Compressed Spectrotemporal Imaging for Multiplexed Protein and Transcript Detection in Single Cells

Résumé fourni par la source

High-dimensional single-cell profiling of proteins and transcripts is essential for understanding cellular heterogeneity, yet broadly accessible imaging approaches remain limited by insufficient multiplexing capacity and a lack of robust signal detection. Here, we introduce compression microscopy (CoM), a spectrotemporal multiplexing strategy that leverages the distinct photobleaching kinetics of conventional fluorophores. Photobleaching-based encoding is difficult to implement reproducibly in three-dimensional cell volumes, where signals can decay before the full volume is captured and out-of-focus excitation distorts intensity trajectories. In CoM, single cells containing target-specific DNA amplicons labeled with ratiometric fluorophore combinations are gently compressed into a thin, stable geometry, reducing the need for z-stack acquisition and minimizing out-of-focus excitation so that fluorophore decay trajectories can be captured reliably. By integrating photobleaching kinetics with ratiometric fluorescence encoding, CoM enables discrimination of up to 40 distinct fluorescence codes using standard widefield epifluorescence microscopy without specialized instrumentation. Applied to leukemia samples, CoM enables simultaneous in situ protein and transcript readouts at the single-cell level and identifies antigen-loss yet transcript-positive tumor cell variants that are difficult to resolve with conventional single-modality assays. CoM provides an accessible and scalable route to high-dimensional single-cell fluorescence profiling.

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