Abstract
Fluorescence microscopy is an established technique for determining the localization and properties of molecules in biological specimens. Obvious advantages of fluorescence are sensitivity, specificity, and spectral characteristics that depend on the environment of the probe. In addition, the low energy content of fluorescence photons in the visible part of the spectrum permits nondestructive measurements in living cells. Imaging the spatial distribution of a molecule using its fluorescence intensity has been complemented with (micro) spectroscopic techniques for studying the physical and chemical properties of the molecular environment of the fluorophore, which allow the observation of biochemical activity in cells. This has typically been achieved by exploiting the steady-state spectral characteristics of fluorescent probes that change their emission energy upon reaction with the environment. With such techniques, an image that is related to the physiological parameter of interest can be calculated from the ratio of intensities obtained at two excitation or emission wavelengths, eliminating the concentration and light path dependence of the fluorescence intensity. To quantify these images, the ratio as a function of the physiological parameter of interest has to be calibrated separately.
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References
Alcala, J. R., E. Gratton, and D. M. Jameson. A multifrequency phase fluorometer using the harmonic content of a mode-locked laser. Anal. Instrum. 14: 225–250, 1985.
Bastiaens, P. I. H., and T. M. Jovin. Microspectroscopic imaging tracks the intracellular processing of a signal transduction protein: Fluorescent labeled protein kinase C bI. Proc. Natl. Acad. Sci. U.S.A. 93: 8407–8412, 1996.
Bastiaens, P. I. H. and A. Squire. Fluorescence lifetime imaging microscopy: Spatial resolution of biochemical processes in the cell. Trends Cell Biol. 9: 48–52, 1999.
Beechem, J. M. Global analysis of biochemical and biophysical data. Methods Enzymol. 210: 37–54, 1992.
Carlsson, K., and A. Liljeborg. Confocal fluorescence microscopy using spectral and lifetime information to simultaneously record four fluorophores with high channel separation. J. Microsc. 185: 37–46, 1997.
Clegg, R. M. Fluorescence resonance energy transfer spectroscopy and microscopy. In: Fluorescence Imaging Spectroscopy and Microscopy, edited by X. F. Wang and B. Herman. New York: Wiley, 1996, pp. 179–251.
Clegg, R. M., and P. C. Schneider. Fluorescence lifetime resolved imaging microscopy: A general description of lifetime-resolved imaging measurements. In: Fluorescence Microscopy and Fluorescence Probes, edited by J. Slavik. New York: Plenum Press, 1996, pp. 15–33.
Draaijer, A., R. Sanders, and H. C. Gerritsen. Fluorescence lifetime imaging, a new tool in confocal microscopy. In: Handbook of Biological Confocal Microscopy, edited by J. B. Pawley. New York: Plenum Press, 1995, pp. 491–505.
French, T., P. T. C. So, D. J. Weaver, T. Coelho-Sampaio, E. Gratton, E. W. Voss, and J. Carrero. Two-photon fluorescence lifetime imaging microscopy of macrophage-mediated antigen processing. J. Microsc. 185: 339–353, 1997.
Gadella, T. W. J., Jr., R. M. Clegg, and T. M. Jovin. Fluorescence lifetime imaging microscopy: Pixel-by-pixel analysis of phase modulation data. Bioimaging 2: 139–159, 1994.
Gadella, T. W. J., Jr., and T. M. Jovin. Oligomerization of epidermal growth-factor receptors on A431 cells studied by time resolved fluorescence imaging microscopy—A stereo-chemical model for tyrosine kinase receptor activation. J. Cell Biol. 129: 1543–1558, 1995.
Gadella, T. W. J., Jr., T. M. Jovin, and R. M. Clegg. Fluorescence lifetime imaging microscopy (FLIM)—Spatial-resolution of microstructures on the nanosecond time-scale. Biophys. Chem. 48: 221–239, 1993.
Gratton, E., and M. Limkeman. A continuously variable frequency cross-correlation phase fluorometer with picosecond resolution. Biophys. J. 44: 315–324, 1983.
Gratton, E., M. Limkeman, J. R. Lakowicz, B. P. Maliwa, H. Cherek, and G. Laczko. Resolution of mixtures of fluorophores using variable-frequency phase and modulation data. Biophys. J. 4: 479–486, 1984.
Kume, H., K. Koyama, K. Nakatsugawa, S. Suzuki, and D. Fatlowitz. Ultrafast microchannel plate photomultipliers. Appl. Opt. 27: 1170–1178, 1988.
Lakowicz, J. R., and K. Berndt. Lifetime-selective fluorescence imaging using an rf phase sensitive camera. Rev. Sci. Instrum. 62: 1727–1734, 1991.
Lakowicz, J. R., G. Laczko, H. Cherec, E. Gratton, and M. Limkeman. Analysis of fluorescence decay kinetics from variable-frequency phase shift and modulation data. Biophys. J. 46: 463–477, 1984.
