Methods, systems and kits for in-pen assays

A non-transitory computer-readable medium comprising executable instructions that, when executed causes the one or more processors of a computer to perform an image processing method for determining a quantity of analyte produced by a biological micro-object or population of biological micro-objects generated therefrom, the method comprising:

• (i) receiving imaging data of a microfluidic device, wherein
• a. the microfluidic device includes a flow region comprising a microfluidic channel, and a plurality of sequestration pens, each comprising an isolation region and a connection region fluidically connecting the isolation region to the microfluidic channel at a single opening,
• b. the imaging data includes an analyte assay image and one or both of a background noise image and a signal reference image,
• c. the biological micro-object or the population of biological micro-objects generated therefrom secretes the analyte into the first fluidic medium within the sequestration pen, and
• d. the analyte assay image comprises an image taken at a time point when the sequestration pen comprises a first fluidic medium and a second fluidic medium has been introduced into the flow region for a first period of time, wherein the second fluidic medium comprises a plurality of reporter molecules, wherein each reporter molecule comprises:
• a binding component configured to bind the analyte, and a detectable label, thus allowing a portion of the plurality of reporter molecules to diffuse into each sequestration pen of the plurality and bind to at least a portion of the analyte secreted therein;
• (ii) defining an area of interest for each sequestration pen, wherein the area of interest:
• a. includes an image area within the microfluidic device, and at least a portion of the sequestration pen, and
• b. extends along an axis of diffusion defined by the sequestration pen and the flow region;
• and
• (iii) determining scores that are indicative of the quantity of analyte in each sequestration pen by analyzing at least a portion of the image area of the area of interest that extends along the axis of diffusion for each sequestration pen, wherein the score is correlated with a spatial distribution of the reporter molecule along the axis of diffusion.