Abstract
Scanning a sample with a focused laser beam and detecting light from the illuminated spot delivers images of superior quality. The images are largely free of lateral scattering, and data can be obtained from a defined plane inside the sample. Moreover, nonlinear optical techniques and optical near-field techniques like multiphoton microscopy, STED or NSOM can be applied to record images from deep sample layers or at a spatial resolution below the diffraction limit. Multi-dimensional TCSPC combines favourably with these techniques. The result is a FLIM technique with excellent spatial resolution, excellent image contrast, optical sectioning capability, near-ideal photon efficiency, excellent time resolution, and resolution of multi-exponential decay profiles in the individual pixels of the image. Moreover, it can be used to record multi-wavelength FLIM data, FLIM data for several excitation wavelengths, spatial mosaics of FLIM data, FLIM Z stacks, and temporally resolved FLIM data of physiological changes on the millisecond time scale. This chapter describes the implementation of multi-dimensional TCSPC in the optical systems of confocal and multiphoton laser scanning microscopes, the extension of the wavelength range into the near infrared, the combination of TCSPC with galvanometer scanners, piezo scanners and polygon scanners, the combination with endoscopic systems and with optical systems for imaging of millimeter and centimeter-size objects, and the use of TCSPC FLIM in near-field optical scanning (NSOM) and stimulated emission-depletion (STED) microscopy systems.
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References
A.M. Adawi, A. Cadby, L.G. Connolly, W.-C. Hung, R. Dean, A. Tahraoui, A.M. Fox, A.G. Cullis, D. Sanvitto, M.S. Skolnick, D.G. Lidzey, Spontaneous emission control in micropillar cavities containing a fluorescent molecular dye. Adv. Mater. 18, 742–747 (2006)
E. Auksorius, B.R. Boruah, C. Dunsby, P.M.P. Lanigan, G. Kennedy, M.A.A. Neil, P.M.W. French, Stimulated emission depletion microscopy with a supercontinuum source and fluorescence lifetime imaging. Opt. Lett. 33, 113–115 (2008)
R.M. Ballew, J.N. Demas, An error analysis of the rapid lifetime determination method for the evaluation of single exponential decays. Anal. Chem. 61, 30 (1989)
E. Beaupaire, M. Oheim, J. Mertz, Ultra-deep two-photon fluorescence excitation in turbid media. Opt. Commun. 188, 25–29 (2001)
Becker & Hickl GmbH, DCS-120 Confocal Scanning FLIM Systems, User Handbook, edition 2012. www.becker-hickl.com
Becker & Hickl GmbH, Modular FLIM Systems for Zeiss LSM 510 and LSM 710 Family Laser Scanning Microscopes, User Handbook, 5th edn (Becker & Hickl GmbH, 2012). www.becker-hickl.com
W. Becker, K. Benndorf, A. Bergmann, C. Biskup, K. König, U. Tirlapur, T. Zimmer, FRET measurements by TCSPC laser scanning microscopy. Proc. SPIE 4431, 94–98 (2001)
W. Becker, Advanced time-correlated single-photon counting techniques (Springer, Berlin, 2005)
W. Becker, B. Su, A. Bergmann, K. Weisshart, O. Holub, Simultaneous fluorescence and phosphorescence lifetime imaging. Proc. SPIE 7903, 790320 (2011)
W. Becker, B. Su, K. Weisshart, O. Holub, FLIM and FCS detection in laser-scanning microscopes: increased efficiency by GaAsP hybrid detectors. Microsc. Res. Technol. 74, 804–811 (2011)
