Extended Data Fig. 1: Calibration of Photopick, an imaging-based method for isolating mammalian cells from pooled culture (Related to Fig. 1).

a. Procedure for registering the DMD and camera pixels. An 11 × 11 grid of spots was projected onto a homogeneous exposure target. The observed locations in the camera were used to develop a piecewise-linear transformation to map DMD pixels onto camera pixels. In this example, the registration reduced the average projection error from 11.6 pixels to 0.22 pixels. b. Fluorescence excitation and emission spectra of three phototaggable FPs, PA-GFP, PA-mCherry, and mEos4a. For mEos4a, the spectra are given in the pre-activation state (green) and post-activation state (red). For the other FPs, the activated spectra are shown. c. Phototransformation efficiency vs. optical dose of 405-nm LED light. The decreased signal under prolonged illumination is due to photobleaching. d. Selective phototagging of mEos4a⁺ cells embedded in PA-mCherry⁺ cells (mEos4a⁺:PA-mCherry⁺ = 1:20; n = 1 trial). Based on the green channel image (i), a mEOS4a mask was created for targeted photoconversion of mEos4a with violet (ii). The red channel image shows that the phototagging was highly specific (iii). The monolayer of cells was then broadly illuminated with violet light (iv) to drive the photoactivation of PA-mCherry⁺ cells (v). Targeted violet illumination of the mEos4⁺ cells resulted in selective phototagging of mEos4a⁺ cells but not surrounding PA-mCherry⁺ cells.