Quantitative fluorescent microscopy

Patterson GF1, Knobel SM, Sharif WD, Rain SR, Piston DW (1997) Use of the green fluorescent protein and its mutants in quantitative fluorescence microscopy. Biophys J 73 2782-2790... 

Rost FWD. Quantitative Fluorescence Microscopy, Cambridge University Press, New York, 1991. 

Xu X, Brzostowski JA, Jin T (2006) Using quantitative fluorescence microscopy and FRET imaging to measure spatiotemporal signaling events in single living cells. Methods Mol Biol 346 281-96... 

Fluorescence microscopy is routinely used to study the location and movement of intracellular species. In general, the fluorescence image reflects the location and concentration of the probe, or that amount of probe remaining in a photobleached sample (Figure 1.8, lower left). Consequently, quantitative fluorescence microscopy... 

Safiejko-Mroczka, B. and Bell, P. B. (1996) Bifunctional protein cross-linking reagents improve labeling of cytoskeletal proteins for qualitative and quantitative fluorescence microscopy. J. Histochem. Cytochem. 44,641-656. 

Different light microscopy techniques, such as autoradiography and quantitative fluorescence microscopy (QFM), have been used to examine the influence of penetration enhancers on percutaneous absorption. 

Patterson GH, Knobel SM, Sharif WD, Kain SR, Piston DW. Use of the green fluorescent protein and its mutants in quantitative fluorescence microscopy. Biophys. J. 1997 73 2782-2790. Kummer AD, Kompa C, Lossau H, POllinger-Dammer F, Michel-Beyerle ME, Silva CM, Bylina EJ, Coleman WJ, Yang MM, Youvan DC. Dramatic reduction in fluorescence quantum yield in mutants of green fluorescent protein due to fast internal conversion. Chem. Phys. 1998 237 183-193. 

Wang, Y.-L, and Taylor, D.L. (1989b). Fluorescence Microscopy of Living Cells in Culture. Part B Quantitative Fluorescence Microscopy- Imaging and Spectroscopy. Academic Press, New York. 

As an alternative to quantitative fluorescence microscopy, a flow cytometer can be used to measure the levels of accumulation (Paschal and Gerace, 1995). The permeabilized cells used in the import assay behave essentially as intact cells and are used unflxed. A standard 20-min incubation reaction time is used and the permeabilized cells are washed once in 4 ml of ice cold transport buffer and resuspended in 0.5 ml of the same buffer. The fluorescence of 10 cells is measured for each sample (Sweet and Gerace, 1996). 

We thank lain Mattaj for advice and support, Paul Fisher for the rapid immunofluorescence method, and Dirk Gdrlich for sharing the latest improvements to the nuclear protein import assay. We particularly thank Rainer Saffrich for constant help and advice on quantitative fluorescence microscopy. Colin Dingwall acknowledges the financial support of the Council for Tobacco Research. 

Fluorescence Microscopy of Living Cells in Culture, Part B Quantitative Fluorescence Microscopy—Imaging and Spectroscopy... 

As in other forms of microscopy, there are three basic kinds of fluorescence microscopy qualitative, quantitative and analytical. Qualitative fluorescence microscopy is concerned with morphology, or with whether something (e.g. an immunological reaction) is present. Quantitative fluorescence microscopy is concerned with finding out how much of a specific substance is present in a specified region of the specimen. Analytical fluorescence microscopy is the characterization of a fluorophore by measurement of excitation and emission spectra or other characteristics such as polarization or decay time. Kinetic studies essentially involve studying the fluorescence parameters described above over a period of time examples include the study of fading rates, enzyme kinetics, time-resolved fluorescence and phosphorescence. 

Wang YL and Taylor LD (1989) Methods in Cell Biology part A Fluorescent analogs, labeling cells and basic microscopy. 29 and part B quantitative fluorescence microscopy imaging and spectroscopy 30 Fluorescence microscopy of living cells in culture. Part A and B, (Eds), Academic Press, Inc., San Diego, New York, Berkely, 498 pp. 

Lee G.M. 1989. Measurement of volume injected into individual cells by quantitative fluorescence microscopy. /. Cell Sci. 94 443-447. 

Velev, O. D. Kaler, E. W. Lenhoff, A. M. Surfactant diffusion into lysozyme crystal matrices Investigated by quantitative fluorescence microscopy. J. Phys. Chem. B2000,104,9267-9275. 

In contrast, when using fluorescence immunocytochemistry to label proteins in tissue sections, the absolute relationship of fluorescence intensity to protein abundance can only be determined through conducting rigorous control experiments, and thus linearity should not be presumed. With that said, because CCD and photomultipHer tube detection systems are linear across a broad range of fluorescence intensities [36], quantitative fluorescence microscopy has been used in numerous smdies to measure relative changes in protein level [33, 35, 36]. In a system where the expression of a protein is only effected in a subpopulation of cells (e.g., GAD67 expression is reduced in a subset of PV+ interneurons in schizophrenia—see ref. 45), quantitative fluorescence immunohistochemistry will be more informative than techniques (e.g.. Western blot) that treat the tissue as a whole. 

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