An Electron Microscope Study of Vitally Stained Single Cells Irradiated With a Ruby Laser Microbeam

Abstract

An electron microscope study has been made of vitally stained single cells whose cytoplasm has been subjected to a localized ruby laser microbeam. Light and moderate laser absorption (the resultant of stain concentration and laser energy density) produced restricted selective damage of mitochondria in cells stained with Janus green B; heavy laser absorption resulted in mitochondrial damage, as well as in nonselective interaction with other cell structures. With four other basic vital stains, the polysomes, ergastoplasm, mitochondria and other organelles at the irradiated site were uniformly damaged. Unstained cells showed no morphological alterations. With light primary damage (that restricted to the irradiation site), no secondary effects of the incident radiation were observed. With moderate primary damage, however, secondary damage of the mitochondria in the unirradiated cell portions was produced, which was reversible within 4 hr after irradiation. Heavy primary lesions caused severe secondary alteration of all cell structures that was irreversible and cell death occurred within 2 hr. Surviving ceils examined 24 hr after light and moderate irradiation could not be distinguished from unirradiated controls. The possible mechanisms involved in the production of laser-induced cellular alterations are discussed.

Morphologic Changes in Photoreceptor Outer Segments Following Photic Injury

“…As observed in the original description [3], the changes were found to be more prominent in the outer segments of cones as compared to the outer segments of rods. Similar alterations of the outer segments of photoreceptor cells have been seen following other experimental injuries, including acute cryopexy [9] and chemical poisoning [5,11], Interestingly, a similar change has also been reported in the mitochondria of single cells that had been focally injured by laser irradiation [1,12], The affected portions of the cells were subsequently sequestered, indicating that they had been irreversibly damaged. This supports the interpretation that the presence of these focal densities observed in the present study in the outer segments of the photoreceptor cells and the apical portions of the retinal pigment epithelium is consistent with a nonspecific coagulation necrosis of cells or portions thereof.…”

Laser Microsurgery in the GFP Era: A Cell Biologist's Perspective

“…The conclusions of this work were that, in essence, nonnaturally pigmented cells did not respond to irradiation unless they were first sensitized by adding an exogenous chromophore (e.g., acridine orange, acridine red, alcian blue, psoralens, coumarins, Janus B green). This conclusion was then confirmed at the EM level, which also revealed that under the appropriate vital staining and laser power conditions, restricted and selective damage was created at the irradiated site (Storb et al, 1966).…”

“…The conclusions of this work were that, in essence, nonnaturally pigmented cells did not respond to irradiation unless they were first sensitized by adding an exogenous chromophore (e.g., acridine orange, acridine red, alcian blue, psoralens, coumarins, Janus B green). This conclusion was then confirmed at the EM level, which also revealed that under the appropriate vital staining and laser power conditions, restricted and selective damage was created at the irradiated site (Storb et al, 1966).In 1969, Michael Berns and his colleagues at the University of California (Irvine) showed, using an argon laser coupled to a phase-contrast microscope, that very small lesions could be easily placed at predetermined sites on selected chromosomes (Berns et al, 1969). Encouraged by their initial successes, this team began a series of studies, based on UV and later visible spectrum laser beams, on how cells react to the selective removal of various structures from the nucleolar organizer/primary constriction (Berns and Cheng, 1971;Berns et al, 1970bBerns et al, , 1972Ohnuki et al, 1972) to the centrosome region (Berns and Richardson, 1977;Peterson and Berns, 1978).…”

LASER MICROSCOPE IRRADIATION OF PHYSARUM POLYCEPHALUM: DYNAMIC AND ULTRASTRUCTURAL EFFECTS

“…Amy and Storb (2) and Storb et al (32) found that, after vital staining of mitochondria with Janus green B, ruby laser radiation caused flocculation of the cytoplasm, distortion and increased electron opacity of the regions irradiated, and a disruption of mitochondria. In tissue culture cells stained with Janus green, at high energies and stain concentrations, Storb et al (32) saw damage remote from the primary injury. In the slime mold we observed no distant changes, except some swelling of tubes, probably a nonspecific response to blockage.…”

“…For microsurgery, a laser beam focused through a microscope can alter or destroy a specific part of a tissue or cell (4-6, 8, 12, 14, 15, 18-20, 24-28, 35). Dyes with specific absorption and localization facilitate differential destruction; for example, Janus green, a vital stain for mitochondria, absorbs the ruby laser wavelength (2,3,25,(32)(33)(34).…”