Multiple-pulse damage thresholds of retinal explants in the ns-time regime

Abstract

The data situation of laser-induced damage measurements after multiple-pulse irradiation in the ns-time regime is limited. Since the laser safety standard is based on damage experiments, it is crucial to determine damage thresholds. For a better understanding of the underlying damage mechanism after repetitive irradiation, we generate damage thresholds for pulse sequences up to N = 20 000 with 1.8 ns-pulses using a square-core fiber and a pulsed Nd:YAG laser. Porcine retinal pigment epithelial layers were used as tissue samples, irradiated with six pulse sequences and evaluated for damage by fluorescence microscopy. The damage thresholds decreased from 31.16 µJ for N = 1 to 11.56 µJ for N = 20 000. The reduction indicates photo-chemical damage mechanisms after reaching a critical energy dose.

Fig. 1.

Dose-response curve for the single exposure evaluation. The probability $p_{\mathrm{S}\mathrm{i}\mathrm{n}\mathrm{g}\mathrm{l}\mathrm{e}}$ of 0.067 is marked schematically in green and indicates the probabilistic summation to produce damage after 10 pulses. The PSM predicts an according pulse energy of 30.63 µJ to be necessary to provoke damage at a tenfold irradiation.

Fig. 2.

Sample preparation of porcine eyes: (a)  Opening (b) Cut (c) Trefoil shape (d) Holding constructions to flatten the sample.

Fig. 3.

Optical setup for laser-induced measurements with top hat profile. The pulsed Nd:YAG is coupled into a multimode fiber to excite mode mixing. The squared beam profile at the distal tip of the fiber is imaged onto the samples. By using a beam splitter, the applied energy can be measured on the samples during irradiation. A camera is used to secure the top hat on the sample by recognition of the squared shape of the image. (a) Schematic illustration of the setup (b) Spatial beam profile at the sample position.

Fig. 4.

Damage thresholds of multiple pulse irradiation with spot edge length d = 319 µm and pulse duration of $\tau$ = 1.8 ns with PRF = 25 Hz. Error bars indicate 95% confidence limits. A decrease of the individual pulse energy of a pulse sequence can be observed with increasing number of pulses.

Fig. 5.

Damage thresholds of multiple pulse irradiation with spot edge length d = 319 µm and pulse duration of $\tau$ = 1.8 ns with PRF = 25 Hz. Error bars indicate 95% confidence limits. A decrease of the individual pulse energy of a pulse sequence can be observed with increasing number of pulses. PSM models (dashed) of the individual multiple-pulse thresholds were generated, but are independent of the starting point and in any case able to describe sufficiently restrictive the reduction.

Fig. 6.

Fluorescence microscope images (a) Activation and excitation of the converted calcein after sub-threshold irradiation: (1) Regular excited, vital hexagonal RPE structures can be recognized. (2) Destroyed cell membranes by intensity modulation peaks in the beam profile. (3) Cells with a higher brightness level, which indicates an increased metabolic activity. (b) Fluorescence microscope image of exposures with an edge length of 319 µm and a pulse duration of 1.8 ns. Hexagonal structure of single RPE cells are visible. Green bright cells represent living cells. Red bright cell nuclei are excited by the PI through destroyed cell membrane.

Fig. 7.

Photo-chemical damage occurs from a dose of about $200\mathrm{J}/\mathrm{c}{\mathrm{m}}^2$ (green line) or lower (considering the decreased slope from N = 1 000). Previous doses that caused the damage based on thermo-mechanical mechanism and have not been of photo-chemical origin. The further slight increase in the effective dose for damage may be due to repair mechanisms.

Fig. 8.

Comparison of integrated damage thresholds with other studies on photo-chemical effects (a) For different emission durations. Experimentally determined damage thresholds of this study (squares) with data from Ham et al. [24] (circles) and NHP experiments from Zhang et al. [28] (triangles). The studies of Ham et al. [24] were examined for different wavelengths of different cw-irradiation durations. Saturation can be identified for the shorter wavelength (b) Wavelength dependence: The data of this work (squares) fit well with the study of Zhang et al. [28] (triangles), for different wavelengths. For longer pulse durations, the dose necessary to cause damage is only slightly higher (probably due to repair mechanisms).