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Wide-Field Fluorescence Lifetime Imaging with Multi-anode Detectors

Part of the Methods in Molecular Biology book series (MIMB,volume 1076)

Abstract

Fluorescence lifetime imaging microscopy (FLIM) has become a powerful and widely used tool to monitor inter- and intramolecular dynamics of fluorophore-labeled proteins inside living cells.

Here, we present recent achievements in the construction of a positional sensitive wide-field single-photon counting detector system to measure fluorescence lifetimes in the time domain and demonstrate its usage in FRET applications.

The setup is based on a conventional fluorescence microscope equipped with synchronized short-pulse lasers that illuminate the entire field of view at minimal invasive intensities, thereby enabling long-term experiments of living cells. The system is capable to acquire single-photon counting images and measures directly the transfer rate of fast photophysical processes as, for instance, FRET, in which it can resolve complex fluorescence decay kinetics.

Key words

  • Time-correlated single-photon counting (TCSPC)
  • Fluorescence lifetime imaging (FLIM)
  • Förster resonance energy transfer (FRET)
  • Positional sensitive photon multiplier
  • Multichannel plate

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References

  1. Hell SW (2007) Far-field optical nanoscopy. Science 316:1153–1158

    CrossRef  PubMed  CAS  Google Scholar 

  2. Straub M, Hell SW (1998) Multifocal multiphoton microscopy: a fast and efficient tool for 3-d fluorescence imaging. Bioimaging 6:177–185

    CrossRef  Google Scholar 

  3. Straub M, Lodemann P, Holroyd P et al (2000) Live cell imaging by multifocal multiphoton microscopy. Eur J Cell Biol 79:726–734

    CrossRef  PubMed  CAS  Google Scholar 

  4. Bewersdorf J, Egner A, Hell SW (2006) Multifocal multi-photon microscopy. Springer, USA, pp 550–560

    Google Scholar 

  5. Andresen V, Egner A, Hell SW (2001) Time-multiplexed multifocal multiphoton microscope. Opt Lett 26:75–77

    CrossRef  PubMed  CAS  Google Scholar 

  6. Hell SW, Nagorni M (1998) 4pi-confocal microscopy with alternate interference. Opt Lett 23:1567–1569

    CrossRef  PubMed  CAS  Google Scholar 

  7. Nagorni M, Hell SW (1998) 4pi-confocal microscopy provides three dimensional images of the microtubule network with 100- to 150-nm resolution. J Struct Biol 123:236–247

    CrossRef  PubMed  CAS  Google Scholar 

  8. Bahlmann K, Jakobs S, Hell SW (2001) 4pi-confocal microscopy of live cells. Ultramicroscopy 87:55–64

    CrossRef  Google Scholar 

  9. Kano H, Jakobs S, Nagorni M et al (2001) Dual-color 4pi-confocal microscopy with 3d-resolution in the 100 nm range. Ultramicroscopy 90:207–213

    CrossRef  PubMed  CAS  Google Scholar 

  10. Hell SW, Stelzer EHK, Lindek S et al (1994) Confocal microscopy with an increased detection aperture: type-B 4Pi confocal microscopy. Opt Lett 19:222–224

    CrossRef  PubMed  CAS  Google Scholar 

  11. Klar TA, Dyba M, Hell SW (2001) Stimulated emission depletion microscopy with an offset depleting beam. Appl Phys Lett 78:393–395

    CrossRef  CAS  Google Scholar 

  12. Hell SW (2003) Toward fluorescence nanoscopy. Nat Biotechnol 21:1347–1355

    CrossRef  PubMed  CAS  Google Scholar 

  13. Gustafsson MG (2000) Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy. J Microsc 198:82–87

    CrossRef  PubMed  CAS  Google Scholar 

  14. Gustafsson MG (2005) Nonlinear structured-illumination microscopy: Wide-field fluorescence imaging with theoretically unlimited resolution. Proc Natl Acad Sci USA 102:13081–13086

    CrossRef  PubMed  CAS  Google Scholar 

  15. Hell SW, Kroug M (1995) Ground-state-depletion: a concept for breaking the diffraction resolution limit. Appl Phys B 60:495–497

    CrossRef  Google Scholar 

  16. Esa A, Edelmann P, Kreth G et al (2000) Three-dimensional spectral precision distance microscopy of chromatin nanostructures after triple-colour DNA labelling: a study of the BCR region on chromosome 22 and the Philadelphia chromosome. J Microsc 199:96–105