W. Becker, The bh TCSPC Handbook, 6th edn (Becker & Hickl GmbH, 2015). www.becker-hickl.com
W. Becker, Fluorescence lifetime imaging—techniques and applications. J. Microsc. 247, 119–136 (2012)
W. Becker, V. Shcheslavskiy, Fluorescence lifetime imaging with near-infrared dyes. Proc. SPIE 8588, 85880R (2013)
W. Becker, V. Shcheslavkiy, S. Frere, I. Slutsky, Spatially resolved recording of transient fluorescence-lifetime effects by line-scanning TCSPC. Microsc. Res. Technol. 77, 216–224 (2014)
W. Becker, Fluorescence lifetime imaging techniques: time-correlated single photon counting, in Fluorescence Lifetime Spectroscopy and Imaging, ed. by L. Marcu, P.W.M. French, D.S. Elson (CRC Press, Boca Raton, 2015)
M.Y. Berezin, H. Lee, W. Akers, S. Achilefu, Near infrared dyes as lifetime solvatochromic probes for micropolarity measurements of biological systems. Biophys. J. 93, 2892–2899 (2007)
M.Y. Berezin, W.J. Akers, K. Guo, G.M. Fischer, E. Daltrozzo, A. Zumbusch, S. Achilefu, Long lifetime molecular probes based on near-infrared pyrrolopyrrole cyanine fluorophores for in vivo imaging. Biophys. J. 97, L22–L24 (2009)
M.Y. Berezin, S. Achilefu, Fluorescence lifetime measurement and biological imaging. Chem. Rev. 110, 2641–2684 (2010)
F. Bestvater, E. Spiess, G. Strobrawa, M. Hacker, T. Feurer, T. Porwolf, U. Berchner-Pfannschmidt, C. Wotzlaw, H. Acker, Two-photon fluorescence absorption and emission spectra of dyes relevant for cell imaging. J. Microsc. 208(Pt. 2), 108–115 (2002)
M. Brambilla, L. Spinelli, A. Pifferi, A. Torricelli, R. Cubeddu, Time-resolved scanning system for double reflectance and transmittance fluorescence imaging of diffusive media. Rev. Sci. Instrum. 79, 013103-1 to -9 (2008)
H.-G. Breunig, H. Studier, K. König, Multiphoton excitation characteristics of cellular fluorophores of human skin in vivo. Opt. Expr. 18(8), 7857–7871 (2010)
J. Bückers, D. Wildanger, G. Vicidomini, L. Kastrup, S.W. Hell, Simultaneous multi-lifetime multi-colour STED imaging for colocalization anlysis. Opt. Expr. 19, 3130–3143 (2011)
A. Cadby, R. Dean, A.M. Fox, R.A.L. Jones, D.G. Lidzey, Mapping the fluorescence decay lifetime of a conjugated polymer in a phase-separated blend using a scanning near-field optical microscope. Nano Lett. 5, 2232–2237 (2005)
A.J. Cadby, R. Dean, C. Elliott, R.A.L. Jones, A.M. Fox, D.G. Lidzey, Imaging the fluorescence decay lifetime of a conjugated-polymer blend by using a scanning near-field optical microscope. Adv. Mater. 19, 107–111 (2007)
A. Chorvatova, D. Chorvat, Tissue fluorophores and their spectroscopic characteristics, in Fluorescence lifetime spectroscopy and imaging, ed. by L. Marcu, P.W.M. French, D.S. Elson (CRC Press, Boca Raton, 2015)
G. Cox, Optical Imaging Techniques in Cell Biology (Taylor & Francis, Boca Raton, 2007)
W. Denk, J.H. Strickler, W.W.W. Webb, Two-photon laser scanning fluorescence microscopy. Science 248, 73–76 (1990)
T. Desmettre, J.M. Devoisselle, S. Mordon, Fluorescence properties and metabolic features of indocyanine green (ICG) as related to angiography. Surv. Ophthalmol. 45, 15–27 (2000)
A. Diaspro, in Building a Two-Photon Microscope Using a Laser Scanning Confocal Architecture, ed. by A. Periasamy. Methods in Cellular Imaging (Oxford University Press, New York, 2001), pp. 162–179
