Seeing deeper with optical pooled screening

Biological research is evolving—combining targeted, hypothesis-driven studies with high-throughput discovery. By applying thousands of perturbations in parallel, pooled screening methods systematically uncover genes and pathways that drive key phenotypes such as survival, proliferation, or drug response. However, most pooled screens rely on sample enrichment based on a single endpoint measure through drug selection or fluorescence-based cell sorting and are limited to single-modality readouts, missing much of biology’s complexity.

Optical pooled screening (OPS)¹ is a powerful solution to this challenge, bridging high-throughput perturbations with high-content imaging to unlock deeper biological insights. In this approach, researchers combine scalability with rich phenotypic information; however, its adoption is limited by complicated, manual workflows, limited plexity, and difficulty in incorporating additional readouts, like RNA and protein expression.

Here, we review the application and share how direct in sample sequencing (DISS) on the AVITI24™ intelligent multiomics system transforms OPS by creating a streamlined, integrated, and automated workflow with data-rich, multimodal results.

What is optical pooled screening?

Optical pooled screening integrates the scalability of pooled perturbation libraries with the rich, single cell phenotypic information typically obtained through microscopy.

In a typical optical pooled screen:

1. Cells are transduced with a library of genetic perturbations, each containing a unique barcode.
2. High-content imaging captures detailed morphology, protein localization, and cell-cell interactions.
3. The identity of the perturbation in each cell is determined through manual in situ sequencing of sgRNAs (<20 bp) by microscopy.

This workflow allows researchers to link complex cellular phenotypes directly to specific genetic perturbations at the single cell level in a massively parallelized manner. OPS presents many advantages over traditional pooled screens by providing rich morphology readout across millions of cells.

Challenges in current optical pooled screening workflows

While optical pooled screening offers many advantages, it introduces a range of workflow challenges that limit its widespread use. One of the primary hurdles is the complexity of the barcode readout. Accurate decoding of single cell perturbations requires labor-intensive, multi-step imaging protocols with intense manual processing. Without readily available commercial workflows, it relies on “homebrew” methods for cycling and in situ readout of the barcodes by microscopy.

Not only is this process time consuming, often taking 2+ weeks, it uses multiple reagent vendors, has limited z-dimension imaging, and only provides <20 bp of in situ sequencing. Because these methods rely on efficient hybridization to the barcode, there is often low detection rate of guides, often <40%.

The massive volume of high-resolution images generated during screens demands significant data storage, computational infrastructure, and sophisticated image analysis pipelines to extract meaningful information.

Together, these workflow challenges highlight the need for innovations that can streamline barcode detection and integrate data into a more seamless, high-fidelity pipeline.

A streamlined OPS workflow powered by AVITI24

DISS on the AVITI24 powers a fully automated, end-to-end workflow for generating OPS data. The pooled cell library can be plated directly on a Teton™ flow cell for fully onboard multiomic detection.

This approach integrates highly accurate in sample sequencing with multiplexed phenotyping capable of profiling cell morphology features, custom protein expression, and 3’ whole transcriptome (available in H2 2025).

See how we performed a 500 gene CRISPR screen, enabling large-scale, high-resolution profiling of perturbation effects and setting the stage for scalable, automated OPS studies. Download the poster.

Applications of optical pooled screening in biology and drug discovery

The versatility of optical pooled screening opens new frontiers across biological research and drug discovery:

• Drug Discovery: Identify genes and pathways that influence subtle phenotypic traits and understand the mechanism of action or resistance of your research compounds.
• Stem Cells and Development: Monitor shifts in differentiation status, uncover regulators of lineage specification, and map the trajectories of stem cells as they commit to specific fates—all by imaging key phenotypic markers.
• Cancer biology: Changes in cell morphology, migration, invasion potential, or immune evasion can be linked to genetic perturbations. For example, uncover regulators of epithelial-mesenchymal transition (EMT) by screening for specific morphological signatures.
• Neuroscience: Studying neurons and glial cells often hinges on fine-grained phenotypic features and OPS provides a scalable way to dissect the genetic regulators of these complex traits.
• Functional Genomics and Systems Biology: Optical pooled screening allows researchers to build high-dimensional phenotypic maps that reveal functional relationships between genes, pathways, and phenotypes, accelerating the creation of comprehensive biological models.

In all these areas, optical pooled screening delivers insights that single readout pooled screens—or even single-gene imaging studies—would miss, opening new avenues for discovery.

The next frontier in functional genomics

As high-throughput screening continues to evolve, streamlined OPS workflows combined with AI-driven phenotyping will transform how we link genotypes to complex phenotypes.

By integrating imaging and transcriptomics at single-cell resolution, these multimodal screens offer unprecedented depth. Efforts toward overcoming current barriers and democratization are making high-content pooled screens accessible to more labs and unlock a new wave of discoveries across biology, drug discovery, and biotechnology.

References

1. Feldman D et al. Optical Pooled Screens in Human Cells. Cell. 2019 Oct 17;179(3):787-799.e17. doi: 10.1016/j.cell.2019.09.016. PMID: 31626775; PMCID: PMC6886477.