    CrossRef  PubMed  CAS  Google Scholar 

  17. Lemmer P, Gunkel M, Baddeley D et al (2008) SPDM: light microscopy with single- molecule resolution at the nanoscale. Appl Phys B 93:1–12

    CrossRef  CAS  Google Scholar 

  18. Betzig E, Patterson GH, Sougrat R et al (2006) Imaging intracellular fluorescent proteins at nanometer resolution. Science 313:1642–1645

    CrossRef  PubMed  CAS  Google Scholar 

  19. Rust MJ, Bates M, Zhuang X (2006) Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM). Nat Methods 3:793–795

    CrossRef  PubMed  CAS  Google Scholar 

  20. Huisken J, Swoger J, Bene D et al (2004) Optical sectioning deep inside live embryos by selective plane illumination microscopy. Science 305:1007–1009

    CrossRef  PubMed  CAS  Google Scholar 

  21. Vois AH, Burns DH, Spelman FA (1993) Orthogonal-plane fluorescence optical sectioning: three dimensional imaging of macroscopic biological specimens. J Microsc 170:229–236

    CrossRef  Google Scholar 

  22. Schönle A, Glatz M, Hell SW (2000) Four-dimensional multiphoton microscopy with time-correlated single-photon counting. Appl Opt 39:6306–6311

    CrossRef  PubMed  Google Scholar 

  23. Koester HJ, Baur D, Uhl R et al (1999) Ca2+ fluorescence imaging with pico- and femtosecond two-photon excitation: signal and photodamage. Biophys J 77:2226–2236

    CrossRef  PubMed  CAS  Google Scholar 

  24. Delic J, Coppey J, Magdelenat H et al (1991) Impossibility of acridine orange intercalation in nuclear dna of the living cell. Exp Cell Res 194:147–153

    CrossRef  PubMed  CAS  Google Scholar 

  25. Fantini S, Franceschini M-A, Maier JS et al (1995) Frequency-domain multichannel optical detector for noninvasive tissue spectroscopy and oximetry. Opt Eng 34:32–42

    CrossRef  CAS  Google Scholar 

  26. Gratton E, Breusegem S, Sutin J et al (2003) Fluorescence lifetime imaging for the two-photon microscope: time-domain and frequency-domain methods. J Biomed Opt 8:381–390

    CrossRef  PubMed  Google Scholar 

  27. Kumar ATN, Raymond SB, Bacskai BJ et al (2008) Comparison of frequency-domain and time-domain fluorescence lifetime tomography. Opt Lett 33:470–472

    CrossRef  PubMed  Google Scholar 

  28. Gadella TWJ (ed) (2008) FRET and FLIM Techniques, vol 33. Elsevier, Amsterdam

    Google Scholar 

  29. Bastiaens PI, Squire A (1999) Fluorescence lifetime imaging microscopy: spatial resolution of biochemical processes in the cell. Trends Cell Biol 9:48–52

    CrossRef  PubMed  CAS  Google Scholar 

  30. Jares-Erijman EA, Jovin TM (2006) Imaging molecular interactions in living cells by FRET microscopy. Curr Opin Chem Biol 10:409–416

    CrossRef  PubMed  CAS  Google Scholar 

  31. Philip J, Carlsson K (2003) Theoretical investigation of the signal-to noise ratio in fluorescence lifetime imaging. J Opt Soc Am A 20:368–379

    CrossRef  Google Scholar 

  32. Becker W (2005) Advanced time-correlated single photon counting techniques. Springer, Berlin

    CrossRef  Google Scholar 

  33. Schmidt-Bocking H (1997) United States Patent 5,686,721

    Google Scholar 

  34. Vitali M, Picazo F, Prokazov Y et al (2011) Long term wide-field FLIM: minimal invasive long term observation of proteins in living cells. PLoS One 6:e15820

    CrossRef  PubMed  CAS  Google Scholar 

  35. Wiza JL (1979) Microchannel plate detectors. Nucl Instrum Methods 162:587–601

    CrossRef  CAS  Google Scholar 

  36. Inami K, Kishimoto N, Enari Y (2006) A 5 ps tof-counter with an mcp-pmt. Nucl Instrum Methods Phys Res A 560:303–308

    CrossRef  CAS  Google Scholar 

  37. Cova S, Lacaita A, Ghioni M et al (1989) 20-ps timing resolution with single-photon avalanche diodes. Rev Sci Instrum 60:1104–1110

    CrossRef  CAS  Google Scholar 

  38. Hallensleben S, Harmer SW, Townsend PD (2000) Optical constants for the s20 photocathode, and their application to increasing photomultiplier quantum efficiency. Opt Commun 180:89–102

