Sublethal Photothermal Stimulation with a Micropulse Laser Induces Heat Shock Protein Expression in ARPE-19 Cells

Figure 2

Establishment of laser parameters for sublethal photothermal stimulation on a cultured cell layer using micropulse-mode laser irradiation. (a) A micropulse-mode laser emits short, intermittent pulses of laser energy. At a 15% duty cycle, the laser was on for 0.3 ms during each 2.0 ms period. When the total laser exposure duration was 1000 ms, the laser cycle was repeated 500 times. (b) Representative phase-contrast microscopy images taken 2 h after laser irradiation with a micropulse laser. The laser had a 15% duty cycle and was set to various laser powers (600–800 mW) and laser exposure durations (500–1000 ms). Multiple spots were made on a single one-cell layer culture plate 1 mm apart in both the and directions. The duration was varied along the -axis, and power was varied along the -axis. The laser spot size remained constant at 200 μm. (c) Areas of morphological change are shown in (b), as measured with NIH Image J software. Data represent the mean ± standard error of 10 independent experiments. Both laser exposure duration and laser power were significantly correlated with coagulation area (Spearman’s correlation coefficient: , ). (d) Representative bright-field (left panels) and fluorescent (center and right panels) images around laser irradiation sites, as captured with confocal microscopy, at the indicated times following laser irradiation. Sublethal photothermal stimulation was performed with the following laser settings: 15% duty cycle, 1000 ms laser exposure duration, and 600 mW laser power. Note that no dead cell signals (red fluorescence) were observed within the 24 h following laser irradiation. (e) Representative phase-contrast microscopy images 2 h after laser irradiation of single-cell layers with a laser in the micropulse (left) and continuous-wave (right) modes. The total laser irradiation energy was 90 mJ in both cases. (f) Mean coagulation area of spots made with a laser in the micropulse and continuous-wave modes shown in (e). Error bars represent one standard error (). The asterisk () indicates a statistically significant difference between means (Mann-Whitney tests, ).

(a)

(b)

(c)

(d)

(e)

(f)