A. Diaspro, M. Corosu, P. Ramoino, M. Robello, Adapting a compact confocal microscope system to a two-photon excitation fluorescence Imaging architecture. Microsc. Res. Technol. 47, 196–205 (1999)
A. Diaspro (ed.), Confocal and Two-Photon Microscopy: Foundations, Applications and Advances (Wiley-Liss, New York, 2001)
R.C. Dunn, Near-field scanning optical microscopy. Chem. Rev. 99, 2891–2927 (1999)
F. Fischer, B. Volkmer, S. Puschmann, R. Greinert, W. Breitbart, J. Kiefer, R. Wepf, Risk estimation of skin damage due to ultrashort pulsed, focused near-infrared laser irradiation at 800Â nm. J. Biomed. Opt. 13(4), 041320 (2008)
F. Fischer, B. Volkmer, S. Puschmann, R. Greinert, W. Breitbart, J. Kiefer, R. Wepf, Assessing the risk of skin damage due to femtosecond laser irradiation. J. Biophoton. 1, 470–477 (2008)
J.J. Freed, J.L. Engle, Development of the vibrating-mirror flying spot microscope for untraviolet spctrophotometry. Ann. N.Y. Acad. Sci. 97, 412–488 (1962)
A. Gaiduk, R. Kühnemuth, S. Felekyan, M. Antonik, W. Becker, V. Kudryavtsev, M. Koenig, C. Sandhagen, C.A.M. Seidel, Time-resolved photon counting allows for new temporal and spacial insights imnto the nanoworld. Proc. SPIE 6372, 637203-1 to -13 (2006)
A. Gaiduk, R. Kühnemuth, S. Felekyan, M. Antonik, W. Becker, V. Kudryavtsev, C. Sandhagen, C.A.M. Seidel, Fluorescence detection with high time resolution: from optical microscopy to simultaneous force and fluorescence spectroscopy. Microsc. Res. Technol. 70, 403–409 (2007)
I. Gannot, I. Ron, F. Hekmat, V. Chernomordik, A. Ganjbakhche, Functional optical detection based on pH dependent fluorescence lifetime. Lasers Surg. Med. 35, 342–348 (2004)
H.C. Gerritsen, M.A.H. Asselbergs, A.V. Agronskaia, W.G.J.H.M. van Sark, Fluorescence lifetime imaging in scanning microscopes: acquisition speed, photon economy and lifetime resolution. J. Microsc. 206, 218–224 (2002)
M. Göppert-Mayer, Über elementarakte mit zwei quantensprüngen. Ann. Phys. 9, 273–294 (1931)
T. Gokus, A. Hartschuh, H. Harutyunyan, M. Allegrini, F. Hennrich, M. Kappes, A.A. Green, M.C. Hersam, P.T. Araujo, A. Jorio, Exiton dynamics in individual carbon nanotubes at room temperature. Appl. Phys. Lett. 92, 153116 (2008)
H.H. Gorris, S.M. Saleh, D.B. Groegel, S. Ernst, K. Reiner, H. Mustroph, O.S. Wolfbeis, Long-wavelength absorbing and fluorescent chameleon labels for proteins, peptides, and amines. Bioconjug Chem. 22, 1433–1437 (2011)
Govindjee, Sixty-three years since kautsky: chlorophyll α fluorescence. Aust. J. Plant Physiol. 22, 131–160 (1995)
S.W. Hell, J. Wichmann, Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy. Opt. Lett. 19, 780–782 (1994)
D. Hu, M. Micic, N. Klymyshyn, Y.D. Suh, H.P. Lu, Correlated topographic and spectroscopic imaging beyond diffraction limit by atomic force microscopy metallic tip-enhanced near-field fluorescence lifetime microscopy. Rev. Sci. Instrum. 74, 3347–3355 (2003)
G.T. Kennedy, H.B. Manning, D.S. Elson, M.A.A. Neil, G.W. Stamp, B. Viellerobe, F. Lacombe, C. Dunsby, P.M.W. French, A fluorescence lifetime imaging scanning confocal endomicroscope. J. Biophoton. 3, 103–107 (2010)
D. Kepshire, N. Mincu, M. Hutchins, J. Gruber, H. Dehghani, J. Hypnarowski, F. Leblond, M. Khayat, B.W. Pogue, A microcomputed tomography guided fluorescence tomography system for small animal molecular imaging. Rev. Sci. Instrum. 80, 043701-1 to -10 (2009)
