SpringerProtocols: Abstract: Quantitative Fluorescence Microscopy Techniques

6. Quantitative Fluorescence Microscopy Techniques

By: Alessandro Esposito², Simon Schlachter², Gabriele S. Kaminski Schierle², Alan D. Elder², Alberto Diaspro³, Fred S. Wouters⁴, Clemens F. Kaminski², Asparouh I. Iliev⁵

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Fluorescence microscopy is a non-invasive technique that allows high resolution imaging of cytoskeletal structures. Advances in the field of fluorescent labelling (e.g., fluorescent proteins, quantum dots, tetracystein domains) and optics (e.g., super-resolution techniques and quantitative methods) not only provide better images of the cytoskeleton, but also offer an opportunity to quantify the complex of molecular events that populate this highly organised, yet dynamic, structure.

For instance, fluorescence lifetime imaging microscopy and Förster resonance energy transfer imaging allow mapping of protein–protein interactions; furthermore, techniques based on the measurement of photobleaching kinetics (e.g., fluorescence recovery after photobleaching, fluorescence loss in photobleaching, and fluorescence localisation after photobleaching) permit the characterisation of axonal transport and, more generally, diffusion of relevant biomolecules.

Quantitative fluorescence microscopy techniques offer powerful tools for understanding the physiological and pathological roles of molecular machineries in the living cell.

Affiliation(s): (2) Department of Chemical Engineering and Biotechnology, University of Cambridge, Cambridge, UK
(3) LAMBS-MicroScoBio, Department of Physics, University of Genoa, Genoa, Italy
(4) Laboratory for Molecular and Cellular Systems, Department of Neuro- and Sensory Physiology, University Medicine Göttingen, Göttingen, Germany
(5) Rudolf Virchow Center & Institute of Pharmacology and Toxicology, University of Würzburg, Würzburg, Germany

Book Title: Cytoskeleton Methods and Protocols

Series: Methods in Molecular Biology | Volume: 586 | Pub. Date: Oct-02-2009 | Page Range: 117-142 | DOI: 10.1007/978-1-60761-376-3_6

Subject: Cell Biology

Key Words: FRET - FLIM - FRAP - FLAP - FLIP - Bioimaging

References

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1.	Diaspro, A. (2001) Confocal and Two-Photon Microscopy: Foundations, Applications, and Advances. Wiley-Liss, New York, p. 576

2.	Lakowicz, J. R. (1999) Principles of Fluorescence Spectroscopy. Kluwer Academic/Plenum Publishers, New York

3.	Wouters, F. S., Verveer, P. J., and Bastiaens, P. I. (2001) Imaging biochemistry inside cells. Trends in Cell Biology 11, 203–211

4.	Clegg, R. M. (1995) Fluorescence resonance energy transfer. Current Opinion in Biotechnology 6, 103–110

5.	Jares-Erijman, E. A., and Jovin, T. M. (2003) FRET imaging. Nature Biotechnology 21, 1387–1395

6.	Jares-Erijman, E. A., and Jovin, T. M. (2006) Imaging molecular interactions in living cells by FRET microscopy. Current Opinion in Chemical Biology 10, 409–416

7.	Elder, A. D., Domin, A., Kaminski-Schierle, G. S., Lindon, C., Pines, A., Esposito, A., and Kaminski, C. F. (2009) A quantitative protocol for dynamic measurements of protein interactions by FRET-sensitized fluorescence emission. Journal of the Royal Society Interface 6(S1), S59–S81

8.	Hoppe, A., Christensen, K., and Swanson, J. A. (2002) Fluorescence resonance energy transfer-based stoichiometry in living cells. Biophysical Journal 83, 3652–3664

9.	Song, W., Wu, J., Ge, G., and Lin, Q. (2008) Two domains of the epidermal growth factor receptor are involved in cytoskeletal interactions. Biochemical and Biophysical Research Communications 370, 589–593

