By Diana P. Bratu (auth.), Douglas J. Taatjes, Brooke T. Mossman (eds.)
A various choice of cutting-edge tools for the microscopic imaging of cells and molecules. The authors disguise a large spectrum of complimentary recommendations, together with such equipment as fluorescence microscopy, electron microscopy, atomic strength microscopy, and laser scanning cytometry. extra quite simply reproducible protocols on confocal scanning laser microscopy, quantitative computer-assisted snapshot research, laser-capture microdissection, microarray photograph scanning, near-field scanning optical microscopy, and mirrored image distinction microscopy around out this eclectic number of state of the art imaging strategies now to be had. The authors additionally talk about preparative equipment for debris and cells via transmission electron microscopy.
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Additional info for Cell Imaging Techniques: Methods and Protocols
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A spectrum analyzer is the most useful ancillary, showing not only the actual wavelength tuned but also the spread of wavelengths present. This is a very effective visual indication of the pulse length, and software is available that will compute an estimated pulse length from the spread, although this does depend on some assumptions about the pulse shape. An autocorrelator will give a more precise measure of the pulse length, but is a fiddly and complex instrument to use, although, again, rapid strides are being made in improving usability for the biologist.
Genetically targeted biosynthetic fluorescent Ca2+ sensors overcome many of the limitations encountered with synthetic and chemiluminescent Ca2+ indicators. First described in 1997 by Tsien and his colleagues, cameleons provide investigators the tools necessary to record Ca2+ signals from specific subcellular compartments (9). Cameleons are fluorescent biosynthetic Ca2+ indicators constructed by inserting a Ca2+ sensor (Xenopus laevis calmodulin and M13, a calmodulin-binding protein) between two mutated forms of green fluorescent protein (GFP).
Mammalian cell transfection reagents. Ca2+ calibration reagents and buffers. 3. 1. Tissue Culture Our experiments are performed on endocrine cell lines and primary cells. For imaging studies using fluorescent synthetic or biosynthetic Ca2+ indicators, we seed cells onto uncoated glass cover slips in six-well tissue culture plates 46 Roe et al. 24–72 h before the experiments; cells are maintained under normal culture conditions (see Note 1). Typically, we seed at a cell density to achieve 60–80% confluence by the day of the experiment in order to image [Ca2+] transients in single cells and also to have sufficient cell-free regions to obtain background fluorescence measurements.