RPE damage thresholds and mechanisms for laser exposure in the microsecond-to-millisecond time regimen

The retinal pigment epithelium (RPE) cells with their strongly absorbant melanosomes form the highest light-absorbing layer of the retina. It is well known that laser-induced retinal damage is caused by thermal denaturation at pulse durations longer than milliseconds and by microbubble formation around the melanosomes at pulses shorter than microseconds. The purpose of this work was to determine the pulse width when both effects merge. Therefore, the RPE damage threshold and mechanism of the damage at single laser pulses of 5-micros to 3-ms duration were investigated.

An argon laser beam (lambda 514 nm) was externally switched by an acousto-optic modulator to achieve pulses with constant power in the time range of 5 micros up to 3 ms. The pulses were applied to freshly prepared porcine RPE samples serving as a model system. After laser exposure RPE cell damage was proved by the cell-viability stain calceinAM. Microbubble formation was detected by acoustic techniques and by reflectometry.

At a pulse duration of 5 micros, RPE cell damage was always associated with microbubble formation. At pulses of 50 micros, mostly thermal denaturation, but also microbubble formation, was detected. At the longer laser pulses (500 micros, 3 ms), RPE cell damage occurred without any microbubble appearance.

At threshold irradiance, the transition time from thermal denaturation to thermomechanical damage of RPE cells is slightly below the laser pulse duration of 50 micros.