Fig. 2: Spatial programming of optogenetic stimulations.
From: Spatiotemporal, optogenetic control of gene expression in organoids
a, Spatial photostimulations: cells are stimulated with a LED array through a photomask, with laser scanning or with a DMD. b, Representative images (n = 3) of LED stimulation of the CaSP1–CasRx system in HEK cells (NG). Magenta, photomask. Scale bar, 500 μm. c, Single-cell Cre–Lox CasRx laser stimulation in HEK cells. The left shows the transmitted light. The right shows the NG. Scale bars, 100 μm. Top right shows a higher magnification. Magenta, photostimulated ROI. d, NG quantification for an ROI covering the photostimulated cell (blue, induced) and for the mean of ROI of the same size tiling the entire FOV 16–18 h after stimulation (gray, other). Each dot is a replicate (n = 9 induced cells in five different experiments), horizontal bars represent the mean and P value for induced versus other comparison is shown (two-sided Wilcoxon–Mann–Whitney). e, Representative image of a complex pattern stimulation of the Cre–Lox CasRx system in HEK cells performed with the DMD setup. The left shows the RFP, the middle shows the NG and the right shows a zoom-in of an individual ROI. Photostimulated ROI and entire DMD FOV (rectangle outside) in magenta. The broader FOV (outside DMD) was imaged 24 h post-photostimulation with a confocal setup. Scale bars, 500 μm. f, Quantification of NG for the DMD ROI (DMD On), the DMD FOV outside the photostimulated ROI (DMD Off) and the imaged region outside the DMD FOV (Outside DMD). Each dot is a replicate (n = 3), horizontal bars represent the mean and P value for the DMD On versus both DMD Off and Outside DMD is shown (two-sided Wilcoxon–Mann–Whitney). g, Organoid photostimulation protocol: four organoids are placed on a glass-bottom dish, immobilized with a Geltrex droplet and photostimulated with laser scanning overnight. h, From left to right: representative (n = 7) live imaging (whole FOV and photostimulated ROI) of four organoids at time 0 and after 16 h of pulsed photostimulation. Magenta, RFP; green, NG. Scale bars, 100 μm. i, Live imaging of a representative control organoid (dark) at 10 days. Scale bar, 250 μm. Magenta, RFP; green, NG. j, Live imaging of a representative time course (4–12 days, n = 3) of an organoid locally photostimulated via laser scanning. Magenta, RFP; green, NG. Scale bars, 100 μm.
