• Alberto Diaspro
  • Giuseppe Chirico
  • Cesare Usai
  • Paola Ramoino
  • Jurek Dobrucki


Thanks to the wide variety of applications, fluorescence microscopy is now one of the most popular imaging techniques in biology (Weber, 1960; Lakowicz, 1999; Periasamy, 2001; Michalet et al., 2003; Tsien, 2003; Bastiens and Hell, 2004; Taroni and Valentini, 2004; Diaspro et al., 2005). Fluorescence microscopy utilizes fluorescently labeled probes of high biochemical affinity to image the molecular composition and dynamics of biological structures. Moreover, the use of probes that change their fluorescence properties in response to specific physiological parameters enables one to analyze the physiological state of cells or tissues (Birks, 1970; Emptage, 2001; Zhang et al., 2002; Lippincott-Schwartz and Patterson, 2003; Stephens and Allan, 2003). Fluorescence is highly specific either as an exogenous label (e.g., 4’,6-diamidino-2-phenylindole (DAPI) bound to DNA) or an endogenous tracker [(e.g., autofluorescence of NADH, or visible fluorescent proteins such as green fluorescent protein (GFP)] providing spatial and functional information through precise photophysical properties such as absorption, emission, lifetime, and anisotropy. Furthermore, sample preparation is relatively simple, allowing non-invasive imaging and three-dimensional (3D) mapping within cells and tissues to be achieved by means of computational optical sectioning, confocal laser-scanning microscopy (CLSM), and two-photon excitation microscopy (TPEM) (Periasamy, 2001). In particular, CLSM and TPEM are two comparatively recent fluorescence microscopy techniques that have improved the quality of biological images (Wilson and Sheppard, 1984; Denk et al., 1990; Pawley, 1995a; Diaspro, 2002, 2004; Matsumoto, 2002; Amos and White, 2003).


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Agard, D.A., Hiraoka, Y., Shaw, P.J., and Sedat, J.W., 1989, Fluorescence microscopy in three-dimensions, Methods Cell Biol. 30:353–378.PubMedGoogle Scholar
  2. Amos, W.B., and White, J.G., 2003, How the confocal laser scanning microscope entered biological research, Biol. Cell 95:335–342.Google Scholar
  3. Arndt-Jovin, D.J., Nicoud, R.M., Kaufmann, J., and Jovin, T.M., 1985, Fluorescence digital-imaging microscopy in cell biology, Science 230:13330–13335.Google Scholar
  4. Axelrod, D., Koppel, D.E., Schlessinger, J., Elson, E., and Webb, W.W., 1976, Mobility measurement by analysis of fluorescence photobleaching recovery kinetics, Biophys. J. 16:1055–1069.Google Scholar
  5. Basche, T., 1998, Fluorescence intensity fluctuations of single atoms, molecules and nanoparticles, J. Lumin. 76–7:263–269.Google Scholar
  6. Bastiaens, P.I., and Hell, S.W., 2004, Recent advances in light microscopy, J. Struct. Biol. 147:1–89.Google Scholar
  7. Benson, D.M., Bryan, J., Plant, A.L., Gotto, A.M. Jr., and Smith, L.C., 1985, Digital image fluorescence microscopy: Spatial heterogeneity of photobleaching rate constants in individual cells, J. Cell Biol. 100:1309–1323.Google Scholar
  8. Berglund, A.J., 2004, Nonexponential statistics of fluorescence photobleaching, J. Chem. Phys. 121:2899–2903.PubMedGoogle Scholar
  9. Bernas, T., Cook, P.R., and Dobrucki, J.W., 2005, Confocal fluorescence imaging of photosensitised DNA denaturation in cell nuclei, Photochem. Photobiol. 81(4):960–969.Google Scholar
