Fig. 5: Spatiotemporal control of 2D and 3D tissues.
A Spatial induction of necroptosis using a 3D-printed photomask mounted in the LPA device using a light intensity of 40 µmol m−2 s−1 (blue LED light). The bottom sample shows the erythromycin control at identical conditions (see Fig. S5, Supplementary Information). Scale bar, 1 mm. The experiment was performed three times with similar results. B Spatial, dynamic, and quantitative control of optogenetic necroptosis induction using the digital mirror device for patterning using a 4x magnification objective in a time series. 8-bit grey shades correspond to 60, 120, 180, and 255. An overlay of EGFP, mCherry, and SYTOX Blue signals is shown. See Fig. S6 and Movie S1–2, Supplementary Information, for controls and movies. Scale bar, 100 µm. C Brightfield and blue light pattern projection (top) and SYTOX Blue signal (bottom) of the experiment in (B) at 33 h. D Spatial induction of necroptosis in a 3D culture of CHO-K1NecrOpto cells using 488 nm laser excitation over a period of 20 h shown as intensity projection of a Z-stack. The tissue sample in the bottom row was supplemented with 2 µg/mL erythromycin. See Fig. S8, Supplementary Information, for individual z-layers. Scale bar, 100 µm. Each experiment was performed once, see Fig. S7, Supplementary Information, for a repeat with higher imaging frequency. E Schematic representation of spheroid blue light illumination and imaging using a custom light sheet microscope. F Spatiotemporal regulation attempts of necroptosis in 3D tissues using light sheet microscopy. 3D spheroid cultures derived from CHO-K1NecrOpto cells were illuminated with a plane (white arrows) of 488 nm laser light for optogenetic activation at the indicated intensities. After 5 h, z-stacks were acquired visualizing constitutive EGFP fluorescence and cell death by staining with SYTOX Red. The experiments were performed four times independently. Scale bar, 50 µm.