T.A. Klar, S.W. Hell, Subdiffraction resolution in far-field fluorescence microscopy. Opt. Lett. 24, 954–956 (1999)
M. Köllner, J. Wolfrum, How many photons are necessary for fluorescence-lifetime measurements? Phys. Chem. Lett. 200, 199–204 (1992)
K. König, P.T.C. So, W.W. Mantulin, B.J. Tromberg, E. Gratton, Two-Photon excited lifetime imaging of autofluorescence in cells during UVA and NIR photostress. J. Microsc. 183, 197–204 (1996)
K. König, Multiphoton microscopy in life sciences. J. Microsc. 200, 83–104 (2000)
K. König, in Cellular Response to Laser Radiation in Fluorescence Microscopes, ed. by A. Periasamy. Methods in Cellular Imaging (Oxford University Press, New York, 2001), pp. 236–254
K. König, in Multiphoton-induced Cell Damage, ed. by B.R. Masters, P.T.C. So. Handbook of Biomedical Nonlinear Optical Microscopy (Oxford University Press, New York, 2008)
K. Koenig, Clinical multiphoton tomography. J. Biophoton. 1, 13–23 (2008)
K. Koenig, A. Uchugonova, in Multiphoton Fluorescence Lifetime Imaging at the Dawn of Clinical Application, ed. by A. Periasamy, R.M. Clegg. FLIM Microscopy in Biology and Medicine (CRC Press, Boca Raton, 2009)
E.S. Kwak, T.J. Kang, A.A. Vanden Bout, Fluorescence lifetime imaging with near-field scanning optical microscopy. Anal. Chem. 73, 3257–3262 (2001)
E. Lapointe, J. Pichette, Y. Berube-Lauziere, A multi-view time-domain non-contact diffuse optical tomography scanner with dual wavelength detection for intrinsic and fluorescence small animal imaging. Rev. Sci. Instrum. 83, 063703-1 to -14 (2012)
M.D. Lesoine, S. Bose, J.W. Petrich, E.A. Smith, Supercontinuum stimulated emission depletion fluorescence lifetime imaging. J. Phys. Chem. B 116, 7821–7826 (2012)
A. Liebert, H. Wabnitz, H. Obrig, R. Erdmann, M. Möller, R. Macdonald, H. Rinneberg, A. Villringer, J. Steinbrink, Non-invasive detection of fluorescence from exogenous chromophores in the adult human brain. NeuroImage 31, 600–608 (2006)
J.J. Mancuso, A.M. Larson, T.G. Wensel, P. Saggau, Multiphoton adaptation of a commercial low-cost confocal microscope for live tissue imaging. J. Biomed. Opt. 14(3), 034048 (2009)
G. McConnel, Confocal laser scanning fluorescence microscopy with a visible continuum source. Op. Expr. 13, 2844–2850 (2004)
G. McConnel, J.M. Girkin, S.M. Ameer-Beg, P.R. Barber, B. Vojnovic, T. Ng, A. Banerjee, T.F. Watson, R.J. Cook, Time-correlated single-photon counting fluorescence lifetime confocal imaging of decayed and sound dental structures with a white-light supercontinuum source. J. Microsc. 225, 126–136 (2007)
D.X. Medina, A. Caccamo, S. Oddo, Methylene blue reduces Aβ levels and rescues early cognitive deficit by increasing proteasome activity. Brain Pathol. 21, 140–149 (2011)
M. Micic, D. Hu, Y.D. Suh, G. Newton, M. Romine, H.P. Lu, Correlated atomic force microscopy and fluorescence lifetime imaging of live bacterial cells. Colloids Surf., B 34, 205–212 (2004)
M. Minsky, US Patent 3013467, 1957
M. Minsky, Memoir on inventing the confocal microscope. Scanning 10, 128–138 (1988)
I.H. Munro, G.R. Jones, M. Tobin, D.A. Shaw, Y. Levine, H. Gerritsen, K. van der Oord, F. Rommerts, Confocal imaging using synchrotron radiation. J. Electron Spectrosc. Relat. Phenom. 80, 343–347 (1996)
