Dynamic autofocus method and system for assay imager

Abstract

A method and system are provided for controlling focus dynamically of a sample imager. The method comprises scanning a sample with an optical assembly that apportions the sample into regions based on a scan pattern. The optical assembly has a focal setting with respect to the sample. The method further comprises shifting the focal setting of the optical assembly during scanning of the sample, and detecting one or more images representative of one of the regions from the sample. The one or more images have associated degrees of focus corresponding to the focal setting of the optical assembly. The method analyzes the image(s) to obtain a focus score or scores corresponding thereto, where the focus scores represent a degree to which the optical assembly was in focus when detecting the images. The method adjusts the focus setting based on the focus score(s).

1 . A method for controlling focus dynamically of a sample imager, comprising:
 scanning a sample with an optical assembly by apportioning the sample into a plurality of regions defined by a scan pattern, the optical assembly having a focal setting with respect to the sample, thereby obtaining images of the plurality of regions;   shifting the focal setting of the optical assembly during scanning of the sample, whereby the images have an associated degree of focus corresponding to the focal setting of the optical assembly;   analyzing the images to obtain at least two focus scores, the focus scores representing a degree to which the optical assembly was in focus when detecting the images; and   adjusting the focus setting based on a function of the at least two focus scores.   
     
     
         2 . The method of  claim 1 , wherein the shifting operation introduces an error signal into a focal position of the optical assembly, the error signal being monitored as a function of the focal position of the optical assembly, the adjusting operation reducing the error signal by adjusting the focal position. 
     
     
         3 . The method of  claim 1 , wherein the regions of the sample are non-overlapping such that the detecting operation detects a series of adjacent images that are separate and distinct from one another. 
     
     
         4 . The method of  claim 1 , wherein the regions comprise rows and columns, the scanning operation comprising scanning an incident beam along the columns in the regions in a raster manner. 
     
     
         5 . The method of  claim 1 , wherein the image comprises an array of pixels and the analyzing operation calculates the focus score based on at least one of contrast, spot size, a signal-to-noise ratio, and a mean-square-error between pixel values for the at least one image being analyzed. 
     
     
         6 . The method of  claim 1 , wherein the image contains at least one of an emission pattern and a transmission pattern produced by the sample, the method further comprising identifying the at least one emission pattern and transmission pattern. 
     
     
         7 . The method of  claim 6 , wherein the identifying and analyzing operations operate upon the same image to identify the focus score and the at least one of the emission pattern and transmission pattern. 
     
     
         8 . The method of  claim 1 , wherein the analyzing operation includes calculating a coefficient of variation in contrast for the image, the coefficients of variation in contrast representing the focus score. 
     
     
         9 . The method of  claim 1 , wherein the analyzing operation includes calculating the full width half maximum (FWHM) measure for a Gaussian spot derived from the image, the FWHM representing the focus score. 
     
     
         10 . The method of  claim 1 , wherein the optical assembly includes a focus lens, the shifting operation including modulating a z-position of the focus lens repeatedly with respect to the sample. 
     
     
         11 . The method of  claim 1 , wherein the shifting operation includes periodically adding a focal offset to the focal setting. 
     
     
         12 . The method of  claim 1 , wherein the shifting, analyzing and adjusting operations are continuously updated during a time delay integration scan using real time information in the image to control a focal position of the optical assembly. 
     
     
         13 . The method of  claim 1 , wherein the shifting, analyzing and adjusting operations are continuously repeated in a control loop to lock in on a desired focal position of the optical assembly. 
     
     
         14 . The method of  claim 1 , wherein the sample emits fluorescence that is captured in the image as a fluorescence spatial emission pattern, the focus score being based on contrast or spot size within the fluorescence spatial emission pattern. 
     
     
         15 . The method of  claim 1 , wherein the sample comprises multiple microparticles that have at least one of first and second labels that emit fluorescence at different first and second wavelengths, the image containing a fluorescence spatial emission pattern of fluorescence emitted at the first and second wavelengths, the method further comprising detecting the first and second labels from the same image utilized to obtain the focus score. 
     
     
         16 . The method of  claim 1 , wherein the sample comprises multiple microparticles that have optically detectable codes that are captured in the image as a coded spatial transmission pattern, the focus score being based on contrast or spot size within the coded spatial transmission pattern. 
     
