Methods and Systems for In Situ Calibration of Imaging in Biological Analysis

Abstract

Software, methods, and systems for calibrating photometric devices are provided. These involve using a non-uniform test illumination field to approximate a photon transfer curve by calculating stable pixel values and statistical dispersions on a pixel-by-pixel basis.

1 - 32 . (canceled) 
     
     
         33 . A method for characterizing a biological instrument, the method comprising:
 providing an instrument comprising:
 a photometric sensor having a plurality of pixels; and 
 a spatially non-uniform pattern, wherein the spatial non-uniformity of the pattern comprises a brightness range; 
   capturing a series of images with the photometric sensor using the spatially non-uniform pattern, wherein the images comprise a range of signal intensities;   estimating a relationship of signal intensity level versus noise level for the photometric sensor using computed values of signal intensity and noise level of the plurality of pixels.   
     
     
         34 . The method of  claim 33 , wherein the spatially non-uniform pattern is a spatially non-uniform illumination source of electromagnetic radiation or a non-uniform test image comprising a surface that scatters electromagnetic radiation in a spatially non-uniformly manner. 
     
     
         35 . The method of  claim 33 , wherein the range of signal intensities spans a dynamic range of the photometric sensor. 
     
     
         36 . The method of  claim 35 , further comprising estimating a relationship of signal intensity level versus noise level for the photometric sensor over the dynamic range of the sensor using the computed values of signal intensity and noise level of the plurality of pixels. 
     
     
         37 . The method of  claim 33 , further comprising determining whether there is a sufficient range of brightness and darkness in the spatially non-uniform pattern to estimate the relationship of signal intensity versus noise level for the photometric sensor over the dynamic range of the sensor. 
     
     
         38 . The method of  claim 33 , further comprising determining if the photometric sensor is within operational limits of detection using the estimated relationship. 
     
     
         39 . The method of  claim 38 , wherein the operational limits of detection for the photometric sensor are based on an operating characteristic of the photometric sensor. 
     
     
         40 . The method of  claim 33 , wherein the photometric sensor provided is selected from a charge coupled device (CCD), a complimentary metal oxide semiconductor device (CMOS) image sensor, a spectrometer, and a line-scan camera. 
     
     
         41 . The method of  claim 33 , wherein the photometric sensor is validated on the basis of the computed signal intensities and noise levels. 
     
     
         42 . A method for characterizing an instrument, the method comprising:
 illuminating a photometric detector comprising a plurality of pixels with an electromagnetic radiation pattern;   for at least some of the pixels, recording a plurality of brightness readings corresponding to a series photometric detector recordings;   calculating signal statistical dispersion values for the at least some of the pixels based on the plurality of brightness readings;   calculating stable intensity values for the at least some of the pixels based on the plurality of brightness readings; and   calculating a photon transfer curve based on the signal statistical dispersion values and the stable intensity values;   wherein the photometric detector is part of a biological instrument.   
     
     
         43 . A biological instrument, comprising:
 a photometric sensor having a plurality of pixels; and   a spatially non-uniform pattern, wherein the spatial non-uniformity of the pattern comprises a brightness range;   a computer system configured to:   a memory coupled to a photometric sensor interface circuit, the memory comprising machine readable instructions comprising:
 read instructions for capturing a series of images with the photometric sensor using the spatially non-uniform pattern, wherein the images comprise a range of signal intensities; 
 read instructions for estimating a relationship of signal intensity level versus noise level for the photometric sensor using computed values of signal intensity and noise level of the plurality of pixels. 
   
     
     
         44 . The biological instrument of  claim 43 , wherein the spatially non-uniform pattern is a spatially non-uniform pattern of electromagnetic radiation or a non-uniform test image comprising a surface that scatters electromagnetic radiation in a spatially non-uniformly manner. 
     
     
         45 . The biological instrument of  claim 43 , wherein the machine readable instructions comprise estimating a relationship of signal intensity level versus noise level for the photometric sensor over a dynamic range of the sensor using the computed values of signal intensity and noise level of the plurality of pixels. 
     
     
         46 . The biological instrument of  claim 43 , wherein the machine readable instructions comprise determining whether there is a sufficient range of brightness and darkness in the spatially non-uniform pattern to estimate the relationship of signal intensity versus noise level for the photometric sensor over the dynamic range of the sensor. 
     
     
         47 . The biological instrument of  claim 43 , wherein the machine readable instructions comprise determining if the photometric sensor is within operational limits of detection using the estimated relationship. 
     
     
         48 . The biological instrument of  claim 47 , wherein the operational limits of detection for the photometric sensor are based on an operating characteristic of the photometric sensor. 
     
     
         49 . The biological instrument of  claim 43 , wherein the spatially non-uniform pattern comprises illumination that varies in spatial intensity over time to produce a time varying non-uniform illumination field. 
     
     
         50 . The biological instrument of  claim 43 , wherein the spatially non-uniform pattern provided is a sample for biological analysis. 
     
     
         51 . The biological instrument of  claim 43 , wherein the photometric sensor provided is selected from a charge coupled device (CCD), a complimentary metal oxide semiconductor device (CMOS) image sensor, a spectrometer, and a line-scan camera.