10.	Cai, X. M., Lietha, D., Ceccarelli, D. F., Karginov, A. V., Rajfur, Z., Jacobson, K., Hahn, K. M., Eck, M. J., and Schaller, M. D. (2008) Spatial and temporal regulation of focal adhesion kinase activity in living cells. Molecular and Cellular Biology 28, 201–214

11.	Gadella, T. W. Jr., Jovin, T. M., and Clegg, R. M. (1993) Fluorescence lifetime imaging microscopy (FLIM) – spatial-resolution of microstructures on the nanosecond time-scale. Biophysical Chemistry 48, 221–239

12.	Becker, W., Bergmann, A., Hink, M. A., Konig, K., Benndorf, K., and Biskup, C. (2004) Fluorescence lifetime imaging by time-correlated single-photon counting. Microscopy Research and Technique 63, 58–66

13.	Esposito, A., Gerritsen, H. C., Wouters, F. S., and Resch-Genger, U. (2007) In: Wolfbeis, O. S. (Ed.) Standardization in Fluorometry: State of the Art and Future Challenges, Springer, Berlin Heidelberg New York

14.	Sanders, R., Draaijer, A., Gerritsen, H. C., Houpt, P. M., and Levine, Y. K. (1995) Quantitative pH imaging in cells using confocal fluorescence lifetime imaging microscopy. Analytical Biochemistry 227, 302–308

15.	Esposito, A., Gralle, M., Dani, M. A. C., Lange, D., and Wouters, F. S. (2008) pHlameleons: a family of FRET-based protein sensors for quantitative pH imaging. Biochemistry 47, 13115–13126

16.	Agronskaia, A. V., Tertoolen, L., and Gerritsen, H. C. (2004) Fast fluorescence lifetime imaging of calcium in living cells. Journal of Biomedical Optics 9, 1230–1237

17.	Webb, S. E. D., Gu, Y., Leveque-Fort, S., Siegel, J., Cole, M. J., Dowling, K., Jones, R., French, P. M. W., Neil, M. A. A., Juskaitis, R., Sucharov, L. O. D., Wilson, T., and Lever, M. J. (2002) A wide-field time-domain fluorescence lifetime imaging microscope with optical sectioning. Review of Scientific Instruments 73, 1898–1907

18.	Esposito, A., Wouters, F. S., Bonifacino, J. S., Dasso, M., Harford, J. B., Lippincott-Schwartz, J., and Yamada, K. M. (2004) Fluorescence lifetime imaging microscopy. In: Bonifacino, J. S., Dasso, M., Harford, J. B., Lippincott-Schwartz, J., and Yamada, K. M. (Eds.) Current Protocols in Cell Biology. Wiley, New York, NY

19.	Elder, A. D., Frank, J. H., Swartling, J., Dai, X., and Kaminski, C. F. (2006) Calibration of a wide-field frequency–domain fluorescence lifetime microscopy system using light emitting diodes as light sources. Journal of Microscopy 224, 166–180

20.	Esposito, A., Dohm, C. P., Kermer, P., Bahr, M., and Wouters, F. S. (2007) a-synuclein and its disease-related mutants interact differentially with the microtubule protein tau and associate with the actin cytoskeleton. Neurobiology of Disease 26, 521–531

21.	Legg, J. W., Lewis, C. A., Parsons, M., Ng, T., and Isacke, C. M. (2002) A novel PKC-regulated mechanism controls CD44-ezrin association and directional cell motility. Nature Cell Biology 4, 399–407

22.	Reits, E. A. J., and Neefjes, J. J. (2001) From fixed to FRAP: measuring protein mobility and activity in living cells. Nature Cell Biology 3, E145–EE47

23.	Stagi, M., Dittrich, P. S., Frank, N., Iliev, A. I., Schwille, P., and Neumann, H. (2005) Breakdown of axonal synaptic vesicle precursor transport by microglial nitric oxide. Journal of Neuroscience 25, 352–362