  10. Bernas, T., Zare_bski, M., Cook, P.R., and Dobrucki, J.W., 2004, Minimizing photobleaching during confocal microscopy of fluorescent probes bound to chromatin: role of anoxia and photon flux, J. Microsc. 215:281–296.Google Scholar
  11. Bianco, B., and Diaspro, A., 1989, Analysis of the three dimensional cell imaging obtained with optical microscopy techniques based on defocusing, Cell Biophys. 15:189–200.PubMedGoogle Scholar
  12. Birks, J.B., 1970, Photophysics of Aromatic Molecules, Wiley Interscience, London.Google Scholar
  13. Bloom, J.A., and Webb, W.W., 1984, Photodamage to intact erythrocyte membranes at high laser intensities: Methods of assay and suppression, J. Histochem. Cytochem. 32:608–616.PubMedGoogle Scholar
  14. Bock, G., Hilchenbach, M., Schauenstein, K., and Wick, G., 1985, Photometric analysis of antifading reagents for immunofluorescence with laser and conventional illumination sources, J. Histochem. Cytochem. 33:699–705.PubMedGoogle Scholar
  15. Bonetto, P., Boccacci, P., Scarito, M., Davolio, M., Epifani, M., Vicidomini, G., Tacchetti, C., Ramoino, P., Usai, C., and Diaspro, A., 2004, Threedimensional microscopy migrates to the Web with PowerUp Your Microscope, Microsc. Res. Tech. 64:196–203.PubMedGoogle Scholar
  16. Braeckmans, K., De Smedt, S.C., Roelant, C., Leblans, M., Pauwels, R., and Demeester, J., 2003, Encoding microcarriers by spatial selective photobleaching, Nat. Mater. 2:169–173.Google Scholar
  17. Braga, J., Desterro, J.M.P., and Carmo-Fonseca, M., 2004, Intracellular macromolecular mobility measured by fluorescence recovery after photobleaching with confocal laser scanning microscopes, Mol. Biol. Cell 15:4749–4760.PubMedGoogle Scholar
  18. Brakenhoff, G.J., Muller, M., and Ghauharali, R.I., 1996, Analysis of efficiency of two-photon versus single-photon absorption of fluorescence generation in biological objects, J. Microsc. 183:140–144.Google Scholar
  19. Cannone, F., Chirico, G., and Diaspro, A., 2003, Two-photon interactions at single fluorescent molecule level, J. Biomed. Opt. 8:391–395.PubMedGoogle Scholar
  20. Chen, R.F., and Scott, C.H., 1985, Atlas of fluorescence spectra and lifetimes of dyes attached to protein, Anal. Lett. 18:393–421.Google Scholar
  21. Chirico, G., Cannone, F., Baldini, G., and Diaspro, A., 2003, Two-photon thermal bleaching of single fluorescent molecules, Biophys. J. 84:588–598.Google Scholar
  22. Chirico, G., Cannone, F., Beretta, S., and Diaspro, A., 2001, Single molecule studies by means of the two-photon fluorescence distribution, Microsc. Res. Tech. 55:359–364.PubMedGoogle Scholar
  23. Chirico, G., Cannone, F., Beretta, S., Diaspro, A., Campanini, B., Bettati, S., Ruotolo, R., and Mozzarelli, A., 2002, Dynamics of green fluorescent protein mutant2 in solution, on spin-coated glasses, and encapsulated in wet silica gels, Protein Sci. 11:1152–1161.Google Scholar
  24. Chirico, G., Cannone, F., Diaspro, A., Bologna, S., Pellegrini, V., Nifosi, R., and Beltram, F., 2004, Multiphoton switching dynamics of single green fluorescent proteins, Phys. Rev. E Stat. Nonlin. Soft. Matter Phys. 70:030901(R).Google Scholar
  25. Cinelli, R.A.G., Ferrari, A., Pellegrini, V., Tyagi, M., Giacca, M., and Beltram, F., 2000, The enhanced green fluorescent protein as a tool for the analysis of protein dynamics and localization: Local fluorescence study at the single-molecule level. Photochem. Photobiol. 71:771–776.PubMedGoogle Scholar
