Optical Pooled Screening using the CELESTA Light Engine
The advent of CRISPR gene editing technology has created the need for large-scale characterization of phenotypes resulting from manipulated genotypes. Optical pooled screening (OPS) is one of several genetic screening techniques developed for this purpose (1). In an optical pooled screen, a genetically diverse library of living cells is imaged and characterized for phenotypic variations without knowing the genotype of the cells. The genotypes are identified in situ after the cells have been fixed or by physical extraction of interesting phenotypes, followed by sequencing (Figure 1). Much of the development of OPS has been carried out in the laboratory of Professor Paul Blainey at Massachusetts Institute of Technology (2). The CELESTA Light Engine is the laser illuminator of choice for both immunofluorescence phenotyping and in situ sequencing. The high power light output of the CELESTA allows exposure times for in situ sequencing to be cut by a factor of 5—10 compared to earlier protocol implementations using a SOLA Light Engine. Since in situ sequencing requires multiple cycles of dye incorporation, imaging and cleavage, the overall time saving is substantial. Recently, Blainey and co-workers expanded the capabilities of OPS to allow analysis of 80,000 CRISPR knockout perturbations from single-cell images of about 10 million cells (3). This provided new insights into previously unresolved changes in the subcellular localization of host and viral proteins during Sendai virus infection of mammalian cells.
Such demanding OPS techniques leverage CELESTA’s optical power, spectral purity and breadth, and stability, depending on robust laser illumination for speed, diverse multiplexing, and consistency.