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Chapter 8:
The Development of Scanning-Slit Confocal Systems for Imaging Live Cells, Tissues, and Organs
Abstract
Chapter 8 will describe and compare a number of confocal microscopes based on slits instead of pinhole or iris apertures. As with conjugate pinholes, the images of two slits (one on the illumination side and one on the detection side) are cofocused on the specimen. A slit has width and length, and these dimensions are different; a pinhole aperture is circularly symmetric. The most important difference is that the axial and transverse resolution will be different in the direction of the slit width and in the perpendicular direction. What is gained in scanning-slit confocal microscopes is a decrease in image acquisition time, since the slit image scans across the specimen, which is equivalent to scanning many points simultaneously (all points along the length of the slit image are scanned in parallel). The use of slits in a Nipkow disk-based confocal microscope is the basis of the Lichtman and Sunderland invention of a new confocal microscope (see his patent in Masters, 1996). This microscope was first developed for imaging acetylcholine receptors on muscle-cell membranes. Because it is conceptually different from the scanning-slit confocal microscopes described in this chapter, it is not discussed further. With the exception of Svishchev, who was motivated to image the live brain cortex, and Baer, who wished to develop a microscope for biological imaging of live cells and tissues, the group of inventors discussed in this chapter were interested in imaging live cells in the living eye. The living eye presents unique problems for in vivo light microscopy; its movement is the principle problem. Specifically they were interested in developing optical microscopes that could image live cells in the unstained cornea. While the initial inventions solved some problems associated with imaging the ex vivo cornea, the progression towards imaging the human cornea in vivo continued until it became a clinical reality. This chapter presents a series of linked technical advances in the development of scanning-slit confocal microscopes, with applications in the fields of biology and ophthalmology. Each technical advance was the partial solution to a problem. The end result is the development of a scanning-slit clinical confocal microscope to examine the human eye in vivo. In this historical context several lessons can be learned. First, a clear statement of the problem is necessary for its eventual solution. Second, curiosity is a strong motivator for technical innovation.
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CHAPTER 8
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