24.	Iliev, A. I., Ganesan, S., Bunt, G., and Wouters, F. S. (2006) Removal of pattern-breaking sequences in microtubule binding repeats produces instantaneous tau aggregation and toxicity. Journal of Biological Chemistry 281, 37195–37204

25.	Gordon, G. W., Berry, G., Liang, X. H., Levine, B., and Herman, B. (1998) Quantitative fluorescence resonance energy transfer measurements using fluorescence microscopy. Biophysical Journal 74, 2702–2713

26.	Esposito, A. (2006) Molecular and Cellular Quantitative Microscopy: Theoretical Investigations, Technological Developments and Applications to the Neurosciences. Utrecht University, The Netherlands

27.	Mao, S., Benninger, R. K. P., Yan, Y. L., Petchprayoon, C., Jackson, D., Easley, C. J., Piston, D. W., and Marriott, G. (2008) Optical lock-in detection of FRET using synthetic and genetically encoded optical switches. Biophysical Journal 94, 4515–4524

28.	Wouters, F. S., Bastiaens, P. I., Wirtz, K. W., and Jovin, T. M. (1998) FRET microscopy demonstrates molecular association of non-specific lipid transfer protein (nsL-TP) with fatty acid oxidation enzymes in peroxisomes. EMBO J 17, 7179–7189

29.	Berney, C., and Danuser, G. (2003) FRET or no FRET: a quantitative comparison. Biophysical Journal 84, 3992–4010

30.	Valentin, G., Verheggen, C., Piolot, T., Neel, H., Coppey-Moisan, M., and Bertrand, E. (2005) Photoconversion of YFP into a CFP-like species during acceptor photobleaching FRET experiments. Nature Methods 2, 801

31.	Piston, D. W., and Kremers, G. J. (2007) Fluorescent protein FRET: the good, the bad and the ugly. Trends in Biochemical Sciences 32, 407–414

32.	Elangovan, M., Wallrabe, H., Chen, Y., Day, R. N., Barroso, M., and Periasamy, A. (2003) Characterization of one- and two-photon excitation fluorescence resonance energy transfer microscopy. Methods 29, 58–73

33.	Feige, J. N., Sage, D., Wahli, W., Desvergne, B., and Gelman, L. (2005) PixFRET, an ImageJ plug-in for FRET calculation that can accommodate variations in spectral bleed-throughs. Microscopy Research and Technique 68, 51–58

34.	Gerritsen, H. C., Asselbergs, M. A., Agronskaia, A. V., and Van Sark, W. G. (2002) Fluorescence lifetime imaging in scanning microscopes: acquisition speed, photon economy and lifetime resolution. Journal of Microscopy 206, 218–224

35.	Esposito, A., Gerritsen, H. C., and Wouters, F. S. (2005) Fluorescence lifetime heterogeneity resolution in the frequency-domain by Lifetime Moments Analysis (LiMA). Biophysical Journal 89, 4286–4299

36.	Colyer, R. A., Lee, C., and Gratton, E. (2007) A novel fluorescence lifetime imaging system that optimizes photon efficiency. Microscopy Research and Technique 71, 201–213

37.	Ghiggino, K. P., Harris, M. R., and Spizzirri, P. G. (1992) Fluorescence lifetime measurements using a novel fiberoptic laser scanning confocal microscope. Review of Scientific Instruments 63, 2999–3002

38.	Buurman, E. P., Sanders, R., Draaijer, A., Gerritsen, H. C., Vanveen, J. J. F., Houpt, P. M., and Levine, Y. K. (1992) Fluorescence lifetime imaging using a confocal laser scanning microscope. Scanning 14, 155–159

39.	Wang, X. F., Uchida, T., and Minami, S. (1989) A fluorescence lifetime distribution measurement system based on phase-resolved detection using an image dissector tube. Applied Spectroscopy 43, 840–845

40.	Morgan, C. G., Mitchell, A. C., and Murray, J. G. (1990) Nanosecond time-resolved fluorescence microscopy: principles and practice. Transactions of the Royal Microscopical Society 1, 463–466

