System 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 different reagent sources to 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;
• an actuator to cause relative vertical motion between the flow cell and the fluorescence microscope;
• a motor to cause relative lateral motion between the flow cell and the fluorescence microscope;
• and
• a control system configured to, as sequential nested loops,
• as a first loop, cause the valve to sequentially couple the flow cell to the plurality of different reagent sources to expose the sample to a plurality of different reagents,
• as a second loop nested within the first loop, for each reagent of the plurality of different reagents, cause the motor to sequentially position the fluorescence microscope relative to sample at a plurality of different fields of view,
• as a third loop nested within the second loop, for each field of view of the plurality of different fields of view, cause the variable frequency excitation light source to sequentially emit a plurality of different wavelengths,
• as a fourth loop nested within the third loop, for each wavelength of the plurality of different wavelengths, cause the actuator to sequentially position the fluorescence microscope relative to sample at all of a predetermined number of different vertical heights, and
• as a fifth loop nested within the fourth loop, for each vertical height of the predetermined number of different vertical heights, obtain an image at the respective vertical height covering the respective field of view of the sample having respective fluorescent probes of the respective reagent as excited by the respective wavelength, and
• after the sequential nested loops, determine a quality metric for each image and, for images having the quality metric below a quality metric threshold, discard the images having the quality metric below the quality metric threshold.