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Fig. 5 Photoactivation of singlet oxygen formation by dL5**-MG2I complex in the CNS neurons of live transgenic zebrafish using 661 nm light. A: When bound to the fluorogen-activating protein dL5**, exposure of the fluorogen MG2I to far-red light results in formation of highly reactive and short-lived singlet oxygen (1O2). B: Normalized spectrum for the far-red LED array used in this experiment. The peak wavelength was 661 nm and half-width at half height was 9.0 nm. C: Tg(eno2:gal4ff); Tg(cox4-cox8-dL5**-mCer3) zebrafish express the fluorogen-activating protein dL5** fused to the fluorescent protein mCerulean3 within neuronal mitochondria. Cyan fluorescence is visible throughout the brain, spinal cord, and peripheral nerves of live transgenic zebrafish (main panels). At high magnification, individual mCer3-labeled mitochondria are seen within axons of the lateral line nerve (inset panels). The upper panels show baseline images, and the lower panels show the same zebrafish following activation of 1O2 production in mitochondria, by exposure to 80 J/cm2 661 nm light at 160 mW/cm2. D: Swimming speed of mito-dL5** zebrafish in response to cyclic illumination. Each graph shows a different experimental group (n = 14–18 zebrafish per group): no MG2I/no light control (black); 661 nm light-only control (blue); MG2I-only control (green); MG2I + 661 nm light 80 J/cm2 delivered at different power (red). Motor responses were elicited using a green safelight that does not activate 1O2 production. Zebrafish were transferred to a 96-well plate for the assay, and movements were captured by infrared videography and measured using automated video tracking software. Responses for each individual zebrafish were averaged over five illumination cycles. For each experimental group, mean frame-to frame larval centroid displacement was scaled to show instantaneous speed (gray traces). The colored markers show group mean ± SE swimming speed within each 1-minute time bin over the 20-minute illumination cycle. E: Mean swimming speed during the dark phase of the visual motor response shown in panel D. Data points show individual zebrafish, bars show mean ± SE; p < 0.0001**** vs. no chemical or light control (1-way ANOVA with Dunnett multiple comparisons test to compare each group to transgene-only controls). Color key for experimental groups is the same as panel D.