![]() ![]() To our knowledge, this is the first correlative FLIM and EM workflow. Correlative imaging of antimycin A-treated fibroblasts shows a decrease in fluorescence lifetime as well as swollen mitochondria with large cavities in STEM tomography. Following chemical fixation, embedding in Epon and cutting with an ultramicrotome, tomograms of selected cells were acquired with a scanning transmission electron microscope (STEM). Human fibroblasts were cultivated in a microscopy slide bearing a coordinate system and FLIM measurement was conducted. This study delineates the correlative imaging of cells with FLIM and electron microscopy (EM). Acta 27:611–624.Fluorescence lifetime imaging microscopy (FLIM) allows the characterization of cellular metabolism by quantifying the rate of free and unbound nicotinamide adenine dinucleotide hydrogen (NADH). Sheppard, C.J.R., and Wilson, T., 1980, Multiple traversing of the object in the scanning microscope, Opt. Determining image position and correcting aberrations, Proc. O’Byrne, J.W., and Cogswell, C.J., 1992, A high resolution confocal transmission microscope: II. Goldstein, S., Hubin, T., Rosenthal, S., and Washburn, C., 1990, A confocal video rate laser beam scanning reflected light microscope with no moving parts, J. Goldstein, S., 1989, A no-moving-parts video rate laser beam scanning type 2 confocal reflected/transmission microscope, J. 165:81–101.ĭixon, A.E., Damaskinos, S., and Atkinson, M.R., 1991a, Transmission and double-reflection scanning stage confocal microscope, Scanning 13:299–306.ĭixon, A.E., Damaskinos, S., and Atkinson, M.R., 1991b, A scanning confocal microscope for transmission and reflection imaging, Nature 351:551–553. 98:633–638.Ĭogswell, C.J., and Sheppard, C.J.R., 1992, Confocal differential interference contrast (DIC) microscopy: Including a theoretical analysis of conventional and confocal DIC imaging, J. SPIE 1660:503–511.Ĭogswell, C.J., and Sheppard, C.J.R., 1989, Imaging using confocal brightfield techniques, Inst. 117:232–242.Ĭogswell, C.J., and O’Byrne, J.W., 1992, A high resolution confocal transmission microscope: I. 59:155a.īrakenhoff, G.J., 1979, Imaging modes in confocal scanning light microscopy (CSLM), J. 69:193–221.Īrt, J.J., Goodman, M.B., and Schwartz, E.A., 1991, Simultaneous fluorescent and transmission laser scanning confocal microscopy, Biophys. This process is experimental and the keywords may be updated as the learning algorithm improves.Īllen, R.D., David, G.B., and Nomarski, G., 1969, The Zeiss-Nomarski differential interference equipment for transmitted-light microscopy, Z. These keywords were added by machine and not by the authors. These can introduce sufficient aberration and deflection into the transmitted wavefront to make it very difficult to keep the transmitted beam aligned with a standard pinhole detector as it scans over the specimen. The fact that confocal transmission modes are not generally available is partly due to the fact that most biological preparations have a complex mixture of optical properties that create regions of varying refractive index (η) and absorption. ![]() This might indeed be considered surprising, since biologists have long known the advantages of observing an enormous variety of specimens (e.g., stained or unstained, preserved or living) using conventional light microscopes with their wide range of transmission modes such as brightfîeld, phase-contrast, or differential interference contrast (DIC). Although confocal reflection (brightfîeld) and fluorescence microscopy have found widespread use in biology, scanning transmission microscopy has been explored and implemented only infrequently, and a single-pass scanning confocal transmission microscope is still not available as a commercial product. ![]()
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