Laser Microirradiation and Computer Video Optical Microscopy in Cell Analysis

Laser light is intense, coherent, monochromatic electromagnetic radiation. Because of these properties it can be a unique probe of cellular structure and function. The damage produced by a focused laser beam may be caused by classical absorption by natural or applied chromophores and the subsequent generation of heat (Berns and Salet, 1972), or it may be caused by a photochemical process. An example of such a process would be the production of monoadducts or diadduct cross-linking in the case of laser light’s stimulated binding of psoralens to nucleic acids (Peterson and Berns, 1978a). However, a third possibility is the generation of damage by an uncommon physical effect that occurs when ultra-high photon densities are achieved in very short periods of time (a few nanoseconds or picoseconds). The resulting nonlinear optical effects such as multiphoton absorption occur when the classic law of reciprocity does not hold. These effects may be responsible for some of the disruption observed in biological material (Berns, 1976). Whichever of the above damage-producing mechanisms is operating, be it “classical” or “uncommon,” the damage often can be confined to a specific cellular or subcellular target in a consistent and controllable way. In addition, once the biophysical mechanism of laser interaction with the molecules is ascertained, the investigator has a method for precise disruption of a specific class of molecules within a strictly delimited region of the living cell. The size of this region may be considerably smaller than the size of the focused laser beam because of the distribution of the target molecules in the target zone. However, the size of the focused laser spot also is of paramount importance because it defines the maximum volume of biological material that will be available for direct interaction with the laser photons. Though the diameter of the focused laser spot is a direct function of the wavelength, the magnification of the focusing objective, and the numerical aperture of the objective, the actual diameter of the “effective” lesion area may be considerably less than the theoretical limit of the focused laser beam. This is because a high-quality laser beam can be generated in the TEM∞ mode, which results in a beam with a gaussian energy profile across it. The profile is carried over to the focused spot, which results in a “hot spot” of energy in the center. It has been demonstrated consistently (Berns, 1974a) that by careful attenuation of the raw laser beam, the damage-producing portion in the focused spot can be confined to the central hot spot (e.g., that is the only region within the focused spot that is above the threshold for damage production). As a result, lesions can be routinely produced less than 0.25 μm in diameter, and frequently down to 0.1 μm in diameter.