH/Musv

Mean 1.07 1.27

t-value 10.25

Max value 1.12 1.4

Min value 1.02 1.2

17%17%

FTIR spectral indicators FTIR spectral indicators (nucleic acids area)(nucleic acids area)

AA10991099/A/A1058 1058 (nucleic acids (nucleic acids

vibrational modes)vibrational modes)

AA11211121/A/A1015 1015 (RNA/DNA)(RNA/DNA)

Wavenumber shift of POWavenumber shift of PO22--

(conformation structure (conformation structure of nucleic acids)of nucleic acids)

AA10281028/A/A1085 1085

(Gluocose /phospate)(Gluocose /phospate)

FTIR spectral indicators (nucleic absorbance)FTIR spectral indicators (nucleic absorbance)T-value 16.1T-value 16.1 147%147%

T-value 13.1T-value 13.1 66%66%

T-value 8.9T-value 8.9 96%96%

T-value 5.7T-value 5.7 43%43%

TransformationTransformation

Morphological changes Morphological changes can be observed by can be observed by microscopemicroscope

Transformation of ATransformation of A11211121/A/A10151015 RNA/DNA RNA/DNA

After 5-6 days we can observe morphological changeAfter 5-6 days we can observe morphological change After 9-10 days we can observe morphological changeAfter 9-10 days we can observe morphological change

After 8-9 days we can observe morphological changeAfter 8-9 days we can observe morphological change

Finding fluorescence differenceFinding fluorescence difference

Aromatic amino acids Aromatic amino acids fluorescence transformationfluorescence transformation

After 5-6 days we can observe After 5-6 days we can observe morphological changemorphological change

After 8-9 days we can observeAfter 8-9 days we can observe morphological changemorphological change

Cluster Analysis of NIH/3T3 fast Cluster Analysis of NIH/3T3 fast transformationtransformation

Cluster Analysis (Ward`s method). According five indicators . Fast transformation

0 1 2 3 4 5 6 7 8 9

Heterogenieity

2 days1 day

3 hoursNIH/3T3

5 days14 days

3 days9 days7 days

NIH/MuSV

Cancerous state classification

Normal state classification

2958 2852

1120 1015

1059 1099

1028 1085

2

A /A

A /A

A /A

A /A

symPO Shift

After 5-6 days we can observe morphological changeAfter 5-6 days we can observe morphological change

0 5 10 15 20 25

Heterogenieity

3 days9 days7 days5 days

14 daysNIH/MuSV

2 days3 hours

1 dayNIH/3T3

Cancerous state classification

Normal state classification

Cluster Analysis ( Ward`s method )Fluorescence classification . Fast transformation

Fluorescence Intensity of Aromatic amino acids

Fluorescence Intensity of NADH

Cluster Analysis of NIH/3T3 fast Cluster Analysis of NIH/3T3 fast transformationtransformation

After 5-6 days we can observe morphological changeAfter 5-6 days we can observe morphological change

2958 2852

1121 1015

1059 1099

1028 1085

A /A

A /A

A /A

A /A

1

2

7

sym

AAA

NADH

W

PO Shif

S

I

I W

Discriminant classification functionDiscriminant classification function

ConclusionsConclusions

1)1) Both spectroscopic technique (FTIR-MSP and LIF) allow to detect Both spectroscopic technique (FTIR-MSP and LIF) allow to detect spectral changes before cell cultures pass complete morphological spectral changes before cell cultures pass complete morphological transformation.transformation.

2) The spectroscopic technique shed light on 2) The spectroscopic technique shed light on carcinogenesiscarcinogenesis metabolic processes:metabolic processes:a) Nucleic acids activitya) Nucleic acids activityb) Glucose / phosphateb) Glucose / phosphatec) Lipids c) Lipids d) Aromatic amino acids d) Aromatic amino acids e) NADHe) NADH

3)3) The transformation tendency can be described by sigmoid functionThe transformation tendency can be described by sigmoid function

AcknowledgementsAcknowledgements

Thanks to my supervisors Thanks to my supervisors Prof. S.Mordechai and Dr Prof. S.Mordechai and Dr M.Hulihel.M.Hulihel.

And all members of our lab And all members of our lab Dr.A.Salaman, Dr.R.Sahu and Ph.D Dr.A.Salaman, Dr.R.Sahu and Ph.D students U.Zeligstudents U.Zelig and Z.Hamodi.and Z.Hamodi.

ThankThank youyou