Fluorescence microscopy measurement

Fluorescence microscopy measurements were performed with a Zeiss Axioplan microscope (Zeiss Co. Germany) equipped with a mercury lamp and a 40X objective. Images were acquired by CCD camera CH250 (Photometrix Co., Germany) cooled at -40°C by a liquid cooling unit CH260 (Photometrix Co., Germany). 

Fig. 2 Normalized fluorescence lifetime distributions (ELDs) for (a) the cGMP-imprinted and (b) non-imprinted thin-layer polymer film obtained from the fluorescence microscopy measurements (adapted from [47, 66])...

Feed ethanol Tracer fluorescent -Rhodamine B Fluorescence microscopy measurements Rectangular microchannel Trachsel et al. [14]... 

Work has also been reported on hnman breast cells (171) and Rhodobacter sphaeroides (172). In these stndies, the feedback mode of SECM was used to look at the regeneration reaction of different mediators when exposed to cells. These studies provide useful information abont the permeability of the membrane to a wide variety of redox couples. A theoretical treatment enabled kinetic information about these processes (173) to be extracted. SECM has also been used to distinguish between normal and malignant human breast cells (174) with corroboration by optical and fluorescence microscopy measurements. 

A wide variety of measurements can now be made on single molecules, including electrical (e.g. scanning tunnelling microscopy), magnetic (e.g. spin resonance), force (e.g. atomic force microscopy), optical (e.g. near-field and far-field fluorescence microscopies) and hybrid teclmiques. This contribution addresses only Arose teclmiques tliat are at least partially optical. Single-particle electrical and force measurements are discussed in tire sections on scanning probe microscopies (B1.19) and surface forces apparatus (B1.20). 

Bullen A, Saggau P (1999) High-speed, random-access fluorescence microscopy II. Fast quantitative measurement with voltage-sensitive dyes. Biophys J 76 2272-2287... 

Gordon, G. W., Berry, G., Liang, X. H., Levine, B. and Herman, B. (1998). Quantitative fluorescence resonance energy transfer measurements using fluorescence microscopy. Biophys. J. 74, 2702-13. 

Khakh, B. S., Fisher, J. A., Nashmi, R., Bowser, D. N. and Lester, H. A. (2005). An angstrom scale interaction between plasma membrane ATP-gated P2X2 and alpha4beta2 nicotinic channels measured with fluorescence resonance energy transfer and total internal reflection fluorescence microscopy. J. Neurosci. 25, 6911-20. 

Schneckenburger, H., Wagner, M., Kretzschmar, M., Strauss, W. S. and Sailer, R. (2004b). Laser-assisted fluorescence microscopy for measuring cell membrane dynamics. Photochem. Photobiol. Sci. 3, 817-22. 

Tsien, R. Y., Rink, T. J. and Poenie, M. Measurement of cytosolic free Ca2+ in individual small cells using fluorescence microscopy with dual excitation wavelengths. Cell Calcium 6 145-157,1985. 

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... 

Fluorescent pH indicators offer much better sensitivity than the classical dyes such as phenolphthalein, thymol blue, etc., based on color change. They are thus widely used in analytical chemistry, bioanalytical chemistry, cellular biology (for measuring intracellular pH), medicine (for monitoring pH and pCC>2 in blood pCC>2 is determined via the bicarbonate couple). Fluorescence microscopy can provide spatial information on pH. Moreover, remote sensing of pH is possible by means of fiber optic chemical sensors. 

There have been rather few studies of the location of probes in whole cells. DPH incorporates into most subcellular fractions (see, e.g, Ref. 64), whereas with TMA-DPH, early after introduction only the plasma membranes appear to be labeled/64,65) There is considerable interest in examining the lipid motional properties of living cells by fluorescence techniques. In this type of study the location of the probe has to be carefully checked before conclusions can be drawn. This is carried out by separate measurements of the recovery of probe from intact labeled cells in isolated subcellular fractions and/or by fluorescence microscopy. 

There has been a continued interest in examining the properties of intact living cells using fluorescence microscopy. This field has seen considerable advances since the application of digital imaging techniques. In examining whole cells, one has to be especially aware of the location(s) of the probe. This is particularly important when bulk measurements are to be made on intact cells. 

C. L. Poglitsch and N. L. Thompson, Interaction of antibodies with Fc receptors in substrate-supported planar membranes measured by total internal reflection fluorescence microscopy, Biochemistry 29, 248-254 (1990). 

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