Fundamental Limits in Confocal Microscopy
The previous chapter described how the confocal approach developed from conventional light microscopy and outlined the basic advantages gained by the use of confocal sampling techniques, primarily that the exclusion of light by the confocal pinhole makes it possible to record data from optical sections. This chapter will discuss the fundamental considerations that limit the performance of all confocal microscopes. Though at present no commercially available equipment approaches these limits, some simple tests will be described to help the user assess how well a given instrument performs. Additional information to help the user to operate the confocal microscope in an optimal manner can be found in Chapter 35, “ATutorial on Confocal Microscopy,” and Chapter 36, “Practical Confocal Microscopy.” These also include methods for measuring resolution and other useful parameters.
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- Gelles, J., Schnapp, B.J., Steur, E., and Scheetz, M.P., 1988, Nanometer scale motion analysis of microtubule-based motor enzymes, Proc. EMSA 46:68–69.Google Scholar
- Oldenbourg, R., Terada, H., Tiberio, R., Inoué, S., 1993, Image sharpness and contrast transfer in coherent confocal microscopy. J. Microscopy 172:31–39.Google Scholar
- Pawley, J.B., 1994, The sources of noise in three-dimensional microscopical data sets. In: Three Dimensional Confocal Microscopy: Volume Investigation of Biological Specimens (J. Stevens, ed.), Academic Press, New York, pp. 47–94.Google Scholar
- Pawley, J.B., 2002, Limitations on optical sectioning in live-cell confocal microscopy, Scanning, 21:241–246.Google Scholar
- Pawley, J.B., and Centonze, V., 1998, Practical laser-scanning confocal light microscopy: Obtaining optimal performance from your instrument. In: Cell Biology: A Laboratory Handbook (J.E. Celis, ed.) Academic Press, New York, pp. 149–169.Google Scholar
- Pawley, J.B., Amos, W.B., Dixon, A., and Brelje, T.C., 1993a, Simultaneous, non-interfering, collection of optimal fluorescent and backscattered light signals on the MRC-500/600. Proc. Microsc. Soc. Am. 51:156–157.Google Scholar
- Pawley, J.B., Hasko, D., and Cleaver, J., 1993, A standard test and calibration specimen for confocal microscopy II. In: Proceedings of the 1993 International Conference on Confocal Microscopy and 3-D Image Processing (C.J.R. Sheppard, ed). Sydney, Australia, p. 35.Google Scholar
- Sandison, D.R., Piston, D.W., Williams, M., Webb, W.W., 1994, Quantitative comparison of background rejection, signal-to-noise ratio, and resolution in confocal and fullfield laser scanning microscopes. Appl. Opt. 1994: 3576–3588.Google Scholar
- Sheppard, J.R., Gu, M., and Roy, M., 1992, Signal-to-noise ratio in confocal microscope systems. J. Microsc. 168:209–218.Google Scholar