M. Oheim, E. Beaurepaire, E. Chaigneau, J. Mertz, S. Charpak, Two-photon microscopy in brain tissue: parameters influencing the imaging depth. J. Neurosci. Methods 111, 29–37 (2001)
J. Pawley (ed.), Handbook of Biological Confocal Microscopy, 3rd edn (Springer Science and Business Media LLC, New York, 2006)
S. Pelet, M.J.R. Previte, P.T.C. So, Comparing the quantification of Förster resonance energy transfer measurement accuracies based on intensity, spectral, and lifetime imaging. J. Biomed. Opt. 11, 034017-1 to -11 (2006)
L. Pires, M.S. Nogueira, S. Pratavieira, L.T. Moriyama, C. Kurachi, Time-resolved fluorescence lifetime for cutaneous melanoma detection. Biomed. Opt. Expr. 5, 3080–3089 (2014)
M. Salah, N. Samy, M. Fadel, Methylene blue mediated photodynamic therapy for resistant plaque psoriasis. J. Drags Dermatol. 8, 42–49 (2009)
R.H. Schirmer, B. Coulibaly, A. Stich, M. Scheiwein, H. Merkle, J. Eubel, K. Becker, H. Becher, O. Müller, T. Zich, W. Schiek, B. Kouyate, Methylene blue as an antimalarial agent. Redox Rep. 8, 272–275 (2003)
P.T.C. So, K.H. Kim, L. Hsu, P. Kaplan, T. Hacewicz, C.Y. Dong, U. Greuter, N. Schlumpf, C. Buehler, Two-photon microscopy of tissues, in Handbook of Biomedical Fluorescence, ed. by M.-A. Mycek, B.W. Pogue (Marcel Dekker, Basel, 2003), pp. 181–208
H. Studier, W. Becker, Megapixel FLIM. Proc. SPIE 8948, 89481K (2014)
K. Suhling, P.M.W. French, D. Phillips, Time-resolved fluorescence microscopy. Photochem. Photobiol. Sci. 4, 13–22 (2005)
P. Theer, M.T. Hasan, W. Denk, Multi-photon imaging using a Ti:sapphire regenerative amplifier. Proc. SPIE 5139, 1–6 (2003)
C.J.R. Van Der Oord, H.C. Gerritsen, F.F.G. Rommerts, D.A. Shaw, I.H. Munro, Y.K. Levine, Micro-volume time-resolved fluorescence spectroscopy using a confocal synchrotron radiation microscope. Appl. Spectrosc. 49, 1469–1473 (1995)
C.J.R. Van der Oord, J.R. Jones, D.A. Shaw, I.H. Munro, Y.K. Levine, H.C. Gerritsen, High-resolution confocal microscopy using synchrotron radiation. J. Microsc. 182, 217–224 (1996)
G.T. Wondrak, NQQO1-activated phenothiazinium redox cyclers for the targeted bioreductive induction of cancer cell apaptosis. Free Radic. Biol. Med. 15, 178–190 (2007)
J.G. White, W.B. Amos, M. Fordham, An evaluation of confocal versus conventional imaging of biological structures by fluorescence light microscopy. J. Cell Biol. 105, 41–48 (1987)
T. Wilson, C. Sheppard, Theory and Practice of Scanning Optical Microscopy (Academic Press, London, 1984)
C. Xu, W.W. Webb, Measurement of two-photon excitation cross sections of molecular fluorophores with data from 690 to 1050 nm. J. Opt. Soc. Am. B 13, 481–491 (1996)
S. Yazdanfar, C. Joo, C. Zhan, M.Y. Berezin, W.J. Akers, S. Achilefu, Multiphoton microscopy with near infrared contrast agents. J. Biomed. Opt. 15(3), 030505-1 to -3 (2010)
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Becker, W., Shcheslavskiy, V., Studier, H. (2015). TCSPC FLIM with Different Optical Scanning Techniques. In: Becker, W. (eds) Advanced Time-Correlated Single Photon Counting Applications. Springer Series in Chemical Physics, vol 111. Springer, Cham. https://doi.org/10.1007/978-3-319-14929-5_2
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