Identifying fiducial markers in fluorescence microscope images

Abstract

Methods, systems, and apparatus, including computer programs encoded on a computer storage medium, for obtaining a fluorescence microscope image that depicts a sample and a plurality of fiducial markers, identifying the plurality of fiducial markers in the image, and using the plurality of fiducial markers to register the image. The sample and the plurality of fiducial markers have a common fluorescence color, and identifying the plurality of fiducial markers in the image includes comparing a spatial intensity distribution of a plurality of fluorescent regions of the image to a reference distribution function.

1 - 20 . (canceled) 
     
     
         21 . A method performed by one or more data processing apparatus, the method comprising:
 obtaining a fluorescence microscope image that depicts a sample and a plurality of fiducial markers, the sample and the plurality of fiducial markers having a common fluorescence color;   identifying the plurality of fiducial markers in the image, comprising:
 identifying a plurality of fluorescent regions of the image; 
 determining a respective area of each respective fluorescent region of the plurality of fluorescent regions of the image; and 
 determining, for each respective fluorescent region, whether the respective fluorescent region corresponds to a fiducial marker based at least in part on the respective area of the respective fluorescent region; and 
   after identifying the plurality of fiducial markers in the image, using the plurality of fiducial markers to register the image.   
     
     
         22 . The method of  claim 21 , further comprising obtaining data defining a range of area values, wherein a fluorescent region that corresponds to a fiducial marker is expected to have an area that included within the range of area values;
 wherein for each fluorescent region, determining whether the fluorescent region corresponds to a fiducial marker comprises:
 comparing the area of the fluorescent region to the range of area values; and 
 determining that the fluorescent region corresponds to a fiducial marker only if the area of the fluorescent region is included in the range of area values. 
   
     
     
         23 . The method of  claim 22 , wherein for one or more fluorescent regions, determining whether the fluorescent region corresponds to a fiducial marker comprises:
 determining that the fluorescent region does not correspond to a fiducial marker in response to determining that the area of the fluorescent region is not included in the range of area values.   
     
     
         24 . The method of  claim 21 , wherein for each fluorescent region, determining the area of the fluorescent region comprises:
 determining a product of: (i) a number of pixels in the fluorescent region, and (ii) an area of each pixel in the image, wherein the area of each pixel in the image depends on a magnification of the image.   
     
     
         25 . The method of  claim 21 , further comprising, prior to identifying the plurality of fiducial markers in the image:
 applying one or more filters to the image to enhance a signal-to-noise ratio (SNR) of the image.   
     
     
         26 . The method of  claim 25 , wherein the one or more filters comprise a two-dimensional Normal filter. 
     
     
         27 . The method of  claim 21 , wherein for one or more fluorescent regions, determining whether the fluorescent region corresponds to a fiducial marker comprises:
 comparing a spatial intensity distribution of the fluorescent region to a reference distribution function; and   determining whether the fluorescent region corresponds to a fiducial marker based at least in part on the comparison of the spatial intensity distribution of the fluorescent region to the reference distribution function.   
     
     
         28 . The method of  claim 27 , wherein comparing the spatial intensity distribution of the fluorescent region to the reference distribution function comprises:
 determining fitted parameter values of the reference distribution function that fit the reference distribution function to the spatial intensity distribution of the fluorescent region.   
     
     
         29 . The method of  claim 28 , wherein determining whether the fluorescent region corresponds to a fiducial marker comprises:
 determining a similarity measure between the reference distribution function with the fitted parameter values and the spatial intensity distribution of the fluorescent region; and   determining whether the fluorescent region corresponds to a fiducial marker based at least in part on the similarity measure between the reference distribution function with the fitted parameter values and the spatial intensity distribution of the fluorescent region.   
     
     
         30 . The method of  claim 29 , wherein determining the similarity measure between the reference distribution function with the fitted parameter values and the spatial intensity distribution of the fluorescent region comprises:
 determining a mean-squared-error (MSE) between the reference distribution function with the fitted parameter values and the spatial intensity distribution of the fluorescent region.   
     
     
         31 . The method of  claim 28 , wherein:
 the reference distribution function is a two-dimensional Normal distribution function;   parameters of the reference distribution function comprise a mean parameter and a covariance parameter.   
     
     
         32 . The method of  claim 21 , wherein the sample comprises RNA molecules and the fiducial markers are beads. 
     
     
         33 . A system comprising:
 one or more computers; and   one or more storage devices communicatively coupled to the one or more computers, wherein the one or more storage devices store instructions that, when executed by the one or more computers, cause the one or more computers to perform operations comprising:   obtaining a fluorescence microscope image that depicts a sample and a plurality of fiducial markers, the sample and the plurality of fiducial markers having a common fluorescence color;   identifying the plurality of fiducial markers in the image, comprising:
 identifying a plurality of fluorescent regions of the image; 
 determining a respective area of each respective fluorescent region of the plurality of fluorescent regions of the image; and 
 determining, for each respective fluorescent region, whether the respective fluorescent region corresponds to a fiducial marker based at least in part on the respective area of the respective fluorescent region; and 
   after identifying the plurality of fiducial markers in the image, using the plurality of fiducial markers to register the image.   
     
     
         34 . The system of  claim 33 , wherein the operations further comprise obtaining data defining a range of area values, wherein a fluorescent region that corresponds to a fiducial marker is expected to have an area that included within the range of area values;
 wherein for each fluorescent region, determining whether the fluorescent region corresponds to a fiducial marker comprises:
 comparing the area of the fluorescent region to the range of area values; and 
 determining that the fluorescent region corresponds to a fiducial marker only if the area of the fluorescent region is included in the range of area values. 
   
     
     
         35 . The system of  claim 34 , wherein for one or more fluorescent regions, determining whether the fluorescent region corresponds to a fiducial marker comprises:
 determining that the fluorescent region does not correspond to a fiducial marker in response to determining that the area of the fluorescent region is not included in the range of area values.   
     
     
         36 . The system of  claim 33 , wherein for each fluorescent region, determining the area of the fluorescent region comprises:
 determining a product of: (i) a number of pixels in the fluorescent region, and (ii) an area of each pixel in the image, wherein the area of each pixel in the image depends on a magnification of the image.   
     
     
         37 . The system of  claim 33 , wherein the operations further comprise, prior to identifying the plurality of fiducial markers in the image:
 applying one or more filters to the image to enhance a signal-to-noise ratio (SNR) of the image.   
     
     
         38 . The system of  claim 37 , wherein the one or more filters comprise a two-dimensional Normal filter. 
     
     
         39 . The system of  claim 33 , wherein for one or more fluorescent regions, determining whether the fluorescent region corresponds to a fiducial marker comprises:
 comparing a spatial intensity distribution of the fluorescent region to a reference distribution function; and   determining whether the fluorescent region corresponds to a fiducial marker based at least in part on the comparison of the spatial intensity distribution of the fluorescent region to the reference distribution function.   
     
     
         40 . One or more non-transitory computer storage media storing instructions that when executed by one or more computers cause the one or more computers to perform operations comprising:
 obtaining a fluorescence microscope image that depicts a sample and a plurality of fiducial markers, the sample and the plurality of fiducial markers having a common fluorescence color;   identifying the plurality of fiducial markers in the image, comprising:
 identifying a plurality of fluorescent regions of the image; 
 determining a respective area of each respective fluorescent region of the plurality of fluorescent regions of the image; and 
 determining, for each respective fluorescent region, whether the respective fluorescent region corresponds to a fiducial marker based at least in part on the respective area of the respective fluorescent region; and 
   after identifying the plurality of fiducial markers in the image, using the plurality of fiducial markers to register the image.