Beyond Static Screens: optical pooled screening of signaling dynamics using time-lapse FLIM

Genetic screens are powerful tools for linking gene perturbations to cellular phenotypes, yet most pooled imaging approaches rely on static endpoint measurements. Here, we extend optical pooled screening to dynamic signaling phenotypes by combining time-lapse Forster resonance energy transfer-fluorescence lifetime imaging microscopy (FRET-FLIM) with optical enrichment and downstream genotyping. To enable high-throughput live-cell screening of dynamic phenotypes, we optimized and benchmarked all major steps in the pipeline. We developed FAST-HIPPOS, a Fiji-based automated analysis pipeline that rapidly segments hundreds of thousands of cells, extracts single-cell lifetime time traces, and identifies phenotypic hits in minutes for targeted photoactivation. We further established a robust phototagging strategy using the photoactivatable dye PA-JaneliaFluor646, providing bright, cloning-free labeling across diverse cell types. Benchmarking further demonstrated high sorting fidelity with low false-positive rates and efficient recovery of guide RNAs from as few as 200 sorted cells with minimal PCR bias. Applying this platform to cAMP signaling in HeLa cells using a custom CRISPR library recovered the expected primary regulators of {beta}-adrenergic signaling--ADRB2, GNAS, and ADCY6--with no gene-level false positives. Together, these optimizations establish a robust general framework for screening dynamic phenotypes that is easily adaptable to other live-cell readouts beyond FRET-FLIM and cAMP.