41.	Gratton, E., Breusegem, S., Sutin, J., Ruan, Q., and Barry, N. (2003) Fluorescence lifetime imaging for the two-photon microscope: time-domain and frequency-domain methods. Journal of Biomedical Optics 8, 381–390

42.	O’Connor, D. V., and Phillips, D. (1984) Time Correlated Single Photon Counting. Academic Press, London

43.	Gerritsen, H. C., Agronskaia, S., Bader, A., and Esposito, A. Time Domain FLIM: theory and Instrumentation in FRET & FLIM Imaging Techniques, Gadella, T. W. G ed. Series Laboratory techniques in biochemistry & molecular biology, van der Vliet PC and Pillai S eds., 33, 95–132

44.	Stiel, H., Teuchner, K., Paul, A., Leupold, D., and Kochevar, I. E. (1996) Quantitative comparison of excited state properties and intensity-dependent photosensitization by rose bengal. Journal of Photochemistry and Photobiology B 33, 245–254

45.	Gadella, T. W., Jr., Clegg, R. M., and Jovin, T. M. (1994) Fluorescence lifetime imaging microscopy: pixel-by-pixel analysis of phase-modulation data. Bioimaging 2, 139–159

46.	Esposito, A., Gerritsen, H. C., Lustenberger, F., Oggier, T., and Wouters, F. S. (2006) Innovating lifetime microscopy: a compact and simple tool for the life sciences, screening and diagnostics. Journal of Biomedical Optics 11, 34016

47.	Squire, A., Verveer, P. J., and Bastiaens, P. I. (2000) Multiple frequency fluorescence lifetime imaging microscopy. Journal of Microscopy 197(Pt 2), 136–149

48.	Hanley, Q. S., and Ramkumar, V. (2005) An internal standardization procedure for spectrally resolved fluorescence lifetime imaging. Applied Spectroscopy 59, 261–266

49.	Hanley, Q. S., Subramaniam, V., Arndt-Jovin, D. J., and Jovin, T. M. (2001) Fluorescence lifetime imaging: multi-point calibration, minimum resolvable differences, and artifact suppression. Cytometry 43, 248–260

50.	van Munster, E. B., and Gadella, T. W., Jr. (2004) Suppression of photobleaching-induced artifacts in frequency-domain FLIM by permutation of the recording order. Cytometry 58A, 185–194

51.	van Munster, E. B., and Gadella, T. W., Jr. (2004) phiFLIM: a new method to avoid aliasing in frequency-domain fluorescence lifetime imaging microscopy. Journal of Microscopy 213, 29–38

52.	Esposito, A., Dohm, C. P., Bahr, M., and Wouters, F. S. (2007) Unsupervised fluorescence lifetime imaging microscopy for high-content and high-throughput screening. Molecular and Cellular Proteomics 68, 1446–1454

53.	Schneider, P. C., and Clegg, R. M. (1997) Rapid acquisition, analysis, and display of fluorescence lifetime-resolved images for real-time applications. Review of Scientific Instruments 68, 4107–4119

54.	Clegg, R. M., Schneider, P. C., and Slavik, J. (1996) Fluorescence lifetime-resolved imaging microscopy: a general description of lifetime-resolved imaging measurements. In: Slavik, J. (Ed.) Fluorescence Microscopy and Fluorescence Probes, Plenum Press, New York, pp. 15–33

55.	Testa, I., Parazzoli, D., Barozzi, S., Garre, M., Faretta, M., and Diaspro, A. (2008) Spatial control of pa-GFP photoactivation in living cells. Journal of Microscopy-Oxford 230, 48–60

56.	Lippincott-Schwartz, J., Altan-Bonnet, N., and Patterson, G. H. (2002) Photobleaching and photoactivation: following protein dynamics in living cells. Nature Biotechnology 20, 87–90

57.	Mazza, D., Braeckmans, K., Cella, F., Testa, I., Vercauteren, D., Demeester, J., De Smedt, S. S., and Diaspro, A. (2008) A new FRAP/FRAPa method for three-dimensional diffusion measurements based on multiphoton excitation microscopy. Biophysical Journal 95, 13

