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Fluorescence Lifetime Imaging (FLIM) of molecular rotors maps microviscosity in cells

Klaus SuhlingKlaus Suhling

DeDepartment of Physicspartment of Physics King’s College King’s College LondonLondon

StrandStrandLondon WC2R 2LSLondon WC2R 2LS

UKUK

Outline / MotivationOutline / Motivation• Optical imaging - background, Fluorescence, Fluorescence

Lifetime Imaging (FLIM)

• Diffusion is relevant for protein mobility in cells, drug delivery

etc

• measure diffusion in cells with Fluorescence Microscopy

• Time-resolved fluorescence spectroscopy

• Fluorescence Lifetime Imaging (FLIM) of molecular rotors

• time-resolved fluorescence anisotropy to measure rotational

mobility

• Summary

Modern Fluorescence Microscopy

• high contrast, exciting light eliminated (Stokes’ shift)

• minimally invasive & non-destructive

• can be performed on live cells and tissue

• tag specific proteins and regions in living cells with

fluorescent labels and locate them

Fluorescent labels for microscopy

• Stain biological specimen with fluorescent dyes, nanodiamonds or quantum dots and observe stained regions

• Use genetically encoded fluorescence proteins, e.g. green fluorescent protein GFP

• Use endogenous fluorescence (“autofluorescence”), e.g. tryptophan, flavins, NaDH, collagen, elastin

What is Fluorescence?What is Fluorescence?

kr

excitedstate S1

groundstate S0

kisc

T1kic

radiative deactivation of the first

electronically excited singlet state

kph / kic

molecular energy levels

Fluorescence can be characterised by:

position

intensity

wavelength

lifetime

polarization

Fluorescence is multi-parameter signalFluorescence is multi-parameter signal

Fluorescence lifetime

average time fluorophore remains in its excited state

=1 / (kr+kisc+kic+[q]kq) = 1 / (kr+knr)

depends on:depends on:

• iintrinsic properties of molecule ntrinsic properties of molecule ((kkrr))

• llocal environment of moleculeocal environment of molecule ( (kknrnr))

use use of of flufluoorophore to probe environmentrophore to probe environmente.g. viscosity, refractive index, pH, Ca2+, polarity, interaction with other molecules ….

Fluorescence Decay – Time Domain

I = I0 e-t/

Excitationpulse

time

inte

nsity Fluorescence

emission

How is the fluorescence decay measured?How is the fluorescence decay measured?

Fluorescence Decay – Frequency Fluorescence Decay – Frequency DomainDomain

excitation fluorescence

phase shift

demodulation M

tan = M=1/(1+ ()2 )1/2

modulation

frequency

The fluorescence lifetime can probe …..

>

K. Suhling et al, Photochem Photobiol Sci 4, 13-22, 2005

Why Fluorescence Lifetime Imaging Why Fluorescence Lifetime Imaging (FLIM)?(FLIM)?

• contrast according to fluorescence lifetimecontrast according to fluorescence lifetime

• aabsolute measurement bsolute measurement iindependent of variations in ndependent of variations in fluorophore concentrationfluorophore concentration, illumination intensity, , illumination intensity, light path length, scatter, photobleachinglight path length, scatter, photobleaching

• directly directly image environment image environment of specific proteins or of specific proteins or dyes in living cellsdyes in living cells

• image molecular inimage molecular interactionteraction, e.g. fluorescence , e.g. fluorescence resonance energy transfer (FRET) to study resonance energy transfer (FRET) to study proximity of proteinsproximity of proteins

• distinguish spectrally similar fludistinguish spectrally similar fluoorophoresrophores

K. Suhling. “Fluorescence Lifetime Imaging.” in Methods Express, Cell Imaging (ed D. Stephens), chapter 11, 219-245, Scion publishing, Bloxham, 2006.

jenlab

Imaging Imaging protein interaction by FRETprotein interaction by FRET

donor fluorescence

lifetime shortened

fluorescence / Förster resonance energy transferoccurs at close proximity of donor and acceptor, <8nm

K. Suhling et al, Photochem Photobiol Sci 4, 13-22, 2005

Roger Tsien

GFP

GFP and mRFP absorption and emission spectraGFP and mRFP absorption and emission spectra

Detect GFP donor

fluorescence

in this spectral window

Identify FRET

by shortened

donor lifetime

GFP-PKCGFP-PKC interacting with ezrin (anti-VSVG-Cy3) interacting with ezrin (anti-VSVG-Cy3) at the tips of filopodia in breast carcinoma cellsat the tips of filopodia in breast carcinoma cells

Tony Ng, Randall Division, King’s College London

Intracellular diffusion

http://nobelprize.org/nobel_prizes/medicine/laureates/1999/illpres/cell.gif

Molecular diffusion is a rate-limiting step in metabolism.

