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Multi-parameter fluorescence imaging

Multi-parameter fluorescence imaging.

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Multi-parameter fluorescence imaging

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  1. Multi-parameter fluorescence imaging Richard Benninger, Pieter de Beule, Peter Lanigan, James McGinty, Clifford Talbot, Bebhinn Treanor, Chris Dunsby, Dan Elson, Neil Galletly,DelisaIbanez-Garcia, Ian Munro Shlomo Nedvetzki,Jose Requejo-Isidro, Jan Siegel, Stephen Webb, Klaus Suhling, Fred Reavell, Mark Neil, Paul French John Lever Dan Davis Bjorn Onfelt David Phillips Praveen Anand Uma Anand Charles Coombs Mike Ferenczi Gordon Stamp Ann Sandison Martin Slade Pat Souter Paul Tadrous Kirill Volynski Andrew Wallace….. Imperial College London Physics Department Bioengineering Department Biology Department Chemistry Department Faculty of Medicine Support from: GSK R&D Ltd; Kentech Instruments Ltd; BBSRC, DTI Beacon Award, EPSRC, HEFCE JIF(V), MRC, EU FP6, Wellcome Trust

  2. Outline • Introduction • Fluorescence lifetime imaging (FLIM) • contrasting different molecular species • imaging different molecular environments • probing local environment of EGFP • intrinsic tissue FLIM contrast using autofluorescence • high-speed (real-time) FLIM • Multi-parameter fluorescence microscopy • Microscopes resolving with respect to x, y, z, l, t, polarisation, q andt, • imaging rotational correlation time • spectro-temporal imaging • imaging fluorescence anisotropy and linear dichroism • Outlook

  3. Imaging (biological tissue) with fluorescence Intensity ~f{h},h=G/(G+k) Wavelength, l ~ hc/(E1-E0) G k E1 Lifetime,t= 1/(G+k) Problems:heterogeneity, scattering and background fluorescence E0 l Difficult to make absolute intensity measurements Solution:imaget– a relative measurement Aim: to detect or image different types or states of molecules using fluorescence to achievecontrast

  4. Fluorescence lifetime imaging (FLIM) Excitation pulse Fluorescenceemission intensity t3 I = I0 e-t/t Delay generator t1 t2 t1 t2 t3 Dichroic mirror time CCD GOI Filter Sample t t Ultrafast laser system t

  5. 4000ps Coumarin 314 DASPI t 3 cm 0ps Influence of the fluorophore environment (viscosity) 200 ps DASPI + ethanol glycerol 100 0 80 20 60 40 40 60 20 80 t 40 ps Macroscopic multi-well-plate imaging – for assays (wide-field FLIM with picosecond diode) Chemically specific imaging = f{G} = f{k}

  6. E-GFP in glycerol/buffer solution % glycerol decreasing n decreasing 2850 ps t 2050 ps Strickler-Berg-plot FLIM to report local environment of E-GFP Application to cell imaging?

  7. 2.5 ns Strickler-Berg-plot 1.4 ns FLIM to report local environment of E-GFP in cells EGFP-tagged MHC protein NK Origin of fluorescence lifetime heterogeneity?

  8. 1440 ps 1218 ps FLIM of muscle cell diffused with Cy3-ATP (NIMR) Thick filament myosin Thin filament actin Contractile filaments: produce force and sliding A band I band I band Intensity image Multiphoton FLIM image 1337 ps – A bands (myosin) 1275 ps – I bands (actin) 10 microM Cy3-EDA-ATP

  9. FLIM microscopy of biological tissue (rat ear) Stained section (Orcein) Intensity image x10 Intensity image x40 Vein Elastic cartilage Artery Vein Fluorescence lifetime images 1750 ps 250 ps <t> <t> 60 ps 270 ps

  10. Benign fibroadenoma Invasive ductal carcinoma Femoral head with subchondral cyst 3576ps 5000ps 2200ps 1580ps 500ps 100ps Fluorescence lifetime imaging of ex vivo tissue H & E FLIM

  11. FLIM microscopy of atherosclerotic plaque excited at 412 nm (1.3 mW) Plaque Intima – normal artery Fluorescence lifetime histograms with normalised pixel count: Fluorescence intensity and lifetime images (x20) of a fixed section of rabbit artery

  12. High speed time domain wide-field FLIM Ultrafast laser system t intensity Delay generator CCD GOI time Sample t Rapid Lifetime Determination (after Wang et al., Applied Spectr. 45, 360 (1991) Two long (ns) gates followed by RLD* Fast delay generator (switches in ~ 1 ns) Frequency domain video rate FLIM:Holub et al., Photosynthetica 38, (2000)581