  26. Cinelli, R.A.G., Pellegrini, V., Ferrari, A., Faraci, P., Nifosi, R., Tyagi, M., Giacca, M., and Beltram, F., 2001, Green fluorescent proteins as optically controllable elements in bioelectronics, Appl. Phys. Lett. 79:3353– 3355.Google Scholar
  27. 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 273:797–801.PubMedGoogle Scholar
  28. Cubitt, A.B., Woollenweber, L.A., and Heim, R., 1997, Understanding structure-function relationship in the Aequoria Victoria Green Fluorescent Protein, In: Green Fluorescent Proteins (K.F. Sullivan and S.A. Kay, eds.), Academic Press, San Diego, California, p. 19.Google Scholar
  29. Davis, S.K., and Bardeen, C.J., 2002, Using two-photon standing waves and patterned photobleaching to measure diffusion from nanometers to microns in biological systems, Rev. Sci. Instrum. 73:2128–2135.Google Scholar
  30. Delon, A., Usson, Y., Derouard, J., Biben, T., and Souchier, C., 2004, Photobleaching, mobility and compartmentalisation: inferences in fluorescence correlation spectroscopy, J. Fluoresc. 14:255–267.Google Scholar
  31. Denk, W., Strickler, J.H., and Webb, W.W., 1990, Two-photon laser scanning fluorescence microscopy, Science 248:73–76.PubMedGoogle Scholar
  32. Deschenes, L.A., and van den Bout, D.A., 2002, Single molecule photobleaching: increasing photon yield and survival time through suppression of two step photolysis, Chem. Phys. Lett. 365:387–395.Google Scholar
  33. Diaspro, A., 2002, Confocal and Two-Photon Microscopy: Foundations, Applications, and Advances, Wiley-Liss, New York.Google Scholar
  34. Diaspro, A., 2004, Confocal and multiphoton microscopy, In: Lasers and Current Optical Techniques in Biology (G. Palumbo and R. Pratesi, eds.), RSC-Royal Society of Chemistry, Cambridge, United Kingdom, pp. 429–478.Google Scholar
  35. Diaspro, A., and Robello, M., 2000, Two-photon excitation of fluorescence for three-dimensional optical imaging of biological structures, J. Photochem. Photobiol. B 55:1–8.PubMedGoogle Scholar
  36. Diaspro, A., Chirico, G., and Collini, M., 2005, Two-photon fluorescence excitation in biological microscopy and related techniques, Quart. Rev. Biophys. In press.Google Scholar
  37. Diaspro, A., Chirico, G., Federici, F., Cannone, F., Beretta, S., and Robello, M., 2001, Two-photon microscopy and spectroscopy based on a compact confocal scanning head, J. Biomed. Opt. 6:300–310.PubMedGoogle Scholar
  38. Diaspro, A., Federici, F., Viappiani, C., Krol, S., Pisciotta, M., Chirico, G., Cannone, F., and Gliozzi, A., 2003, Two-photon photolysis of 2-nitrobenzaldehyde monitored by fluorescent-labeled nanocapsules, J. Phys. Chem. B 107:11008–11012.Google Scholar
  39. Dickson, R.M., Cubitt, A.B., Tsien, R.Y., and Moerner, W.E., 1997, On/off blinking and switching behaviour of single molecules of green fluorescent protein, Nature 388:355–358.PubMedGoogle Scholar
  40. Dittrich, P.S., and Schwille, P., 2001, Photobleaching and stabilization of fluorophores used for single molecule analysis with one- and two-photon excitation, Appl. Phys. B 73:829–837.Google Scholar
  41. Dixit, R., and Cyr, R., 2003, Cell damage and reactive oxygen species production induced by fluorescence microscopy: Effect on mitosis and guidelines for non-invasive fluorescence microscopy, Plant J. 36: 280–290.PubMedGoogle Scholar
