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™ 5D multiomics system transforms OPS by creating a streamlined, integrated, and automated workflow with data-rich, multimodal results.
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:
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.
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.
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.
The versatility of optical pooled screening opens new frontiers across biological research and drug discovery:
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.
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 increasing democratization are making high-content pooled screens accessible to more labs, unlocking a new wave of discoveries across biology, drug discovery, and biotechnology.