Multiplexed tissue imaging

A method for analyzing a biological sample, the method comprising:

• obtaining a biological sample mounted on a first substrate;
• affixing a second substrate to the first substrate to form an enclosed chamber on the first substrate with the biological sample positioned within an interior volume of the enclosed chamber, the first and second substrates defining an imaging window through the interior volume, wherein the second substrate comprises a first port formed by a first aperture extending through a thickness of the second substrate and a second port formed by a second aperture extending through the thickness of the second substrate;
• binding capture agents to the biological sample by introducing a composition comprising the capture agents into the interior volume through the first port, wherein the composition comprises multiple different types of capture agents, each of the different types of capture agents comprising a different type of binding agent that selectively binds to a different sample component, and an oligonucleotide that is specific to each different type of binding agent;
• and
• performing multiple imaging cycles, wherein each imaging cycle comprises:
• (a) binding a probe to the biological sample;
• (b) obtaining an image of the bound probe in the biological sample within the imaging window, the image having a field of view of at least 500 mm2;
• and
• (c) removing at least a portion of the probe from the biological sample,
• wherein the binding the capture agents to the biological sample is performed prior to performing the multiple imaging cycles.