  42. Dobrucki, J.W., 2001, Interaction of oxygen-sensitive luminescent probes Ru(phen)3 2+ and Ru(bipy)32+ with animal and plant cells in vitro. Mechanisms of phototoxicity and conditions for non-invasive oxygen measurements. J. Photochem. Photobiol. B. 65:136–144.Google Scholar
  43. Dobrucki, J.W., and Darzynkiewicz, Z., 2001, Chromatin condensation and sensitivity of DNA in situ to denaturation during cell cycle and apoptosis — a confocal microscopy study, Micron. 32:645–652.PubMedGoogle Scholar
  44. Drummond, D.R., Carter, N., and Cross, R.A., 2002, Multiphoton versus confocal high-resolution z-sectioning of enhanced green fluorescent microtubules: Increased multiphoton photobleaching within the focal plane can be compensated using a Pockels cell and dual widefield detectors, J. Microsc. 206:161–169.Google Scholar
  45. Edidin, M., Zagyansky, Y., and Lardner, T.J., 1976, Measurement of membrane protein lateral diffusion in single cells, Science. 191:466–468.PubMedGoogle Scholar
  46. Eggeling, C., Brand, L., and Seidel, C.A.M., 1997, Laser-induced fluorescence of coumarin derivatives in aqueous solution: Photochemical aspects for single molecule detection, Bioimaging. 5:105–115.Google Scholar
  47. Eggeling, C., Widengren, J., Rigler, R., and Seidel, C.A.M., 1998, Photobleaching of fluorescent dyes under conditions used for single molecule detection: Evidence of two step photolysis, Anal. Chem. 70:2651–2659.Google Scholar
  48. Ellenberg, J., Lippincott-Schwartz, J., and Presley, J.F., 1998, Two-color green fluorescent protein time-lapse imaging, Biotechniques. 25:838–842, 844–846.Google Scholar
  49. Emptage, N.J., 2001, Fluorescent imaging in living systems, Curr. Opin. Pharmacol. 1:521–525.PubMedGoogle Scholar
  50. Garcia-Parajo, M.F., Segers-Nolten, G.M.J., Veerman, J.-A., Greve, J., and van Hulst, N.F., 2000, Real-time light-driven dynamics of the fluorescence emission in single green fluorescent protein molecules, Proc. Nat. Acad. Sci. USA 97:7237–7242.PubMedGoogle Scholar
  51. Greenbaum, L., Rothmann, C., Lavie, R., and Malik, Z., 2000, Green fluorescent protein photobleaching: a model for protein damage by endogenous and exogenous singlet oxygen, Biol. Chem. 381:1251–1258.Google Scholar
  52. Harper, I.S., 2001, Fluorophores and their labeling procedures for monitoring various biological signals, In: Methods in Cellular Imaging (A. Periasamy, ed.), Oxford University Press, New York, pp. 20–39.Google Scholar
  53. He, J.A., Hu, Y.Z., and Jiang, L.J., 1997, Photodynamic action of phycobiliproteins: In situ generation of reactive oxygen species, Biochim. Biophys. Acta 1320:165–174.Google Scholar
  54. Heim, R., Prasher, D.C., and Tsien, R.Y., 1994, Wavelength mutations and posttranslational autoxidation of green fluorescent protein, Proc. Natl. Acad. Sci. USA 91:12501–12504.PubMedGoogle Scholar
  55. Hines, M.A., and Guyot-Sionnest, P., 1996, Synthesis and characterization of strongly luminescing ZnS-Capped CdSe nanocrystals, J. Phys. Chem. 100:468–471.Google Scholar
  56. Hirschfeld, T., 1976, Quantum efficiency independence of the time integrated emission from a fluorescent molecule, Appl. Opt. 15:3135–3139.Google Scholar
  57. Hockberger, P.E., Skimina, T.A., Centonze, V.E., Lavin, C., Chu, S., Dadras, S., Reddy, J.K., and White, J.G., 1999, Activation of flavin-containing oxidases underlies light-induced production of H2O2 in mammalian cells, Proc. Nat. Acad. Sci. USA 96:6255–6260.PubMedGoogle Scholar
