Figure 4.

Single cell resolution and spatial control of intracellular R-GFP delivery. (a) Red polygons define regions irradiated by the NIR laser. The blue oval (ROI 2) was not exposed to NIR. Fluorescence intensity increased for the irradiated ROIs after laser. The cell in ROI 1 was separated from the control cell ROI 2 by ∼5 μm. Scale bar is 50 μm. (b) Line profiles taken along the cell in ROI 1 shows R-GFP fluorescence intensity increased up to 8-fold after laser, whereas the neighboring control cell in ROI 2 was unchanged. (c) Single photon confocal microscopy of R-GFP-HGN fluorescence before and after NIR laser exposure in the region denoted by the red square. Scale bar is 25 μm. (d) Intensity profile along lines 3 and 4 in panel c showing the GFP fluorescence increase after laser. Nucleoli staining observed after laser irradiation (‡). Subcellular release of R-GFP was elicited mostly (▲), while the cellular area (•), not exposed, showed only a slight increase. Laser power density was ∼8 × 10³ W/cm² (0.1 mJ/cm²), and ∼300 μs exposure time in the region is denoted by the red square. (e) Quantification of corrected total cell fluorescence percent increase in selected cells (outlines shown in panel f) above the initial intensity. N = 6 for cells completely outside the ROI, n = 5 for all other cases. Mean intensity for cells completely inside the ROI was significantly higher than cells completely outside (Kolmogorov–Smirnov test; p < 0.005; ***). Error bars represent standard error of the mean (SEM).