Visual Cell Sorting: A High-throughput, Microscope-based Method to Dissect Cellular Heterogeneity

Abstract

Microscopy is a powerful tool for characterizing complex cellular phenotypes, but linking these phenotypes to genotype or RNA expression at scale remains challenging. Here, we present Visual Cell Sorting, a method that physically separates hundreds of thousands of live cells based on their visual phenotype. Visual Cell Sorting uses automated imaging and phenotypic analysis to direct selective illumination of Dendra2, a photoconvertible fluorescent protein expressed in live cells; these photoactivated cells are then isolated using fluorescence-activated cell sorting. First, we use Visual Cell Sorting to assess the effect of hundreds of nuclear localization sequence variants in a pooled format, identifying variants that improve nuclear localization and enabling annotation of nuclear localization sequences in thousands of human proteins. Second, we use Visual Cell Sorting to recover cells that retain normal nuclear morphologies after paclitaxel treatment, then derive their single cell transcriptomes to identify multiple pathways associated with paclitaxel resistance in human cancers. Unlike alternative methods, Visual Cell Sorting depends on inexpensive reagents and commercially available hardware. As such, it can be readily deployed to uncover the relationships between visual cellular phenotypes and internal states, including genotypes and gene expression programs.

Pooled image-base screening of mitochondria with microraft isolation distinguishes pathogenic mitofusin 2 mutations

“…In contrast to methods that use ISS 19 , 23 , Raft-seq physically isolates the cells which can be sequenced using commercial technology. Raft-seq associates a sequence with the specific image of cell(s), something not possible in methods that sort using FACS-Seq 18 , 20 , 27 . Previous screening experiments using microrafts 30 isolated fixed cells, so Raft-Seq’s ability to capture individual live cells for growth alongside their original microscopy data is unique among pooled screening platforms.…”

“…Beyond enrichment, simple phenotypes measurable by flow cytometry 10 – 12 , or phenotypes measurable by sequencing 13 – 15 are possible. There are now platforms that use an imaging-based approach in pooled genetic perturbation screens and have been demonstrated on relatively simple phenotypes 16 – 18 or on precise phenotypes that were known in advance 19 , 20 .…”

“…Some platforms use in situ sequencing (ISS) 23 – 25 to generate sequencing results that contain positional data of a specific cell 19 , 26 using specialized non-commercial sequencing rigs. Other platforms photoactivate endogenous fluorophores in targeted cells which are then put through FACS-seq 18 , 20 , 27 .…”

Rewriting Human History and Empowering Indigenous Communities with Genome Editing Tools

“…Many computational approaches to predicting variant effects have been developed, but they can only identify a small fraction of pathogenic variants with a high confidence [86,87]. There is therefore an undeniable need for cellular and in vivo studies to functionally validate population-specific variation [1,[88][89][90].…”