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Fig. 1
Evaluation of KillerRed optical targeting in vivo using epifluorescence and SPIM. (a) Schematic showing perivascular renin cell location in 3 dpf zebrafish. Mural renin cells are shown in red, arterial vessels in green, veins in blue, and the glomerulus (GL), nephron and cloaca in yellow. The dorsal aorta (DA) is distinguished from the anterior mesenteric artery (AMA) by using dark and light green to represent them, respectively. (b) ren:mem-KillerRed cells along the AMA budding off the DA in 3 dpf ren:mem-KillerRed;kdrl:GFP fish were illuminated using either an epifluorescence microscope (top panel, mCherry filter, 1hr), or the SPIM light-sheet at 3 mW or 4 mW (bottom panels, maximum intensity projections, 561 nm laser, 1hr). Representative images from 0, 10, 20 and 30 min illumination are shown. (c) Graphs showing the fluorescence intensity as a percentage of maximum intensity (arbitrary fluorescence, a.u.), plotted against time and averaged over n = 6 fish (with 95% confidence intervals, indicated by the filled dashed lines). Arrows and rotated times at foot of arrows indicate the 1/e value. Inset: percentage of fish surviving following illumination by epifluorescence or light-sheet microscopy for 1 hr (n = 18). (d) Morphological changes of ren:mem-KillerRed cells were assessed using multiphoton microscopy (MPM). MPM images of 3 dpf ren:mem-KillerRed;αSMA:GFP renin-expressing cells were taken prior to- and post-treatment using the SPIM light-sheet. Images are presented as single depth slices. Red arrows indicate renin cells. (e) Endothelial cell structure prior to- and post-light treatment using the SPIM light-sheet was assessed using MPM in 3 dpf ren:mem-KillerRed;kdrl:GFP fish. Images are presented as maximum intensity projections. (f) Apoptosis of ren:mem-KillerRed cells was confirmed using an Apop-Tag in-situ TUNEL staining kit on 15 μm sections of 3 dpf ren:mem-KillerRed;kdrl:GFP larvae illuminated with the SPIM and fixed immediately. White arrows designate apoptosis-positive post-treatment KillerRed+ cells. Images show a maximum intensity projection over 5 μm using confocal light scanning microscopy. All scale bars represent 30 μm.