  58. Jaiswal, J.K., and Simon, S., 2004, Potentials and pitfalls of fluorescent quantum dots for biological imaging, Trends Cell Biol. 14:497–504.PubMedGoogle Scholar
  59. Jung, G., Wiehler, J., Göhde, W., Tittel, J., Basché, Th., Steipe, B., and Bräuchle, C., 1998, Confocal microscopy of single molecules of the green fluorescent protein, Bioimaging 6:54–61.Google Scholar
  60. Kao, F.J., Wang, Y.M., Chen, J.C., Cheng, P.C., Chen, R.W., and Lin, B.L., 2002, Photobleaching under single photon and multi-photon excitation: Chloroplasts in protoplasts from Arabidopsis thaliana, Opt. Commun. 201:85–91.Google Scholar
  61. Kawano, H., Nabekawa, Y., Suda, A., Oishi, Y., Mizuno, H., Mijawaki, A., and Midorikawa, K., 2003, Attenuation of photobleaching in two-photon excitation fluorescence from green fluorescent protein with shaped excitation pulses, Biochem. Biophys. Res. Commun. 311:592–596.PubMedGoogle Scholar
  62. Ko, D.S., 2004, Photobleaching time distribution of a single tetramethylrhodamine molecule in agarose gel, J. Chem. Phys. 120:2530–2531.PubMedGoogle Scholar
  63. Kohen, E., Reyftmann, J.P., Morliere, P., Santus, R., Kohen, C., Mangel, W.F., Dubertret, L., and Hirschberg, J.G., 1986, A microspectrofluorometric study of porphyrin-photosensitized single living cells. II. Metabolic alterations, Photochem. Photobiol. 44:471–475.PubMedGoogle Scholar
  64. König, K., and Tirlapur, U.K., 2002, Cellular and subcellular perturbations during multiphoton microscopy, In: Confocal and Two-Photon Microscopy: Foundations, Applications and Advances (A. Diaspro, ed.), Wiley-Liss, New York, pp. 191–205.Google Scholar
  65. Krenik, K.D., Kephart, G.M., Offord, K.P., Dunnette, S.L., and Gleich, G.J., 1989, Comparison of antifading agents used in immunofluorescence, J. Immunol. Methods 117:91–97.PubMedGoogle Scholar
  66. Kriete, A., 1992, Visualization in Biomedical Microscopies, VCH, Weinheim, Germany.Google Scholar
  67. Kubitschek, U., Kückmann, O., Kues, T., and Peters, R., 2000, Imaging and tracking of single GFP molecules in solution, Biophys. J. 78: 2170–2179.Google Scholar
  68. Lakowicz, J.R., 1999, Principles of Fluorescence Microscopy, Plenum Press, New York.Google Scholar
  69. Landon, T.M., 1997, Alexa dyes, tracers and conjugates, Bioprobes 26:1–6.Google Scholar
  70. Larson, D.R., Zipfel, W.R., Williams, R.M., Clark, S.W., Bruchez, M.P., Wise, F.W., and Webb, W.W., 2003, Water-soluble quantum dots for multiphoton fluorescence imaging in vivo, Science 300:1434–1436.PubMedGoogle Scholar
  71. Lippincott-Schwartz, J., and Patterson, G.H., 2003, Development and use of fluorescent protein markers in living cells, Science 300:87–91.PubMedGoogle Scholar
  72. Maher, R.C., Cohen, L.F., and Etchegoin, P., 2002, Single molecule photobleaching observed by surface enhanced resonant Raman scattering (SERRS), Chem. Phys. Lett. 352:378–384.Google Scholar
  73. Manitto, P., Speranza, G., Monti, D., and Gramatica, P., 1987, Singlet oxygen reactions in aqueous solution. Physical and chemical quenching rate constants of crocin and related carotenoids, Tetrahedron Lett. 28: 4221–4224.Google Scholar
  74. Mathies, R.A., and Stryer, L., 1986, Single-molecule fluorescence detection: A feasibility study using phycoerythrin, In: Applications of Fluorescence in the Biomedical Sciences (D.L. Taylor, A.S. Waggoner, R.F. Murphy, F. Lanni, and R.R. Birge, eds.), Alan R. Liss, New York, pp. 129–140.Google Scholar