58.	Teissie, J., Tocanne, J. F., and Baudras, A. (1978) A fluorescence approach of the determination of translational diffusion coefficients of lipids in phospholipid monolayer at the air–water interface. European Journal of Biochemistry 83, 77–85

59.	Iliev, A. I., and Wouters, F. S. (2007) Application of simple photobleaching microscopy techniques for the determination of the balance between anterograde and retrograde axonal transport. Journal of Neuroscience Methods 161, 39–46

60.	Sprague, B. L., Pego, R. L., Stavreva, D. A., and McNally, J. G. (2004) Analysis of binding reactions by fluorescence recovery after photobleaching. Biophysical Journal 86, 3473–3495

61.	Lippincott-Schwartz, J., Snapp, E., and Kenworthy, A. (2001) Studying protein dynamics in living cells. Nature Reviews. Molecular cell biology 2, 444–456

62.	Grailhe, R., Merola, F., Ridard, J., Couvignou, S., Le Poupon, C., Changeux, J. P., and Laguitton-Pasquier, H. (2006) Monitoring protein interactions in the living cell through the fluorescence decays of the cyan fluorescent protein. Chemphyschem 7, 1442–1454

63.	Bunt, G., and Wouters, F. S. (2004) Visualization of molecular activities inside living cells with fluorescent labels. International Review of Cytology 237, 205–277

64.	Palamidessi, A., Frittoli, E., Garre, M., Faretta, M., Mione, M., Testa, I., Diaspro, A., Lanzetti, L., Scita, G., and Di Fiore, P. P. (2008) Endocytic trafficking of Rac is required for the spatial restriction of signaling in cell migration. Cell 134, 135–147

65.	Takanishi, C. L., Bykova, E. A., Cheng, W., and Zheng, J. (2006) GFP-based FRET analysis in live cells. Brain Research 1091, 132–139

66.	Zal, T., and Gascoigne, N. R. J. (2004) Photobleaching-corrected FRET efficiency imaging of live cells. Biophysical Journal 86, 3923–3939

67.	Digman, M., Caiolfa, V. R., Zamai, M., and Gratton, E. (2007) The Phasor approach to fluorescence lifetime imaging analysis. Biophysical Journal 94, L14–L16

68.	Hanley, Q. S., and Clayton, A. H. (2005) AB-plot assisted determination of fluorophore mixtures in a fluorescence lifetime microscope using spectra or quenchers. Journal of Microscopy 218, 62–67

69.	Wouters, F. S., and Esposito, A. (2008) Quantitative analysis of fluorescence lifetime imaging made easy. HFSP Journal 2, 7

70.	Verveer, P. J., and Bastiaens, P. I. (2003) Evaluation of global analysis algorithms for single frequency fluorescence lifetime imaging microscopy data. Journal of Microscopy 209, 1–7

71.	Verveer, P. J., Squire, A., and Bastiaens, P. I. (2000) Global analysis of fluorescence lifetime imaging microscopy data. Biophysical Journal 78, 2127–2137

72.	Vermeer, J. E. M., Van Munster, E. B., Vischer, N. O., and Gadella, T. W. J. (2004) Probing plasma membrane microdomains in cowpea protoplasts using lipidated GFP-fusion proteins and multimode FRET microscopy. Journal of Microscopy-Oxford 214, 190–200

73.	Cole, N. B., Smith, C. L., Sciaky, N., Terasaki, M., Edidin, M., and Lippincott-Schwartz, J. (1996) Diffusional mobility of golgi proteins in membranes of living cells. Science 273, 797

74.	Domin, A., Hooper, R., Rauch, U., Lindon, A. C., and Kaminski, C. F. (2003) Linked-fluorophore FRET calibration and FRET studies of the cyclin-CDK switch in mammalian cells. Proceedings of SPIE 5139, 7

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