1. Page 1 Klaus Suhling Department of Physics Kings College London Strand London WC2R 2LS UK Fluorescence Lifetime Imaging (FLIM) of molecular rotors maps microviscosity in cells Klaus Suhling Department of Physics Kings College London Strand London WC2R 2LS UK

2. Page 2 Outline / 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

3. Page 3 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

4. Page 4 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

5. Page 5 What is Fluorescence? krkr excited state S 1 ground state S 0 k isc T1T1 k ic radiative deactivation of the first electronically excited singlet state k ph / k ic molecular energy levels

6. Page 6 Fluorescence can be characterised by: position intensity wavelength lifetime polarization Fluorescence is multi-parameter signal

7. Page 7 Fluorescence lifetime average time fluorophore remains in its excited state =1 / (k r +k isc +k ic +[q]k q ) = 1 / (k r +k nr ) depends on: intrinsic properties of molecule (k r )intrinsic properties of molecule (k r ) local environment of molecule (k nr )local environment of molecule (k nr ) use of fluorophore to probe environment e.g. viscosity, refractive index, pH, Ca 2+, polarity, interaction with other molecules ….

8. Page 8 Fluorescence Decay – Time Domain I = I 0 e -t/ Excitation pulse time intensity Fluorescence emission How is the fluorescence decay measured?

9. Page 9 Fluorescence Decay – Frequency Domain excitation fluorescence phase shift demodulation M tan = M=1/(1+ ( ) 2 ) 1/2 modulation frequency

10. Page 10 The fluorescence lifetime can probe ….. > K. Suhling et al, Photochem Photobiol Sci 4, 13-22, 2005

11. Page 11 Why Fluorescence Lifetime Imaging (FLIM)? contrast according to fluorescence lifetimecontrast according to fluorescence lifetime absolute measurement independent of variations in fluorophore concentration, illumination intensity, light path length, scatter, photobleachingabsolute measurement independent of variations in fluorophore concentration, illumination intensity, light path length, scatter, photobleaching directly image environment of specific proteins or dyes in living cellsdirectly image environment of specific proteins or dyes in living cells image molecular interaction, e.g. fluorescence resonance energy transfer (FRET) to study proximity of proteinsimage molecular interaction, e.g. fluorescence resonance energy transfer (FRET) to study proximity of proteins distinguish spectrally similar fluorophoresdistinguish spectrally similar fluorophores

12. Page 12 K. Suhling. Fluorescence Lifetime Imaging. in Methods Express, Cell Imaging (ed D. Stephens), chapter 11, 219-245, Scion publishing, Bloxham, 2006. jenlab

13. Page 13 Imaging protein interaction by FRET donor fluorescence lifetime shortened fluorescence / Förster resonance energy transfer occurs at close proximity of donor and acceptor, <8nm K. Suhling et al, Photochem Photobiol Sci 4, 13-22, 2005

14. Page 14 Roger Tsien GFP GFP and mRFP absorption and emission spectra Detect GFP donor fluorescence in this spectral window Identify FRET by shortened donor lifetime

15. Page 15 GFP-PKC interacting with ezrin (anti-VSVG-Cy3) at the tips of filopodia in breast carcinoma cells Tony Ng, Randall Division, Kings College London

16. Page 16 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?

17. Page 17 Methods for intracellular diffusion measurements 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 INTENSITY TIME VISCOSITY

18. Page 18 BODIPY-based molecular rotor – a viscosity probe 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

19. Page 19 How does a molecular rotor work? 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

20. Page 20 Bodipy absorption spectrum

21. Page 21 Bodipy fluorescence emission spectra in methanol / glycerol solution Fluorophore concentration constant Φ = A η x Φ - fluorescence quantum yield A, x - constant η - viscosity (Förster & Hoffmann, Z Phys Chem 1971, 75, 63–69)

22. Page 22 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

23. Page 23 Sample TCSPC Card 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

24. Page 24 Increase in viscosity Time / ns Emission intensity Fluorescence decays of bodipy-based molecular rotor in methanol / glycerol solutions

25. Page 25 Fluorescence lifetime τ is a function of viscosity η τ = k 0 -1 A η x log ( τ) = x log ( η ) + log (A/k 0 ) Plot log ( τ) vs log ( η ) straight line according to theory – serves as calibration

26. Page 26 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

27. Page 27 Bodipy-based molecular rotors in cells show punctate distribution SK-OV-3 cells incubated with 1 μM solution of bodipy molecular rotors (DMSO delivery)

28. Page 28 FLIM of bodipy-based molecular rotors in cells average fluorescence lifetime 1.6ns, apparent microviscosity 100cp Impossible to learn this from intensity measurements alone

29. Page 29 1850ps J. Levitt et al, J Phys Chem C, 113, 11634–11642, 2009

30. Page 30 Time-resolved Fluorescence Anisotropy Molecular tumbling characterised by rotational correlation time by rotational correlation time For a spherical molecule : viscosity molecular volume Excite with linearly polarized light log intensity time information about rotational diffusion of molecules

31. Page 31 Rotational correlation time θ versus viscosity η in solution

32. Page 32 Time-resolved fluorescence anisotropy imaging parallel perpendicular rotational correlation time 590 ± 110 ps, ~60 cP

33. Page 33 Combine fluorescence lifetime τ and rotational correlation time τ = k 0 -1 A η x = ηV / kT Therefore τ = k 0 -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

34. Page 34 log τ vs log of bodipy in cells solution cells Cell data in good agreement with solution data gradient=0.53±0.09

35. Page 35 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. Commercially available Molecular Rotors Measure intensity and determine viscosity?

36. Page 36 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 = 0.30 streak camera Measurements (Junle Qu, Institute of Optoelectronics, Shenzhen University, China

37. Page 37 DCVJ in YTS NK Cells Fluorescence Brightfield Inherent optical sectioning due to multiphoton excitation Punctate distribution targeting vesicles?

38. Page 38 0.966 ns 0.099 ns 650 cP! FLIM of DCVJ in YTS NK cells Double exponential decay – fluorescence intensity measurements unreliable

39. Page 39 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

40. Page 40 HeLa cell division 470 nm excitation 0 mins3 mins10 mins20 mins40 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

41. Page 41 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

42. Page 42 Porphyrin-based molecular rotor in cells blue = low viscosity (50cp), orange = high viscosity (300cp) Transmitted lightinitialadvanced 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

43. Page 43Conclusions 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

44. Page 44 Acknowledgements Prof Tony Ng, Dr James Levitt, post-doc, Pei-Hua Chung, PhD student, Kings College London, UK Dr Marina Kuimova, Dr Gokhan Yahioglu, Chemistry Department Imperial College London, UK Prof Harry Andersons 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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