Spectral–Spatial Encoded Microscopy Enables Simultaneous Multi-Cell-Type and Multiplexed Subcellular Imaging

Understanding how organelle dynamics vary across different cell types is essential for dissecting cellular heterogeneity, yet current spectral imaging methods cannot simultaneously resolve multiple organelles in mixed populations. Here we present spectral spatial encoded microscopy (S 2 eM), a fluorescence imaging strategy that embeds cell identity into unique combinations of fluorescent proteins and organelle markers, enabling simultaneous multi-cell-type classification and multiplexed organelle imaging within a single field of view. Using S 2 eM, we distinguished four barcoded cell types and concurrently tracked the ER, mitochondrial, lysosomal, and lipid-droplet dynamics at high temporal resolution. This approach revealed cell-type-specific differences in osmotic stress responses and recovery kinetics, and uncovered ORP8-dependent alterations in LD–lysosome–mitochondria contact-site dynamics. Because all cell types are imaged under identical conditions, S 2 eM provides internally controlled comparisons that minimize experimental variability. These results establish S 2 eM as a broadly applicable platform for probing organelle behavior and interorganelle communication across heterogeneous cell populations.