Major factor in determining mass transport for signalling, reactions, and drug delivery.

Influenced by factors including crowding of macromolecules and viscosity of intracellular media.

Aqueous regions with η ~1-2cP

How is microscopic intracellular diffusion measured?

Methods for intracellular diffusion measurements

r

kTwD D

64

21

2

RT

V

Time-resolved Fluorescence Anisotropy

Rotational diffusion

Measure fluorescence decays with polarization parallel and perpendicular to that of the excitation beam

Fluorescence Recovery After Photobleaching (FRAP)Translational diffusion

• Bleach a region of interest• Monitor recovery of fluorescence in ROI due to diffusion of unbleached fluorophores

INT

EN

SIT

Y

TIME

t

rtr exp)( 0

VISCOSITY

BODIPY-based molecular rotor – a viscosity probe

N N

BF2

OC12H25

Lipophilic chain - not soluble in water

Not a molecular rotor

M. Kuimova et al, J Am Chem Soc 130(21), 6672–6673, 2008

Boron dipyrromethane

How does a molecular rotor work?

N N

BF2

OC12H25

Two states - bright and dark

bright state is excited

This is more difficult (takes longer) to

reach in viscous solvents as excited

state only lasts nanoseconds

Torsional motion of phenyl ring

around single bond

certain conformation allows non-

radiative deactivation of the excited

state

Bodipy absorption spectrum

Bodipy fluorescence emission spectra inmethanol / glycerol solution

Fluorophore

concentration constant

Φ = A ηx

Φ - fluorescence quantum yield

A, x - constant

η - viscosity

(Förster & Hoffmann, Z Phys

Chem 1971, 75, 63–69)

Quenching and concentration effects cannot be distinguished in fluorescence intensity measurements

If fluorophore concentration is NOT constant, there is no way

to distinguish between quenching and concentration

Better solution - use

fluorescence lifetime

Possible solution - ratiometric

measurements of molecular rotor in

combination with unquenched

fluorophore, eg Luby-Phelps et al,

Biophys J 65, 236–242, 1993.

- but requires mono-exponential

fluorescence decay

Sample

TCSPCCard

Fluorescence Decay or Anisotropy Analysis

Pulsed Laser

Dichroic Beamsplitter

Fluorescence Lifetime Image

Emission Filter

Polariser

Detection PMT

short

long

Scanner

Time-Correlated Single Photon Counting (TCSPC) - based confocal FLIM set up

0 5 10 15 20

1E-3

0.01

0.1

1Increase in viscosity

Time / ns

Em

issi

on

inte

nsi

ty

Fluorescence decays of bodipy-based molecular Fluorescence decays of bodipy-based molecular

rotor in methanol / glycerol solutions rotor in methanol / glycerol solutions

Fluorescence lifetime τ is a function of viscosity η

τ = k0-1 A ηx

log (τ) = x log (η) + log (A/k0)

Plot log (τ) vs log (η)

straight line according to theory – serves as calibration

Log fluorescence lifetime τ plotted vs log viscosity η

gradient = 0.50±0.03

Straight line - as expected

M. Kuimova et al, J Am Chem Soc 130(21), 6672–6673, 2008

Bodipy-based molecular rotors in cells show punctate distribution

SK-OV-3 cells incubated

with 1 μM solution of bodipy

molecular rotors

(DMSO delivery)

FLIM of bodipy-based molecular rotors in cells

average

fluorescence

lifetime

≈1.6ns,

apparent

microviscosity

≈100cp

Impossible to learn this from intensity measurements alone

1850ps

J. Levitt et al, J Phys Chem C, 113, 11634–11642, 2009

Time-resolved FluorescenceTime-resolved Fluorescence A Anisotropynisotropy

tIztI

tItItr

||

||

Molecular tumbling characteriMolecular tumbling characterissed ed by rotational correlation timeby rotational correlation time

t

ertr

0)(

For a spherical molecule :For a spherical molecule :

kT

V

viscosityviscosity

molecular volumemolecular volume

Excite with linearly polarized lightExcite with linearly polarized light

log in

ten

sity tIII

tI

time

information about information about rotational diffusionrotational diffusion of molecules of molecules

Rotational correlation time θ versus viscosity η

kT

V

in solution

Time-resolved fluorescence anisotropy imaging

parallel

perpendicular

rotational correlation

time 590 ± 110 ps,

~60 cP

Combine fluorescence lifetime τ and rotational correlation time

τ = k0-1 A ηx

= ηV / kT

Therefore

τ = k0-1 A (kT / V)x

Plot log τ vs log - straight line

is function of rotational mobility only, but in cells τ

could be affected by other quenching mechanisms

log τ vs log of bodipy in cells

1 10

1

10

log

)

log()

• solution• cells

Cell data in good agreement with solution data

gradient=0.53±0.09

Twisted Intramolecular Charge Transfer (TICT) states formed upon photoexcitation.

