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 measurements, 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 effects. We have investigated these conditions the hearing organ of the guinea pig. Two dyes were used, namely, calcein-AM, which is metabolized in vital cells to a fluorescent product in the cytoplasm, and a lipophilic membrane dye. The effect of the dyes on cell function was tested in the intact hearing organ, maintained in the isolated temporal bone, by measuring the electrophysiological 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 cochlea. In two preparations, the specimens viewed in the confocal system were fixed and processed for electron microscopy. Identified cells were followed before, during, and after laser exposure and could ultimately be examined at the ultrastructural level. Exposure to the laser beam did not cause damage in unstained cells, even at high intensities. In stained tissue, confocal microscopy could safely be performed at normal beam intensity without causing ultrastructural changes. At high intensities, about 100 times normal for 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 subsurface cisternae, contortion of the cell wall, and shrinkage. The styryl dyes, in particular, which selectively stain the sensory and neuronal cells in the organ of Corti, could be exploited for phototoxic use.