Optical enrichment enables accurate cell identification and isolation. (a and b) Cells can be activated into different fluorescent intensity levels that are clearly distinguished by FACS. Example images of cells (hTERT-RPE1 PA-mCherry) that have undergone various photoactivation times (a) and their corresponding FACS results (b; number of cells analyzed for each condition is ∼10,000, replicate number = 2). Scale bar: 100 µm. (c) Schematic of the experiment to measure precision. mIFP-positive (hTERT-RPE1 PA-mCherry H2B-mGFP mIFP-NLS) and mIFP-negative cells (hTERT-RPE1 PA-mCherry H2B-mGFP) were mixed and analyzed under GFP and mIFP channels separately. mIFP expression was used as a phenotype to indicate cells of interest (mIFP-positive cells). An activation mask was generated for each mIFP-positive cell. Cells identified by FACS to be mIFP and mCherry double-positive are true positives, while mCherry-positive cells without mIFP fluorescence result from accidental activation (false-positive cells). (d and e) Cells of interest can be identified by automated image analysis followed by photoactivation and distinguished through FACS with high accuracy. (d) Example images of image analysis (GFP channel, green; mIFP channel, pink), before and after photoactivation (mCherry channel, red) are shown. Scale bar: 100 µm. (e) Example FACS data (number of cells analyzed = 662). (f) Different ratios of mIFP-positive cells and mIFP-negative cells were mixed to measure precision at different percentages of hits.