This chapter describes a variety of confocal microscopes, based on several types of scanning techniques, that were invented by different individuals in several countries around the world. Marvin Minsky invented a confocal microscope in which the specimen was mechanically scanned with respect to the illumination light. PetrÃ n invented a confocal microscope based on the rotating Nipkow disk for scanning and descanning the light with respect to the specimen. Guoqing Xiao and Gordon Kino used a similar rotating Nipkow-type disk, but only used one side of the disk for scanning and descanning the light on and from the specimen. Svishchev used a two-sided mirror for the same process. Finally, laser scanning confocal microscopes are described.
In many cases, the motivation was a research problem that was not accessible with existing types of optical microscopes. Many of these problems were in the domain of in vivo microscopy. For example, PetrÃ n investigated the living brain cortex and the live retina, and Svishchev studied the brain cortex in a living animal. These diverse inventors had a common requirement: an optical microscope that could image live, unstained, thick, highly scattering specimens. There is one striking exception. Minsky was attempting to use the light microscope to observe thick, fixed, Golgi-stained brain slices.
Instead of the low-contrast, blurred images from these specimens, these researchers dreamed of a microscope with depth discrimination that could be used to observe such specimens as the live brain cortex and living retina. Many of the early inventions of various types of confocal microscopes were driven by the limitations of existing optical microscopes.
7.1 The Confocal Microscope: The Problem and Its Solution
The lack of depth discrimination or optical sectioning capability is the major limitation of the conventional (nonconfocal) fluorescence microscope. In the past, the common solution was to use very thin specimens such as cells in tissue culture monolayers or thin smears of cells for pathology. Nevertheless, this limitation precluded the use of the light microscope for thick, highly scattering specimens, e.g., in vivo human skin, live embryos, intravital microscopy of organs, brain imaging, and studies of hard tissues such as teeth and bone. Similar problems occurred when such specimens were observed with reflected light in an optical microscope.
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