    CrossRef  CAS  Google Scholar 

  39. Battistoni G, Campana P, Chiarella V et al (1982) Resistive cathode transparency. Nucl Instrum Methods Phys 202:459–464

    CrossRef  Google Scholar 

  40. Jagutzki O, Lapington JS, Worth LBC et al (2002) Position sensitive anodes for mcp read-out using induced charge measurement. Nucl Instrum Methods Phys Res A 477:256–261

    CrossRef  CAS  Google Scholar 

  41. Bastiaens PI, van Hoek A, Wolkers WF et al (1992) Comparison of the dynamical structures of lipoamide dehydrogenase and glutathione reductase by time-resolved polarized flavin fluorescence. Biochemistry 31:7050–7060

    CrossRef  PubMed  CAS  Google Scholar 

  42. van den Berg PA, Mulrooney SB, Gobets B et al (2001) Exploring the conformational equilibrium of E. coli thioredoxin reductase: characterization of two catalytically important states by ultrafast flavin fluorescence spectroscopy. Protein Sci 10:2037–2049

    CrossRef  PubMed  Google Scholar 

  43. Szabelski M, Ilijev D, Sarkar P (2009) Collisional quenching of erythrosine B as a potential reference dye for impulse response function evaluation. Appl Spectrosc 63:363–368

    CrossRef  PubMed  CAS  Google Scholar 

  44. Harris JM, Lytle FE (1977) Measurement of subnanosecond fluorescence decays by sampled single. Rev Sci Instrum 48:1469–1476

    CrossRef  CAS  Google Scholar 

  45. Hanley QS, Subramaniam V, Arndt-Jovin DJ et al (2001) Fluorescence lifetime imaging: multi-point calibration, minimum resolvable differences, and artifact suppression. Cytometry 43:248–260

    CrossRef  PubMed  CAS  Google Scholar 

  46. Zuker M, Szabo AG, Bramall L et al (1985) Delta function convolution method (DFCM) for fluorescence decay experiments. Rev Sci Instrum 56:14–22

    CrossRef  CAS  Google Scholar 

  47. Van Den Zegel M, Boens N, Daems D et al (1986) Possibilities and limitations of the time-correlated single photon counting technique: a comparative study of correction methods for the wavelength dependence of the instrument response function. Chem Phys 101:311–335

    CrossRef  Google Scholar 

  48. Boens N, Qin W, Basarić N et al (2007) Fluorescence lifetime standards for time and frequency domain fluorescence spectroscopy. Anal Chem 79:2137–2149

    CrossRef  PubMed  CAS  Google Scholar 

  49. Luchowski R, Gryczynski Z, Sarkar P (2009) Instrument response standard in time-resolved fluorescence. Rev Sci Instrum 80:033109-1–033109-6

    CrossRef  Google Scholar 

  50. Davis CC, King TA (1970) Correction methods for photon pile-up in lifetime determination by single-photon counting. J Phys A Gen Phys 3:101–109

    CrossRef  Google Scholar 

  51. Nair DK, Jose M, Kuner T et al (2006) Fret-Flim at nanometer spectral resolution from living cells. Opt Express 14:12217–12229

    CrossRef  PubMed  Google Scholar 

  52. Jose M, Nair DK, Reissner C et al (2007) Photophysics of clomeleon by film: discriminating excited state reactions along neuronal development. Biophys J 2:2237–2254

    CrossRef  Google Scholar 

  53. van der Meer BW, Coker G, Chen SY (1994) Resonance energy transfer theory and data. VCH, New York

    Google Scholar 

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Acknowledgments

This work was supported by the following grants: DFG FOR 521 HA3498/1-3; BMBF “Quantum” VDI 13N10077 and DFG SFB 854 TPZ (W.Z.). The authors thank Thomas Kuner, Institute of Anatomy and Cell Biology, University of Heidelberg, Germany, Fernando Picazo and Juan Llopis, University of Castilla-La Mancha, Albacete, Spain for providing FRET constructs.

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Hartig, R., Prokazov, Y., Turbin, E., Zuschratter, W. (2014). Wide-Field Fluorescence Lifetime Imaging with Multi-anode Detectors. In: Engelborghs, Y., Visser, A. (eds) Fluorescence Spectroscopy and Microscopy. Methods in Molecular Biology, vol 1076. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-649-8_20

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  • DOI: https://doi.org/10.1007/978-1-62703-649-8_20

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