Processing of images from fluorescent in-situ hybridization using multiplexed fluorescent switching

A computer program product, tangibly embodied in a non-transitory computer-readable medium comprising instructions to cause one or more computers to:

• obtain from a fluorescent in-situ hybridization imaging system a first image in a first color channel of a sample in which first readout probes and second readout probes were bound to first encoding probes and second encoding probes, respectively, so as to both emit light in the first color channel;
• obtain from the fluorescent in-situ hybridization imaging a second image in the first color channel of a sample in which first readout probes were bound to the first encoding probes so as to emit light in the first color channel and the second encoding probes substantially did not emit light in the first color channel;
• register the first image and the second image to form a plurality of registered images;
• identify one or more pixels having a first intensity exceeding a first threshold in the first image and having a second intensity exceeding a second threshold in the second image, and classify such pixels as activated in a first readout call;
• and
• identify one or more pixels having the first intensity exceeding the first threshold in the first image and the second intensity below the second threshold in the second image, and classify such pixels as activated in a second readout call.

A computer program product, tangibly embodied in a non-transitory computer-readable medium comprising instructions to cause one or more computers to:

• store a codebook comprising a plurality of code words, each code word including a plurality of bits and an associated indication of an analyte, wherein each code word includes multiple bits corresponding to a same first color channel from within a same hybridization step, and wherein different combinations of values for the multiple bits correspond to different analytes;
• obtain from a fluorescent in-situ hybridization imaging system a plurality of image slices, wherein multiple image slices from the plurality of image slices correspond to the same color channel from within the same hybridization step;
• register the plurality of images to form a stack of registered images;
• determine a pixel word comprising a plurality of values with each intensity value corresponding to a registered pixel in an image of the stack of registered images,
• compare the pixel word to each code word of the plurality of code words to find a best matching code word;
• and
• determine the analyte corresponding to the best matching code word.