Competing radiative and non-radiative de-excitation pathways.

Electron-transfer from julolidine nitrogen to nitrile group TICT state.

Rotation around julolidine-vinyl bond.Steric hindrance of rotation governed

by solvent.

Viscosity-dependent.Viscosity-dependent.

Commercially available Molecular Rotors

Measure intensity and determine viscosity?

0.0 0.5 1.0 1.5 2.0 2.5 3.0

0.40

0.45

0.50

0.55

0.60

0.65

0.70

0.75

0.80

0.85

log

log

0.0

0.5

1.0

1.5

2.0

2.5

3.0

log

DCVJ lifetime vs viscosity calibration

Log-log plots from 2 groups in different viscosity regimes support Förster-Hoffman equation. Different values.

So we can measure viscosity

using the measured fluorescence lifetime of

DCVJ

= 0.48

= 0.30streak camera

Measurements (Junle Qu,

Institute of Optoelectronics,

Shenzhen University, China

DCVJ in YTS NK Cells

Fluorescence

Brightfield

Inherent optical sectioning due to multiphoton excitation

Punctate distributiontargeting vesicles?

0.966 ns

0.099 ns ≡ 650 cP!

FLIM of DCVJ in YTS NK cells

Double

exponential decay –

fluorescence

intensity

measurements

unreliable

3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5

1

10

100

1000

10000

Inte

ns

ity

Time / ns

FWHM = 70 ps

No afterpulse

Short lifetime is still ~100 ps while IRF of SPAD is 70 ps Lifetime from PMT detector deconvolution is correct!

Autofluorescence can be excluded.Genuinely high viscosity region or bound rotor?

FLIM of DCVJ in YTS cells using fast Single Photon Avalanche Diode (SPAD) detector

HeLa cell division 470 nm excitation

0 mins 3 mins 10 mins 20 mins 40 mins

What next?

Can we measure changes in viscosity during mitosis?

Need to know exactly where the DCVJ resides in the cell(unlike bodipy, DCVJ does not have lipophilic chain)

• Monitor uptake mechanisms

Porphyrin-based molecular rotor for photodynamic therapy (PDT)

Twisted form emits at 710nm,

planar form at 780nm - calibration

Put into cells

M. Kuimova et al, Organic & Biomolecular Chemistry 7, 889-896, 2009

Porphyrin-based molecular rotor in cells

blue = low viscosity (50cp), orange = high viscosity (300cp)

Transmitted light initial advanced

Independent singlet oxygen decay measurements at 1270nm

show an increasing decay time – consistent with slower

diffusion due to higher viscosity

ratiometric images: viscosity increases upon irradiation of sensitiser and subsequent cell death

M. Kuimova at al, Nature Chemistry 1, 69-73, 2009

ConclusionsConclusions• FLIM is minimally invasive, non-destructive and can be

performed on live cells and tissue• FLIM of FRET can directly image the local environment of

fluorophores and interaction of proteins in live cells• Modified hydrophobic bodipy is a fluorescent molecular rotor,

its fluorescence lifetime is function of viscosity• Cellular uptake with punctate and uniform distribution• FLIM reveals high apparent viscosity in cells – relevant for

diffusion• Time-resolved fluorescence anisotropy measurements are

consistent with high apparent viscosity in hydrophobic region cells

• DCVJ has a biexponential decay profile in cells with both lifetimes corresponding to very high viscosities > 600 cP!?

• Porphyrin-based molecular rotor allows monitoring increasing viscosity during cell death

AcknowledgementsAcknowledgements

Prof Tony Ng, Dr James Levitt, post-doc, Pei-Hua Chung, PhD student, King’s College London, UK

Dr Marina Kuimova, Dr Gokhan Yahioglu, Chemistry Department Imperial College London, UK

Prof Harry Anderson’s group, Chemistry Department, Oxford University, UK

Prof Peter Ogilby, Department of Chemistry, University of Aarhus, Denmark

Dr Stan Botchway, Prof Tony Parker, Rutherford Appelton Labs, UK

Prof Junle Qu, Institute of Optoelectronics, Shenzhen University, China

Thank you for your attention

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