Abstract
A key difference between confocal microscopy and widefield microscopy is that the aim of confocal is to explore the structure and structural relationships along the optical (Z) axis as well as in the X-Y plane. In other words, the investigation of spatial relationships is being evaluated in at least three dimensions. Often, this involves the collection of a series of planar images along the Z axis and the reconstruction of the data into an image that depicts all three dimensions. However, even when the interest is solely in a single X-Y plane, the goal is to separate as thin a plane of information as possible from the planes directly above and below. Obviously, in order to acquire and analyze three-dimensional (3D) structural information, the 3D structural relationships must be preserved during the preparation of the sample. In contrast, in standard widefield microscopy information along the Z-axis is merged into a single 2D image. Because of the added need to preserve the 3D structure for confocal images, methods suitable for preparing samples for widefield microscopy must often be altered to accommodate the additional demand of preserving relationships in the third dimension.
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References
Beckerle, M. 1984. Microinjected fluorescent polystyrene beads exhibit saltatory motion in tissue culture cells. J Cell Biol. 98:2126–2132.
Berglund, D., and J. Starkey. 1991. Introduction of antibody into viable cells using electroporation. Cytometry. 12:64–67.
Germroth, P.G., R.G. Gourdie and R.P. Thompson. 2005. Confocal microscopy of thick sections from acrylamide gel embedded embryos. Microscopy Res Tech. 30(6):513–520.
Molé-Bajer, 1968 Studies of selected endosperm cells with the light and electron microscope. The technique. Cellule. 67, 257–265.
Piston, D., and S. Knobel. 1999. Real-time analysis of glucose metabolism by microscopy. Trends Endocrinol Metab. 10:413–417.
Slayer, E.M. 1976 Optical Methods in Biology/Robert Krieger Publishing Co. Huntington, NY. pp288–302.
Waters, J. 2007. Live-cell fluorescence imaging. Methods Cell Biol. 81:115–140.
Williams, R., D. Piston, and W. Webb. 1994. Two-photon molecular excitation provides intrinsic 3-dimensional resolution for laser-based microscopy and microphotochemistry. FASEB J. 8:804–813.
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Jerome, W.G., Fuseler, J., Price, R.L. (2011). Specimen Preparation. In: Price, R., Jerome, W. (eds) Basic Confocal Microscopy. Springer, New York, NY. https://doi.org/10.1007/978-0-387-78175-4_4
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DOI: https://doi.org/10.1007/978-0-387-78175-4_4
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