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

Abstract

A fluorescent in-situ hybridization imaging system includes a flow cell to contain a sample, a fluorescence microscope, and a control system. The fluorescence microscope includes a variable frequency excitation light source to illuminate the sample, a plurality of emission bandpass filters on a filter wheel, an actuator to rotate the filter wheel, and a camera positioned to receive fluorescently emitted light from the sample. The control system is configured to cause the variable frequency excitation light source to emit a light beam having a selected wavelength, cause the actuator to rotate the filter wheel to position a selected filter in a light path between the sample and the camera, obtain an image from the camera, and coordinate the variable frequency excitation light source and filter wheel such that the selected filter has an emission bandpass associated with emission by the fluorescent probes when excited by the selected wavelength.

What is claimed is: 
     
         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 fluorescence microscope including 
 a variable frequency excitation light source to illuminate the sample, 
 a plurality of emission bandpass filters on a filter wheel, 
 a first actuator to rotate the filter wheel, 
 a camera positioned to receive fluorescently emitted light from the sample that has passed through a filter on the filter wheel; 
   a control system configured to 
 cause the variable frequency excitation light source to emit a light beam having a selected wavelength, 
 cause the first actuator to rotate the filter wheel to position a selected filter in a light path between the sample and the camera, 
 obtain an image from the camera, and 
 coordinate the variable frequency excitation light source and filter wheel such that the selected filter has an emission bandpass associated with emission by the fluorescent probes when excited by the selected wavelength when the image is obtained, 
 wherein the control system comprises a first microcontroller cause the first actuator to rotate the filter wheel in response to a first trigger signal and to generate a second trigger signal, and a second microcontroller to cause the variable frequency excitation light source to emit the light beam having the selected wavelength in response to the second trigger signal. 
   
     
     
         2 . The system of  claim 1 , wherein the second microcontroller is configured to send a third trigger signal to the camera to cause image acquisition. 
     
     
         3 . The system of  claim 1 , comprising a dichroic filter in the light path between the sample and the camera. 
     
     
         4 . The system of  claim 3 , wherein the dichroic filter is positioned in the light path between the sample and the filter wheel. 
     
     
         5 . The system of  claim 3 , wherein the dichroic mirror is a multi-pass dichroic mirror having a pass band for each emission wavelength for the fluorescent probes. 
     
     
         6 . The system of  claim 1 , wherein each filter of the plurality of emission bandpass filters has a unique band and is associated with a unique wavelength for the variable frequency excitation light source. 
     
     
         7 . The system of  claim 1 , wherein the variable frequency excitation light source is configured to illuminate the sample with excitation light normal to the sample. 
     
     
         8 . The system of  claim 1 , comprising a second actuator to cause relative vertical motion between the flow cell and the fluorescence microscope. 
     
     
         9 . The system of  claim 8 , wherein the second microcontroller is configured to control the second actuator to select an imaging height of the fluorescence microscope above the flow cell. 
     
     
         10 . The system of  claim 8 , wherein the actuator comprises a piezoelectric actuator. 
     
     
         11 . The system of  claim 1 , comprising a valve to control flow from one of a plurality of reagent sources to the flow cell. 
     
     
         12 . The system of  claim 11 , wherein the control system includes a computer to control the valve. 
     
     
         13 . The system of  claim 12 , wherein the first microcontroller receives input from the computer. 
     
     
         14 . The system of  claim 12 , further comprising a motor to cause to cause relative lateral motion between the flow cell and the fluorescence microscope. 
     
     
         15 . The system of  claim 14 , 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. 
     
     
         16 . The system of  claim 14 , wherein the computer is configured to control the motor. 
     
     
         17 . 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. 
     
     
         18 . 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 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 the plurality of images for the field of view.