FIGURE 1.12 • Structure of the confocal microscope and diagram of the beam path. The light source for the confocal microscope comes from a laser. The laser beam (red line) travels to the tissue sample via a dichroic beam splitter and then to two movable scanning mirrors; these mirrors scan the laser beam across the sample in both x and y directions. Finally, the laser beam enters the fluorescence microscope and travels through its optical system to illuminate an examined tissue sample. The emitted light by the illuminated tissue sample (blue line) travels back through the optical system of the microscope, through both scanning mirrors, passes through the beam splitter, and is focused onto the pinhole. The light that passes through the pinhole is received and registered by the detector attached to a computer that builds the image one pixel at a time.
has the capability to obtain exceptional resolution (0.2 to 0.5 m) and clarity from a thin section of a biologic sample simply by rejecting out-of-focus light. The confocal microscope uses an illuminating laser light system that is strongly convergent and therefore produces a high-intensity excitation light in the form of a shallow scanning spot. A mirror system is used to move the laser beam across the specimen, illuminating a single spot at a time (Fig. 1.12). Many single spots in the same focal plane are scanned, and a computer software program reconstructs the image from the data recorded during scanning. In this aspect, confocal microscopy resembles the imaging process in a computed axial tomography (CAT) scan.
Furthermore, by using only the narrow depth of the in-focus image, it is possible to create multiple images at varying depths within the specimen. Thus, one can literally dissect layer by layer through the thickness of the specimen. It is also possible to use the computer to make three-dimensional reconstructions of a series of these images. Because each individual image located at a specific depth within the specimen is extremely sharp, the resulting assembled three-dimensional image is equally sharp. Moreover, once the computer has assembled each sectioned image, the reconstructed three-dimensional image can be rotated and viewed from any orientation desired (see Fig. 1.4).
The ultraviolet microscope uses quartz lenses with an ultraviolet light source.
The image in the ultraviolet (UV) microscope depends on the absorption of UV light by molecules in the specimen. The UV source has a wavelength of approximately 200 nm. Thus, the UV microscope may achieve a resolution of 0.1 m. In principle, UV microscopy resembles the workings of a spectrophotometer; the results are usually recorded photographically. The specimen cannot be inspected directly through an ocular because the UV light is not visible and is injurious to the eye.
The method is useful in detecting nucleic acids, specifically the purine and pyrimidine bases of the nucleotides. It is also useful for detecting proteins that contain certain amino acids. Using specific illuminating wavelengths, UV spectrophotometric measurements are commonly made through the UV microscope to determine quantitatively the amount of DNA and RNA in individual cells. As described in the Folder 1.2 on page 7, Feulgen microspectrophoto metry is used clinically to evaluate the degree of ploidy (multiples of normal DNA quantity) in sections of tumors.
The polarizing microscope uses the fact that highly ordered molecules or arrays of molecules can rotate the angle of the plane of polarized light.
The polarizing microscope is a simple modification of the light microscope in which a polarizing filter (the polarizer) is located between the light source and the specimen, and a second polarizer (the analyzer) is located between the objective lens and the viewer.