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Transcript of Page 1 Klaus Suhling Department of Physics Kings College London Strand London WC2R 2LS UK...
Page 1
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
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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
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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
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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
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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
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Fluorescence can be characterised by:
position
intensity
wavelength
lifetime
polarization
Fluorescence is multi-parameter signalFluorescence is multi-parameter signal
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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 ….
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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?
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Fluorescence Decay – Frequency Fluorescence Decay – Frequency DomainDomain
excitation fluorescence
phase shift
demodulation M
tan = M=1/(1+ ()2 )1/2
modulation
frequency
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The fluorescence lifetime can probe …..
>
K. Suhling et al, Photochem Photobiol Sci 4, 13-22, 2005
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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
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K. Suhling. “Fluorescence Lifetime Imaging.” in Methods Express, Cell Imaging (ed D. Stephens), chapter 11, 219-245, Scion publishing, Bloxham, 2006.
jenlab
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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
Page 14Roger 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
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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
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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?
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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
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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
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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
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Bodipy absorption spectrum
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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)
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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
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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
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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
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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
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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
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Bodipy-based molecular rotors in cells show punctate distribution
SK-OV-3 cells incubated
with 1 μM solution of bodipy
molecular rotors
(DMSO delivery)
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FLIM of bodipy-based molecular rotors in cells
average
fluorescence
lifetime
≈1.6ns,
apparent
microviscosity
≈100cp
Impossible to learn this from intensity measurements alone
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1850ps
J. Levitt et al, J Phys Chem C, 113, 11634–11642, 2009
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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
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Rotational correlation time θ versus viscosity η
kT
V
in solution
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Time-resolved fluorescence anisotropy imaging
parallel
perpendicular
rotational correlation
time 590 ± 110 ps,
~60 cP
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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
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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
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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?
Page 360.0 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
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DCVJ in YTS NK Cells
Fluorescence
Brightfield
Inherent optical sectioning due to multiphoton excitation
Punctate distributiontargeting vesicles?
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0.966 ns
0.099 ns ≡ 650 cP!
FLIM of DCVJ in YTS NK cells
Double
exponential decay –
fluorescence
intensity
measurements
unreliable
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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
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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
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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
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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
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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
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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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