6.1 The Problem with Thick Specimens in Light Microscopy
It was evident to users of the light microscope that there were still unsolved problems with thick, highly scattering specimens. The use of the fluorescent light microscope together with fluorescent, thick specimens was difficult; moreover, light from above and below the focal plane contributed to a blurring of the image and a general loss of contrast. These problems were also evident during in vivo microscopy of embryos, tissues, and organs. On the other hand, these problems did not exist for very thin, fluorescent specimens.
Real biological specimens have internal structures that vary with depth and position. Prior to the use of three-dimensional computer reconstructions, in order to obtain a valid understanding of the heterogeneous specimen, it was necessary to use the light microscope to image many focal planes from the top to the lower surface, and then to reconstruct either a mental three-dimensional visualization of the specimen, or use computer techniques to make this visualization. This was the technique used by RamÃ³n y Cajal in his seminal microscopic studies of the vertebrate nervous system.
The laser was invented by Theodore Maiman in 1960. Two years earlier, Arthur Schawlow, Charles Townes, and, independently, Alexander Prokhorov showed that it was possible to amplify stimulated emission in the optical and infrared regions of the spectrum.
The Minsky patent for his confocal microscope was issued in 1961. Prior to these milestones, there were many technical innovations that aimed to increase the resolution of the light microscope. The laser is not a requirement for the confocal microscope, since usable light sources include the sun, white light arc lamps, and a 12V halogen lamp. We now discuss some of these innovations and their role in the development of light microscopy.
6.2 Some Early Attempts to Solve These Problems
A series of creative technical innovations in the field of light microscopy resulted in technical improvements and a deepened theoretical understanding of confocal light microscopy. The basic advances will be briefly discussed and classified into common groupings: advances in fluorescence microscopy and in light sources and point scanning.