System for acquisition and processing of multiplexed fluorescence in-situ hybridization images
In a fluorescent in-situ hybridization imaging system performs, as nested loops, the following: (1) a valve sequentially couples a flow cell to a plurality of different reagent sources to expose the sample to a plurality of different reagents, (2) for each reagent of the plurality of different reagents, a motor sequentially positions the fluorescence microscope relative to sample at a plurality of different fields of view, (3) for each field of view of the plurality of different fields of view, a variable frequency excitation light source sequentially emits a plurality of different wavelengths, (4) for each wavelength of the plurality of different wavelengths, an actuator sequentially positions the fluorescence microscope relative to sample at a plurality of different vertical heights, and (5) for each vertical height of the plurality of different vertical heights, an image is obtained.
1. 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.
2. The system of claim 1 , wherein the actuator comprises a piezoelectric actuator.
3. The system of claim 1 , wherein the variable frequency excitation light source comprises a plurality of laser modules and the control system is configured to sequentially activate the laser modules to sequentially emit the plurality of different wavelengths.
4. The system of claim 3 , wherein the control system includes a computer to control the valve and the motor and a microcontroller to control the laser modules and the actuator.
5. The system of claim 3 , further comprising a filter wheel positioned in a light path between the sample and the camera, wherein the filter wheel includes a plurality of emission bandpass filters, and wherein the control system is configured to coordinate the variable frequency excitation light source and filter wheel such that selected filter has an emission bandpass associated with emission by the fluorescent probes when excited by the wavelength of the plurality of different wavelengths when the image is obtained.
6. The system of claim 1 , comprising a stage to support the sample in the flow cell, and wherein the motor comprises a pair of linear actuators configured to drive the stage along a pair of perpendicular axes.
7. The system of claim 1 , wherein the camera comprises a CMOS camera.
8. The system of claim 1 , further comprising a data processing system configured to, for each field of view of at least two fields of view of the plurality of different fields of view and for each pixel of at least two pixels shared in images having the quality metric above the quality metric threshold of the field of view, 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 for the pixel in a plurality of images having the quality metric above the quality metric threshold for the field of view.
9. The system of claim 1 , wherein the quality metric is a sharpness quality value, and the quality metric threshold is a sharpness quality threshold.
10. A computer program product for control of a fluorescent in-situ hybridization imaging system having a flow cell to hold a sample, a variable frequency excitation light source to illuminate the sample, and a fluorescence microscope, the computer program product comprising a non-transitory computer readable media having instructions to cause one or more processors to:
as sequential nested loops
as a first loop, cause a valve to sequentially couple the flow cell to a 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 a motor to sequentially position the fluorescence microscope relative to the 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 to illuminate the said each field of view,
as a fourth loop nested within the third loop, for each wavelength of the plurality of different wavelengths, cause an 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.
11. The system of claim 10 , wherein the quality metric is a sharpness quality value, and the quality metric threshold is a sharpness quality threshold.