  75. Matsumoto, B., 2002, Cell Biological Applications of Confocal Microscopy, 2nd ed., Academic Press, San Diego, California.Google Scholar
  76. Mattoussi, H., Mauro, J.M., Goldman, E.R., Anderson, G.P., Sundar, V.C., Mikulec, F.V., and Bawendi, M.G., 2000, Self-assembly of CdSe-ZnS quantum dot bioconjugates using an engineered recombinant protein, J. Am. Chem. Soc. 122:12142–12150.Google Scholar
  77. McNally, J.G., and Smith, C.L., 2002, Photobleaching by confocal microscopy. In: Confocal and Two-Photon Microscopy: Foundations, Applications, and Advances (A. Diaspro, ed.), Wiley-Liss, Inc., New York, pp. 525–538.Google Scholar
  78. Mertz, J., 1998, Molecular photodynamics involved in multi-photon excitation fluorescence, Eur. Phys. J. D3:53–66.Google Scholar
  79. Michalet, X., Kapanidis, A.N., Laurence, T., Pinaud, F., Doose, S., Pflughoefft, M., and Weiss, S., 2003, The power and prospects of fluorescence microscopies and spectroscopies, Annu. Rev. Biophys. Biomol. Struct. 32:161–182.PubMedGoogle Scholar
  80. Moerner, W.E., Peterman, E.J.G., Brasselet, S., Kummer, S., and Dickson, R.M., 1999, Optical methods for exploring dynamics of single copies of green fluorescent protein, Cytometry 36:232–238.PubMedGoogle Scholar
  81. Molski, A., 2001, Statistics of the bleaching number and the bleaching time in single-molecule fluorescence spectroscopy, J. Chem. Phys. 114: 1142–1147.Google Scholar
  82. Ness, J.M., Akhtar, R.S., Latham, C.B., and Roth, K.A., 2003, Combined tyramide signal amplification and quantum dots for sensitive and photostable immunofluorescence detection, J. Histochem. Cytochem. 5:981–987.Google Scholar
  83. Nifosì, R., Ferrari, A., Arcangeli, C., Tozzini, V., Pellegrini, V., and Beltram, F., 2003, Photoreversible dark state in a tristable green fluorescent protein variant, J. Chem. Phys. B 107:1679–1684.Google Scholar
  84. Ono, M., Murakami, T., Kudo, A., Isshiki, M., Sawada, H., and Segawa, A., 2001, Quantitative comparison of anti-fading mounting media for confocal laser scanning microscopy, J. Histochem. Cytochem. 49:305–311.PubMedGoogle Scholar
  85. Ormo, M., Cubitt, A.B., Kallio, K., Gross, L.A., Tsien, R.Y., and Remington, S.J., 1996, Crystal structure of the Aequorea victoria green fluorescent protein, Science 273:1392–1395.PubMedGoogle Scholar
  86. Patterson, G.H., and Piston, D.W., 2000, Photobleaching in two-photon excitation microscopy, Biophys. J. 78:2159–2162.Google Scholar
  87. Pawley, J.B., 1995a, Handbook of Biological Confocal Microscopy, Plenum Press, New York.Google Scholar
  88. Pawley, J.B., 1995b, Fundamental limits in confocal microscopy, In: Handbook of Biological Confocal Microscopy (J.B. Pawley, ed.), Plenum Press, New York, pp. 19–37.Google Scholar
  89. Periasamy, A., 2001, Methods in Cellular Imaging, Oxford University Press, New York.Google Scholar
  90. Peterman, E.J.G., Brasselet, S., and Moerner, W.E., 1999, Optical methods for exploring dynamics of single copies of green fluorescent protein, J. Phys. Chem. A 103:10553–10560.Google Scholar
  91. Peters, R., Peters, J., Tews, K.H., and Bahr, W., 1974, Amicrofluorimetric study of translational diffusion in erythrocyte membranes, Biochem. Biophys. Acta 367:282–294.PubMedGoogle Scholar
  92. Poo, M., and Cone, R.A., 1974, Lateral diffusion of rhodopsin in the photoreceptor membrane, Nature 247:438–441.PubMedGoogle Scholar
