Fig. 1: The SPACECAT protocol enables spatially precise, temporally stable fluorescence signals in arbitrary regions of interest.

a Synthetic schema for calcein NVOC. b HEK293T cells were stained with calcein NVOC and imaged before photoactivation and afterwards for 3 h. Cells in schematic adapted with permission from ref. ⁶⁶. c Representative time course images from a single field of view. Presented images of calcein NVOC were taken before photoactivation (calcein NVOC Pre), 10 min after (10’), 60 min after (60’), and 180 min (180’) after photoactivation. Images of CellTrace Red and Hoechst stains were taken 180 min after photoactivation. Dashed line: photoactivated region boundary; scale bar = 100 μm. See Supplementary Fig. 6a for quantitative time courses across n = 10 fields of view. d Contour plots representing the spatial distribution of fluorescence changes in photoactivated regions (green lines, n = 68 cells across 8 fields of view) and regions outside of photoactivation mask (black lines, n = 1347 cells across 8 fields of view). ΔF/F₀ is calculated as a cell’s change in mean fluorescence 180 min after photoactivation, divided by the cell’s pre-photoactivation mean fluorescence. e Composite image of HEK293T cells stained and photo-uncaged within the “MIT” logo region. By sequential addition of 3 photoactivatable probes (calcein NVOC, PA-JF549, PA-JF646) and leveraging different photoactivation thresholds (10 s for calcein NVOC, 0.5 s for PA-JF549 and PA-JF646), 5-color encoding is achieved. Scale bar = 100 μm. Green: uncaged calcein NVOC; yellow: uncaged PA-JF646, red: uncaged PA-JF549. Multiplexed encoding scheme repeated a total of n = 2 times.