Laser scanning confocal microscopy of the hearing organ: fluorochrome-dependent cellular damage is seen after overexposure

In order to combine laser confocal microscopy with physiological measuremen ts, a number of conditions have to be met: the dye must not be toxic to the cells the laser light itself must not damage the cells; and the excitation of the fluorochrome during imaging must not generate products with toxic e ffects. We have investigated these conditions the hearing organ of the guin ea pig. Two dyes were used, namely, calcein-AM, which is metabolized in vit al cells to a fluorescent product in the cytoplasm, and a lipophilic membra ne dye. The effect of the dyes on cell function was tested in the intact he aring organ, maintained in the isolated temporal bone, by measuring the ele ctrophysiological potentials generated by the sensory cells in response to tone pulses. The loading of the cells with the dyes had no adverse effects. The effect of the laser beam was Explored on isolated coils from the cochl ea. In two preparations, the specimens viewed in the confocal system were f ixed and processed for electron microscopy. Identified cells were followed before, during, and after laser exposure and could ultimately be examined a t the ultrastructural level. Exposure to the laser beam did not cause damag e in unstained cells, even at high intensities. In stained tissue, confocal microscopy could safely be performed at normal beam intensity without caus ing ultrastructural changes, At high intensities, about 100 times normal fo r 60 times as long, irradiation damage was seen that was selective in that the cells stained with the different dyes exhibited damage at the different sites corresponding to the subcellular location of the dyes. Cells stained with calcein showed lysis of mitochondria and loss of cytoplasmic matrix, whereas cells stained with the styryl membrane dye showed swelling of subsu rface cisternae, contortion of the cell wall, and shrinkage. The styryl dye s, in particular, which selectively stain the sensory and neuronal cells in the organ of Corti, could be exploited for phototoxic use.