  93. Reits, E.A., and Neefjes, J.J., 2001, From fixed to FRAP: measuring protein mobility and activity in living cells, Nat. Cell Biol. 3:E145–E147.Google Scholar
  94. Reyftmann, J.P., Kohen, E., Morliere, P., Santus, R., Kohen, C., Mangel, W.F., Dubertret, L., and Hirschberg, J.G., 1986, A microspectrofluorometric study of porphyrin-photosensitized single living cells. I. Membrane alterations, Photochem. Photobiol. 44:461–469.PubMedGoogle Scholar
  95. Rizzuto, R., Brini, M., DeGiorgi, F., Rossi, R., Heim, R., Tsien, R.Y., and Pozzan, T., 1996, Double labelling of subcellular structures with organelletargeted GFP mutants in vivo, Curr. Biol. 6:183–188.Google Scholar
  96. Schneider, M.B., and Webb, W.W., 1981, Measurements of submicron laser beam radii, Appl. Opt. 20:1382–1388.Google Scholar
  97. Sokolov, K., Aaron, J., Hsu, B., Nida, D., Gillenwater, A., Follen, M., MacAulay, C., Adler-Storthz, K., Korgel, B., Descour, M., Pasqualini, R., Arap, W., Lam, W., and Richards-Kortum, R., 2003, Optical systems for in vivo molecular imaging of cancer, Technol. Cancer Res. Treat. 2:491–504.PubMedGoogle Scholar
  98. Song, L., Hennink, E.J., Young, I.T., and Tanke, H.J., 1995, Photobleaching kinetics of fluorescein in quantitative fluorescence microscopy, Biophys. J. 68:2588–2600.Google Scholar
  99. Song, L., Varma, C.A., Verhoeven, J.W., and Tanke, H.J., 1996, Influence of the triplet excited state on the photobleaching kinetics of fluorescein in microscopy, Biophys. J. 70:2959–2968.Google Scholar
  100. Souchier, L.A., Ffrench, M., and Bryon, P.A., 1993, Comparison of antifading agents used in fluorescence microscopy: image analysis and laser confocal microscopy study, J. Histochem. Cytochem. 41:1833–1840.PubMedGoogle Scholar
  101. Stavreva, D.A., and McNally, J.G., 2004, Fluorescence recovery after photobleaching (FRAP) methods for visualizing protein dynamics in living mammalian cell nuclei, Methods Enzymol. 375:443–455.PubMedGoogle Scholar
  102. Stelzer, E.H.K., 1998, Contrast, resolution, pixelation, dynamic range and signal to noise ratio: fundamental to its resolution in fluorescence light microscopy, J. Microsc. 189:15–24.Google Scholar
  103. Stephens, D.J., and Allan, V.J., 2003, Light microscopy techniques for live cell imaging, Science 300:82–86.PubMedGoogle Scholar
  104. Straub, T., 2003, Heterochromatin dynamics, PLoS Biol. 1:E14.PubMedGoogle Scholar
  105. Taroni, P., and Valentini, G., 2004, Fluorescence spectroscopy and imaging (non microscopic). Part I: Basic principles and techniques, In: Lasers and Current Optical Techniques in Biology (G. Palumbo and R. Pratesi, eds.), RSC-Royal Society of Chemistry, Cambridge, United Kingdom, pp. 259–290.Google Scholar
  106. Tsien, R.Y., 1998, The green fluorescent protein, Annu. Rev. Biochem. 67:509–544.PubMedGoogle Scholar
  107. Tsien, R.Y., 2003, Imagining imaging’s future, Nat. Rev. Mol. Cell Biol. 4:SS16–SS21.Google Scholar
  108. Tsien, R.Y., and Waggoner, A.S., 1995, Fluorophores for confocal microscopy. Photophysics and photochemistry, In: Handbook of Biological Confocal Microscopy (J.B. Pawley, ed.), Plenum Press, New York, pp. 267–279.Google Scholar
  109. Voura, E.B., Jaiswal, J.K., Mattoussi, H., and Simon, S.M., 2004, Tracking metastatic tumor cell extravasation with quantum dot nanocrystals and fluorescence emission-scanning microscopy, Nat. Med. 10:993–998.Google Scholar
