Abstract: A system and method of performing deep cell body segmentation on a biological sample is provided. The method includes receiving a first and a second stained image. The first image is processed using a trained machine learned model that outputs locations of a plurality of cell nuclei in the first stained image. Seed points are then determined based on the locations of the plurality of cell nuclei. The second image is then processed using the seed points to determine a plurality of cell membranes using a watershed segmentation. The second image is then post-processed and an output image is produced. The output image is then analyzed and gene sequencing is performed.

Abstract: A method of generating a codebook includes obtaining a plurality of gene-identifying code words for the codebook. Each gene-identifying code word is represented by a sequence of N bits that correspond to a best match to a pixel data value identifying a gene. A plurality of negative control code words is generated, and each negative control code word is represented by a sequence of N bits. The negative control code words have an equal number of on-values. On-values of the plurality of negative control code words are evenly distributed across the N bits such that each ordinal position in the sequence of N bits has a same total number of on-bits from the plurality of negative control code words, and a Hamming distance between each negative control code word and each gene-identify code word is at least a distance threshold.

Abstract: A method of method of spatial transcriptomics includes receiving a plurality of images of a sample from an mFISH imaging system and generating a pixel word represented by a sequence of N intensity values. For each pixel, the pixel word is compared to a codebook and a closest matching code word of a plurality of code words is identified. Each code word is represented by a sequence of N bits. The plurality of code words include a plurality of gene-identifying code words and a plurality of negative control code words, the plurality of negative control code words have an equal number of on-values, and on-values of the plurality of negative control code words are evenly distributed across the N bits such that each ordinal position in the sequence of N bits has a same total number of on-bits from the plurality of negative control code words.

Abstract: Disclosed herein are methods for performing assays, including general functional assays, on a biological cell. The methods can include contacting a biological cell with a test agent for a period of time; lysing the biological cell while the biological cell is disposed within a sequestration pen located within an enclosure of a microfluidic device; and allowing RNA molecules released from the lysed biological cell to be captured by capture oligonucleotides linked to a capture object disposed within the sequestration pen of the microfluidic device. Each capture oligonucleotide can include a priming sequence that binds a primer, and a capture sequence. Each cDNA transcribed from a captured RNA can have an oligonucleotide sequence complementary to the captured RNA molecule, with the complementary oligonucleotide sequence being covalently linked to one of the capture oligonucleotides of the capture object.