Simultaneous spatio-temporal measurement of gene expression and cellular activity

A method for identifying location and abundance of an analyte in a biological sample, the method comprising:

• (a) contacting the biological sample with a substrate comprising a plurality of capture probes, wherein a capture probe of the plurality of the capture probes comprises (i) a spatial barcode and (ii) a capture domain that binds specifically to a sequence present in the analyte;
• (b) recording a cellular activity or an intracellular gene expression of a nucleic acid of the biological sample, wherein the biological sample is located within a perfusion chamber comprising a gasket and the substrate, wherein the gasket comprises (i) a plurality of apertures, (ii) a plurality of input channels being fluidly connected to the plurality of apertures, respectively, and (iii) a plurality of output channels being fluidly connected to the plurality of apertures, respectively;
• (c) hybridizing the analyte to the capture domain of the capture probe;
• (d) extending the capture probe using the analyte as a template, thereby generating an extended capture probe;
• (e) amplifying the extended capture probe to produce a plurality of extended capture probes;
• and
• (f) determining (i) all or part of the sequence of the spatial barcode, or a complement thereof, and (ii) all or part of the sequence of the analyte, or a complement thereof, and using the determined sequences of (i) and (ii) to identify the location and abundance of the analyte in the biological sample.