System and method for acquisition and processing of multiplexed fluorescence in-situ hybridization images

A fluorescent in-situ hybridization imaging system, comprising:

• a flow cell to contain a sample to be exposed to fluorescent probes in a reagent;
• a valve to control flow from one of a plurality of reagent sources the flow cell;
• a pump to cause fluid flow through the flow cell;
• a fluorescence microscope including a variable frequency excitation light source and a camera positioned to receive fluorescently emitted light from the sample;
• and
• a control system configured to
• receive a plurality of images of the sample from the fluorescence microscope, the plurality of images representing a common field of view,
• perform registration of the plurality of images to generate a plurality of registered images,
• for each pixel of a plurality of pixels of the registered images, decode the pixel by identifying a code word from a plurality of code words in a code book that provides a best match to data values in the plurality of registered images for the pixel,
• for each code word identified as a best match for one or more pixels and each pixel of the one or more pixels, determine whether a bit ratio for an image word for the pixel meets a threshold for the code word, the image word being formed from the data values in the plurality of registered images for the pixel, and
• for at least one pixel that is determined to meet the threshold determine a gene associated with the code word and store an indication that the gene is expressed at the pixel, and screen pixels for which the bit ratio does not meet the threshold.