  13. Smith & Nephew Wide-field RLD endoscopic FLIM 6.00 29 Hz: (RLD) 0.00 t (ns) 6.00 0.00 6.00 6.00 29 Hz: (RLD) 7.2 Hz: (RLD) 0.00 t (ns) 0.00 6.00 0.00 C++ & post-processing using RLD algorithm FLIM image is 344 x 256 pixels High-speed FLIM Wide-field macro FLIM 6.00 ~ 0.1 Hz: 0.00

  14. Smith & Nephew Wide-field conventional endoscopic FLIM 4.00 4.00 RC RC (ns) (ns) t t M M C C C C RP RP 0.50 0.50 High-speed FLIM Wide-field RLD (7.2 Hz) endoscopic FLIM Sheep’s kidney RC: renal cortex; RP: Renal pelvis M: medulla; C: Calyces

  15. High-speed RLD FLIM of moving samples Wide-field macro FLIM of multiwell plate array (Stilbene 1 and Coumarin 314) FLIM images acquired and displayed at 6.25 Hz (LabView)

  16. High speed time domain wide-field FLIM Ultrafast laser system t1 t2 t3 t4 t intensity Delay generator CCD GOI time Sample Solution is single-shot FLIM with 4-channel GOI t Rapid Lifetime Determination (after Wang et al., Applied Spectr. 45, 360 (1991) Two long (ns) gates followed by RLD* Sequential image acquisition leads to motion artefacts [2-channel: Agronskaia et al., J. Appl. Phys. D 36, (2003) 1655 ]

  17. Gated Segmented Imager 49% 31% 12% 8% intensity t2 t1 t3 t4 time Optical Insights 4-channel imager Kentech GSI Segmented photocathode CCD MCP Hamamatsu ORCA ER Primary image plane Secondary image plane

  18. High-speed single-shot FLIM of moving samples Wide-field macro FLIM of multiwell plate array (Stilbene 1 and Coumarin 314) acquired at 20 fps, played at 8 fps Autofluorescence of human pancreas section x10 magnification, acquired at 10 fps, played at 30 fps FLIM image is 185x165 pixels

  19. Wide-field 2D (FLIM) microscopy F F F • , polarisation using 2-channel imager(Optical Insights, Inc) Broadly tunable excitation source (Spectra-Physics Hurricane + OPA) ~ 200 fs, 300-1200 nm @ 5 kHz Resolving fluorescence w.r.t. x, y, z, t , l, polarisation, FRET x, y, t(FLIM)using GOI(Kentech Instruments Ltd)

  20. Spectrally-resolved FLIM (of rat heart tissue) FLIM image pairs 400 ps < t < 1000 ps F F F (Optical Insights, Inc) <t> <t> <t> 539 ps 554 ps 532 ps 663 ps 536 ps 486 ps t t t 450-505 nm 450-505 nm 450-505 nm 505-800 nm 505-575 nm 575-800 nm Wide-field (high-speed) imaging Combine FLIM with spectral (or polarisation) resolution using 2-channel imager Spectrally-resolved FLIM applicable to FRET

  21. Polarisation-resolved wide-field fluorescence imaging Polarized Excitation F F F Polarisation-resolved FLIM Excitation axis R6G + Methanol t R6G + Ethylene Glycol Wide-field (high-speed) imaging Wide-field polarisation-resolved imaging using 2-channel imager Polarising beamsplitter filter

  22. Polarisation-resolved wide-field fluorescence imaging F F F R6G + Methanol log intensity t logintensity R6G + Ethylene Glycol t Wide-field (high-speed) imaging Wide-field polarisation-resolved imaging using 2-channel imager Polarising beamsplitter filter Polarisation-resolved FLIM Excitation axis R6G + Methanol t R6G + Ethylene Glycol

  23. Polarisation-resolved wide-field fluorescence imaging F F F logintensity For a spherical molecule : with rotational correlation time viscosity t Wide-field (high-speed) imaging Wide-field polarisation-resolved imaging using 2-channel imager Polarising beamsplitter filter Anisotropy is defined by:

  24. Fluorescence Lifetime and Anisotropy Imaging (FLAIm):Fluorescein in NaOH/glycerol I +2I 10,000 ps 0 ps Wide-field FLIM image Wide-field map of rotational correlation time % glycerol weight in wells t(ps) q(ps)

  25. Fluorescence Lifetime and Anisotropy Imaging (FLAIm): in solution and in cells Fluorescence (total) lifetime map (merged with intensity) 6800ps Rotational correlation time map (merged with intensity) 0ps now in 3D Frequency domain: rFLIM, Clayton et al., Biophys. J. 83, (2002) 1631

