Fig. 2

Computer-assisted microscopy isolation (CAMI) opens the door to new types of high-throughput single-cell molecular analysis through non-disruptive collection of individual cells from fixed tissue and selection of cells by phenotypic morphology or location. a Coronal sections of rat brain labeled with mouse-anti-NeuN antibody (blue) and rabbit anti-nNOS antibody (yellow) were imaged with a high-throughput microscope. b High-resolution detail of a region of the somatosensory cortex indicated in a. Outlines show nuclear segmentations and phenotype classifications predicted by our software. Cells outlined in yellow are predicted to be nNOS+, cells outlined in magenta are nNOS−, and gray indicates cells that should be discarded (e.g., due to artifacts). Dotted lines indicate cells that were targeted for extraction. c The same region after extracting two nNOS+ and two nNOS− cells. d Individual cells automatically selected and extracted using CAMI, nNOS-expressing interneurons on the left and nonexpressing cells on the right. e Expression levels measured by dPCR show that CAMI reliably separates cells. Cells identified as nNOS+ show significantly higher expression (7.96 ± 0.48) than those identified as nNOS− (0.48 ± 0.95), two-sampled t-test p = 0.0061. Expression levels of housekeeping gene S18 did not vary significantly between cells identified as nNOS+ (116.37 ± 16.54) and nNOS− (103.98 ± 10.29), two-sampled t-test p = 0.1992. f Whole-transcriptome gene expression profiles of nNOS− cells (two 50-cell replicates and one 300-cell) and astrocytes (50 cells) extracted by CAMI and sequenced by Ion Torrent PGM. Analysis reveals strong correlations (Pearson’s R) between the nNOS− replicates, and weak correlations between the astrocytes and nNOS− cells. g CAMI also enables a novel, cost-effective alternative to RNAi screening. Cells with interesting phenotypes are identified and extracted from mixed populations of stable shRNA-expressing silenced cell lines. After UV exposure, cells normally recruit polymerase η to repair DNA damage, which is visualized as foci by our green fluorescent marker. Absence of an upstream regulator can disrupt the foci formation and lead to homogeneous polymerase η expression. h CAMI identified 150 foci-forming and 150 homogeneous cells and extracted them. i Extracted cells were sequenced using next-generation sequencing (NGS). The ratio between the two populations revealed known upstream regulators of polymerase η (BRCA2, RAD18, and SPARTAN) and identified promising new regulators, Rad52 and FANCA