Fig. 6. Spatially resolved transduction of single cells using a conventional confocal microscope.

(A) Experimental workflow. A-431 cells containing 1% fluorescently labeled (CFSE or eFluor670) cells were seeded on a gridded coverslip. Forty-eight hours after seeding, cells were incubated with 50 nM PhyB-DARPin_EGFR in PBS supplemented with 10% FCS under 740-nm light for 10 min. After washing, OptoAAVs_GFP or OptoAAVs_mScarlet in PBS supplemented with 10% FCS were added and a bright field and CFSE or eFluor670 image was acquired under 740-nm illumination (200 μmol m⁻² s⁻¹). After switching the 740-nm light off, a single CFSE or eFluor670-positive cell was illuminated with the 633-nm laser of the confocal microscope. Following 2-hour incubation in the dark, cells were washed and incubated for 46 hours in medium under 740-nm light. Last, cells were fixed, DAPI-stained, and imaged by confocal microscopy. (B) Light-controlled transduction of a single CFSE-stained A-431 cell. The experiment was performed as in (A) with OptoAAV_mScarlet (MOI: 4.9 × 10⁴). The area illuminated with the 633-nm laser is encircled in red. Representative images from n = 9 successful experiments (out of 15). All experiments are shown in fig. S14A. Scale bar, 100 μm.