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Gene and compound functions are often interrogated by perturbation. However, we have limited methods to capture associated phenotypes in an unbiased and holistic manner. Here, we describe Fluopack screening as a novel platform enabling the profiling of subcellular phenotypes associated with perturbation. Our approach leverages imaging of a panel of fluorescent chemical probes to survey cellular processes in an unbiased and high throughput fashion. Segmentation-free, whole image analysis applied to Fluopack images identifies probes revealing distinct phenotypes upon perturbation, thereby informing on the function and mechanism of action of perturbagens. This chemical biology approach allows to interrogate phenotypes that tend to be overlooked by other methods, such as lipid trafficking and ion concentration inside the cell. Fluopack screening is a powerful approach to study orphan protein function, as exemplified by the characterization of TMEM41B as novel regulator of lipid mobilization.

Fluopack screening platform for unbiased cellular phenotype profiling