  26. Wide-field 3D (FLIM) microscopy F F F • , polarisation using 2-channel imager(Optical Insights, Inc) Broadly tunable excitation source (Spectra-Physics Hurricane + OPA) ~ 200 fs, 300-1200 nm @ 5 kHz Resolving fluorescence w.r.t. x, y, z, t , l, polarisation, FRET x, y, t(FLIM)using GOI(Kentech Instruments Ltd) x, y, z, using structured illumination

  27. Wide-field 3-D imaging using structured light CCD Actuator L +L/3 +2L/3 Opt. Lett. 22 (1997) 1905 Only zero spatial frequency does not attenuate with defocus  spatially modulate whole-field image using grid

  28. Wide-field 3-D imaging using structured light CCD Actuator L Sectioned image Conventional image Opt. Lett. 22 (1997) 1905 Only zero spatial frequency does not attenuate with defocus  spatially modulate whole-field image using grid

  29. Wide-field optical sectioning FLIM of E-GFP tagged MHC protein in human B cells Conventional FLIM image Sectioned FLIM image Conventional intensity image Sectioned intensity image

  30. Wide-field 3-D imaging with multi-foci, multi-photon microscope x, y, z, t (GOI): 3-D FLIM x, y, z, t, polarisation (2-channel imager):q rotational correlation time x, y, z, t (GOI), l(2-channel imager/line scan + spectrograph) : 5-D FLIM/FRET Broadly tunable fs Ti:S source (S-P Tsunami + SHG) 680-1000 &, 340-500 nm, ~100 fs, 80 MHz Multi-beam multi-photonmicroscope(LaVision Biotec GmbH: (64 beams scanned at < 400 Hz) EM-CCD detector Resolving fluorescence w.r.t. x, y, z, t , l , polarisation, FRET x, y, z, l(2-channel imager ): spectral 3-D x, y, z, l(line scan + spectrograph): hyperspectral 3-D x, y, z, polarisation (2-channel imager): 3-D imaging of fluorophore orientation

  31. Wide-field sectioned spectral-temporal imaging with multi-foci, multi-photon microscope Change in mean l over decay Time-gated intensity Time-gated mean l Change in Dl over decay Time-gatedDl 7700 ps 8500 ps 12000 ps Fixed rabbit artery section

  32. Wide-field 3-D imaging of fluorescence anisotropy with multi-foci, multi-photon microscope Vertically polarised excitation Horizontally polarised excitation Human B cells with lipids labelled with BODIPY pe Polarisation-resolved imaging

  33. Wide-field 3-D imaging of linear dichroism with multi-foci, multi-photon microscope Fluorescence anisotropy Linear dichroism Human B cells with lipids labelled with BODIPY pe Dichroism is useful to study average molecular (dipole) orientation - independent of rotational diffusion or energy transfer Anisotropy can then report dynamic properties

  34. Wide-field 3-D imaging of linear dichroism with multi-foci, multi-photon microscope Human B cells with lipids labelled with BODIPY pe Linear dichroism Dichroism is useful to study average molecular (dipole) orientation - independent of rotational diffusion or energy transfer Mean tilt of dipole indicates degree of disorder in membrane

  35. Wide-field 3-D imaging of dichroism with multi-foci, multi-photon microscope Mean tilt as a function of time after adding cyclodextrane to BODIPY pe stained cells Cyclodextrane removes cholesterol from membrane, increasing disorder in lipid bilayer – so increasing mean tilt angle of dipoles

  36. Wide-field 3-D imaging of nanotubes Multi-foci, multi-photon microscope linear dichroism image …work in progress… Confocal microscope Human B cells transfected with GPI-GFP Following Rustom et al., SCIENCE 303 13 FEBRUARY 2004

  37. Outlook for MFI • Multi-dimensional functional imaging using fluorescence • Up to 6-D wide field fluorescence imaging (resolve t, l, x, y, z and polarization) • Image binding via change in rotational correlation time • Resolve rotational diffusion, viscosity, pH….. in 3D • Apply multi-parameter fluorescence imaging to: • cell biology: intracellular and intercellular signalling • FLIM-FRET e.g. to measure receptor conformation changes at the cell surface • high throughput screening and rapid assays ,e.g. for toxicology, drug discovery • real-time monitoring of chemical processes, e.g. microfluidics… • assess photo-physical properties of polymers, semiconductor wafers,… • Develop and deploy clinical FLIM instrumentation • Develop endoscopic FLIM • Develop tools to analyse complex fluorescence decays • Investigate origin of fluorescence contrast • in vivo diagnostic imaging: detecting disease and monitoring therapies

  38. That’s all folks Thank you for your attention

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