Lakowicz, J. R., H. Szmacinski, W. J. Lederer, M. S. Kirby, M. L. Johnson, and K. Nowaczyk. Fluorescence lifetime imaging of intracellular calcium in COS cells using Quin-2. Cell Calcium 15: 7–27, 1994.
Ng, T., A. Squire, G. Hansra, E Bornancin, C. Prevostel, A. Hanby, W. Harris, D. Barnes, S. Schmidt, H. Mellor, P. I. H. Bastiaens, and P. J. Parker. Imaging protein kinase Ca activation in cells. Science 283: 2085–2089, 1999.
Periasamy, A., P. Wodnicki, X. F. Wang, S. Kwon, G. W. Gordon, and B. Herman. Time resolved fluorescence lifetime imaging microscopy using a picosecond pulsed tunable dye-laser system. Rev. Sci. Instrum. 67: 3722–3731, 1996.
Piston, D. W., G. Marriott, T. Radivoyevich, R. M. Clegg, T. M. Jovin, and E. Gratton. Wideband acoustooptic light-modulator for frequency-domain fluorometry and phosphorimetry. Rev. Sci. Instrum. 60: 2596–2600, 1989.
Press, W. H., S. A. Teukolky, and W. T. Vetterling. Numerical Recipes in C—The Art of Scientific Computing, 2nd ed. Cambridge: Cambridge University Press, 1992.
Sanders, R., A. Draaijer, H. C. Gerritsen, P. M. Houpt, and Y. K. Levine. Quantitative Ph imaging in cells using confocal fluorescence lifetime imaging microscopy. Anal. Biochem. 227: 302–308, 1995.
Schlick, T., and A. Fogelson. TNPACK—A truncated Newton minimization package for large scale problems: I. Algorithm and usage. ACM Trans. Math. Soft. 18: 46–70, 1992.
Schneider, P. C., and R. M. Clegg. Rapid acquisition, analysis, and display of fluorescence lifetime—resolved images for real-time applications. Rev. Sci. Instrum. 68: 4107–4119, 1997.
Scully, A. D., A. J. MacRobert, S. Botchway, P. O’Neill, A. W. Parker, R. B. Ostler, and D. Phillips Development of a laser-based fluorescence microscope with subnanosecond time resolution. J. Fluoresc. 6: 119–125, 1996.
Squire, A., and P. I. H. Bastiaens. Three dimensional image restoration in fluorescence lifetime imaging microscopy. J. Microsc. 193: 36–49, 1999.
Squire, A., R. J. Verveer, and P. I. H. Bastiaens. Multiple frequency fluorescence lifetime imaging microscopy. J. Microsc. 197: 136–149, 2000.
Straume, M., S. G. Frasier-Cadore, and M. L. Johnson. Least-squares analysis of fluorescence data. In: Topics in Fluorescence Spectroscopy, edited by J. R. Lakowicz. New York: Plenum Press, 1991.
Sytsma, J., J. M. Vroom, C. J. Degrauw, and H. C. Gerritsen. Time gated fluorescence lifetime imaging and microvolume spectroscopy using two-photon excitation. J. Microsc. 191: 39–51, 1998.
Szmancinski, H., and J. R. Lakowicz. Possibility of simultaneously measuring low and high calcium concentrations using Fura-2 and lifetime-based sensing. Cell Calcium 18: 64–75, 1995.
Tsien, R. Y. The green fluorescent protein. Annu. Rev. Biochem. 76: 509–538, 1998.
Tsien, R. Y., B. J. Bacskai, and S. R. Adams. FRET for studying intracellular signaling. Trends Cell Biol. 3: 242–245, 1993.
Verkman, A. S., M. Armijo, and K. Fushimi. Construction and evaluation of a frequency domain epifluorescence microscope for lifetime and anisotropy decay measurements in subcellular domains. Biophys. Chem. 40: 117–125, 1991.
Verveer, P. J., A. Squire, and P. I. H. Bastiaens. Global analysis of fluorescence lifetime imaging microscopy data. Biophys. J. 78: 2127–2137, 2000.
Watkins, A. N., C. M. Ingersoll, G. A. Baker, and F. V. Bright. A parallel multiharmonic frequency-domain fluorometer for measuring excited-state decay kinetics following one-, two-, or three-photon excitation. Anal. Chem. 70: 3384–3396, 1998.
Wouters, F. S., P. I. H. Bastiaens. Fluorescence lifetime imaging of receptor tyrosine kinase activity in cells. Curr. Biol. 9: 1127–1130, 1999.
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© 2001 American Physiological Society
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Verveer, P.J., Squire, A., Bastiaens, P.I.H. (2001). Frequency-Domain Fluorescence Lifetime Imaging Microscopy: A Window on the Biochemical Landscape of the Cell. In: Periasamy, A. (eds) Methods in Cellular Imaging. Methods in Physiology. Springer, New York, NY. https://doi.org/10.1007/978-1-4614-7513-2_16
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DOI: https://doi.org/10.1007/978-1-4614-7513-2_16
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