     
         17 . The method of  claim 1 , wherein the sample comprises multiple microparticles that have optically detectable codes, the microparticles having chemical probes attached thereto, each of the chemical probes being associated with a corresponding one of the codes, the image containing optically detectable codes spatially distributed across the image, the method further comprising detecting the codes from the same image utilized to obtain the focus score. 
     
     
         18 . An optical imaging system, comprising:
 a sample holder to receive a sample;   an optical assembly to scan the sample, the optical assembly apportioning the sample into regions defined by a scan pattern, the optical assembly having a focal setting with respect to the sample;   a focus control module to introduce a shift by a predetermined extent into the focal setting of the optical assembly;   a detector to detect images representative of at least two regions from the sample, the images each having an associated degree of focus corresponding to the focal setting of the optical assembly; and   an image analysis module to analyze the images to obtain at least two focus scores, each of the focus scores representing a degree to which the optical assembly was in focus when detecting the image, and to determine a desired focal setting based on a function of the at least two focus scores, wherein the focus control module adjusts the focus setting based on the desired focal setting.   
     
     
         19 . A method for controlling focus dynamically of a sample imager, comprising:
 (a) detecting a first region of a sample with an optical assembly, the optical assembly having a first focal setting with respect to the sample, thereby obtaining a first image;   (b) analyzing the first image to obtain a first focus score; the first focus score representing a degree to which the optical assembly was in focus when detecting the first image;   (c) shifting the focal setting of the optical assembly by a predetermined extent to a second focal setting;   (d) detecting a second region of the sample with the optical assembly at the second focal setting, thereby obtaining a second image;   (e) analyzing the second image to obtain a second focus score; the second focus score representing a degree to which the optical assembly was in focus when detecting the second image;   (f) determining a desired focal setting for the optical assembly based on a function of at least the first focus score and the second focus score; and   (g) repeating steps (a) through (e) under conditions wherein the first focal setting is adjusted based on the desired focal setting.   
     
     
         20 . The method of  claim 19 , wherein the function comprises a difference between the first focus score and the second focus score. 
     
     
         21 . The method of  claim 20 , wherein the first focus score and the second focus score are based on contrast or spot size within the images. 
     
     
         22 . The method of  claim 19 , wherein the first region and the second region are adjacent regions of the sample. 
     
     
         23 . The method of  claim 22 , wherein the first image and the second image are obtained by continuous scanning. 
     
     
         24 . The method of  claim 19 , further comprising performing a second repetition of the method, wherein the first focal setting is adjusted to a lesser extent in the second repetition. 
     
     
         25 . The method of  claim 22 , further comprising changing the relative locations of the optical assembly and the sample prior to the shifting of the focal setting. 
     
     
         26 . A method for controlling focus dynamically of a sample imager, comprising:
 (a) detecting a plurality of images of a sample, the plurality of images including information relating to detected light signals from the sample, the plurality of images including first and second images;   (b) analyzing the first and second images to obtain respective focus scores, the focus scores representing a degree to which the optical assembly was in focus when detecting the first and second images, wherein the focus scores of the first and second images are different;   (c) comparing the focus scores of the first and second images; and   (d) relatively shifting the sample with respect to the optical assembly based upon said comparison of the focus scores.   
     
     
         27 . The method of  claim 26  wherein the first image corresponds to light signals within a first spectral band that are emitted from a first label in the sample, and the second image corresponds to light signals within a second spectral band that are emitted from a second label in the sample, wherein the first and second spectral bands are different, the optical assembly having different optimal focal planes for the first and second labels. 
     
     
         28 . The method of  claim 26  wherein the first and second images are of adjacent scan regions, the focal setting of the optical assembly being shifted a predetermined extent before obtaining the second image. 
     
     
         29 . A method for controlling focus dynamically of a sample imager, comprising:
 (a) obtaining first and second images of a scan region of a sample, the sample being positioned relative to an optical assembly, the first and second images including information relating to detected light signals from first and second labels in the sample, respectively;   (b) analyzing the first and second images to obtain first and second focus scores; the focus scores representing a degree to which the optical assembly was in focus when detecting the first and second images;   (c) comparing the first and second focus scores; and   (d) relatively shifting the sample with respect to the optical assembly based upon said comparison of the first and second focus scores, the sample having a modified position relative to the optical assembly.