  110. Watson, A., Wu, X., and Bruchez, M., 2003, Lighting up cells with quantum dots, Biotechnology 34:296–303.Google Scholar
  111. Weber, G., 1960, Enumeration of components in complex systems by fluorescence spectrophotometry, Nature 190:27–29.Google Scholar
  112. Weber, G., and Teale, F.W.J., 1958, Fluorescence excitation spectrum of organic compounds in solution, Trans. Faraday Soc. 54:640–648.Google Scholar
  113. Wennmalm, S., and Rigler, R., 1999, On death numbers and survival times of single dye molecules, J. Phys. Chem. B 103:2516–2519.Google Scholar
  114. White, J., and Stelzer, E., 1999, Photobleaching GFP reveals protein dynamics inside live cells, Trends Cell Biol. 9:61–65.PubMedGoogle Scholar
  115. Widengren, J., and Rigler, R., 1996, Mechanisms of photobleaching investigated by fluorescence correlation spectroscopy, Bioimaging 4:149–157.Google Scholar
  116. Wilson, T., and Sheppard, C.J.R., 1984, Theory and Practice of Scanning Optical Microscopy, Academic Press, London.Google Scholar
  117. Wu, X., Liu, H., Liu, J., Haley, K.N., Treadway, J.A., Larson, J.P., Ge, N., Peale, F., and Bruchez, M.P., 2003, Immunofluorescent labeling of cancer marker Her2 and other cellular targets with semiconductor quantum dots, Nat. Biotechnol. 21:41–46.Google Scholar
  118. Xie, X.S., and Trautman, J.K., 1998. Optical studies of single molecules at room temperature, Annu. Rev. Phys. Chem. 49:441.PubMedGoogle Scholar
  119. Xu, C., 2002, Cross-section of fluorescent molecules in multiphoton microscopy, In: Confocal and Two-Photon Microscopy: Foundations, Applications, and Advances (A. Diaspro, ed.), Wiley-Liss, New York, pp. 75–99.Google Scholar
  120. Zehetmayer, P., Hellerer, Th., Parbel, A., Scheer, H., and Zumbusch, A., 2002, Spectroscopy of single phycoerythrocyanin monomers: dark state identification and observation of energy transfer heterogeneities, Biophys. J. 83:407–415.Google Scholar
  121. Zhang, J., Campbell, R.E., Ting, A.Y., and Tsien, R.Y., 2002, Creating new fluorescent probes for cell biology, Nat. Rev. Mol. Cell Biol. 3:906–918.PubMedGoogle Scholar
  122. Zhao, K.H., and Scheer, H., 1995, Type I and type II reversible photochemistry of phycoerythrocyanin a-subunit from Mastigocladus laminosus both involve Z, E isomerization of phycoviolobilin chromophore and are controlled by sulfhydryls in apoprotein, Biochem. Biophys. Acta 1228:244–253.Google Scholar
  123. Zimmer, M., 2002, Green fluorescent protein (GFP): Applications, structure, and related photophysical behavior, Chem. Rev. 102:759–781.Google Scholar
  124. Zondervan, R., Kulzer, F., Kol’chenko, M.A., and Orrit, M., 2004, Photobleaching of rhodamine 6G in poly(vinyl alcohol) at the ensemble and single-molecule levels, J. Phys. Chem. A 108:1657–1665.Google Scholar

Copyright information

© Springer Science+Business Media, LLC 2006

Authors and Affiliations

  • Alberto Diaspro
    • 1
    • 2
  • Giuseppe Chirico
    • 2
    • 3
  • Cesare Usai
    • 4
  • Paola Ramoino
    • 5
  • Jurek Dobrucki
    • 6
  1. 1.LAMBS-IFOM/MICROSCOPIO, Department of PhysicsUniversity of GenoaGenoaItaly
  2. 2.The National Institute for the Physics of MatterItaly
  3. 3.University of Milano BicoccaMilanItaly
  4. 4.National Research Council (CNR)GenoaItaly
  5. 5.University of GenoaGenoaItaly
  6. 6.Cell Biophysics DivisionJagiellonian UniversityKrakowPoland

Personalised recommendations