Methods and systems for biological instrument calibration

Abstract

In one exemplary embodiment, a method for calibrating an instrument is provided. The instrument includes an optical system capable of imaging fluorescence emission from a plurality of reaction sites. The method includes performing a region-of-interest (ROI) calibration to determine reaction site positions in an image. The method further includes performing a pure dye calibration to determine the contribution of a fluorescent dye used in each reaction site by comparing a raw spectrum of the fluorescent dye to a pure spectrum calibration data of the fluorescent dye. The method further includes performing an instrument normalization calibration to determine a filter normalization factor. The method includes performing an RNase P validation to validate the instrument is capable of distinguishing between two different quantities of sample.

What is claimed is: 
     
         1 . A method for calibrating an instrument, wherein the instrument includes an optical system capable of imaging florescence emission from a plurality of reaction sites, the method comprising:
 performing a region-of-interest (ROI) calibration to determine reaction site positions in an image;   performing a pure dye calibration to determine the contribution of a fluorescent dye used in each reaction site by comparing a raw spectrum of the fluorescent dye to a pure spectrum calibration data of the fluorescent dye;   performing an instrument normalization calibration to determine a filter normalization factor; and   performing an RNase P validation to validate the instrument is capable of distinguishing between two different quantities of sample.   
     
     
         2 . The method of  claim 1 , wherein the ROI calibration comprises:
 estimating initial region of interest (ROI) from fluorescence thresholds from each sample well;   estimating the center locations of each ROI;   estimating the size of each ROI;   determining the average size of the ROIs from the plurality of reaction sites;   deriving global gridding models;   applying the global gridding models to the ROIs, wherein the application of the global gridding models improve the precision of the ROI center locations;   recovering missing ROIs; and   adjusting the radius of the ROIs, wherein the adjustment improves the signal-to-noise ratio of the optical system.   
     
     
         3 . The method of  claim 1 , wherein the ROI calibration improves reaction site determination errors by minimizing at least one of the following group: dye saturation within the plurality of reaction sites, grid rotation, variation of magnification factors, and optical radial distortion. 
     
     
         4 . The method of  claim 1 , wherein the pure dye calibration comprises:
 imaging a sample holder, loaded into the instrument, at more than one channel, the sample holder comprising a plurality of reaction sites and more than one dye type, each dye occupying more than one reaction site;   identifying a peak channel for each dye on the sample holder;   normalizing each channel to the peak channel for each dye; and   producing a dye matrix comprising a set of dye reference values.   
     
     
         5 . The method of  claim 1 , wherein the optical system comprises a plurality of excitation filters and a plurality of emission filters, and wherein the instrument normalization calibration comprises:
 determining a first correction factor for each of the excitation filters and emission filters;   calculating a second correction factor for a pair of filters, wherein each pair of filters comprises one excitation filter and one emission filter; and   applying the second correction factors to filter data.   
     
     
         6 . The method of  claim 1 , wherein the RNase P validation comprises:
 receiving amplification data from a validation plate to generate a plurality of amplification curves, wherein the validation plate includes a sample of a first quantity and a second quantity, and each amplification curve includes an exponential region;   determining a set of fluorescence thresholds based on the exponential regions of the plurality of amplification curves;   determining, for each fluorescence threshold of the set, a first set of cycle threshold (C t ) values of amplification curves generated from the samples of the first quantity and a second set of C t  values of amplification curves generated from the samples of the second quantity; and   calculating if the first and second quantities are sufficiently distinguishable based on C t  values at each of the plurality of fluorescence thresholds.   
     
     
         7 . The method of  claim 1 , further comprising:
 performing an auto-dye correction for real-time spectral calibration of the multi-component data;   performing a plate detection to determine whether there is a plate loading error;   performing an auto-background calibration to compensate for background changes; and   performing instrument normalization using a reflective material to detect any changes or variability in fluorescent emissions.   
     
     
         8 . A computer readable storage medium encoded with processor-executable instructions for calibrating an instrument, wherein the instrument includes an optical system capable of imaging florescence emission from a plurality of reaction sites, the instructions comprising instructions for:
 performing a region-of-interest (ROI) calibration to determine reaction site positions in an image;   performing a pure dye calibration to determine the contribution of a fluorescent dye used in each reaction site by comparing a raw spectrum of the fluorescent dye to a pure spectrum calibration data of the fluorescent dye;   performing an instrument normalization calibration to determine a filter normalization factor; and   performing an RNase P validation to validate the instrument is capable of distinguishing between two different quantities of sample.   
     
     
         9 . The computer readable storage medium of  claim 8 , wherein the instructions for ROI calibration comprise instructions for:
 estimating initial region of interest (ROI) from fluorescence thresholds from each sample well;   estimating the center locations of each ROI;   estimating the size of each ROI;   determining the average size of the ROIs from the plurality of reaction sites;   deriving global gridding models;   applying the global gridding models to the ROIs, wherein the application of the global gridding models improve the precision of the ROI center locations;   recovering missing ROIs; and   adjusting the radius of the ROIs, wherein the adjustment improves the signal-to-noise ratio of the optical system.   
     
     
         10 . The computer readable storage medium of  claim 8 , wherein the ROI calibration improves reaction site determination errors by minimizing at least one of the following group: dye saturation within the plurality of reaction sites, grid rotation, variation of magnification factors, and optical radial distortion. 
     
     
         11 . The computer readable storage medium of  claim 8 , wherein the instructions for pure dye calibration comprise instructions for:
 imaging a sample holder, loaded into the instrument, at more than one channel, the sample holder comprising a plurality of reaction sites and more than one dye type, each dye occupying more than one reaction site;   identifying a peak channel for each dye on the sample holder;   normalizing each channel to the peak channel for each dye; and   producing a dye matrix comprising a set of dye reference values.   
     
     
         12 . The computer readable storage medium of  claim 8 , wherein the optical system comprises a plurality of excitation filters and a plurality of emission filters, and wherein the instructions for instrument normalization calibration comprise instructions for:
 determining a first correction factor for each of the excitation filters and emission filters;   calculating a second correction factor for a pair of filters, wherein each pair of filters comprises one excitation filter and one emission filter; and   applying the second correction factors to filter data.   
     
     
         13 . The computer readable storage medium of  claim 8 , wherein the instructions for RNase P validation comprise instructions for:
 receiving amplification data from a validation plate to generate a plurality of amplification curves, wherein the validation plate includes a sample of a first quantity and a second quantity, and each amplification curve includes an exponential region;   determining a set of fluorescence thresholds based on the exponential regions of the plurality of amplification curves;   determining, for each fluorescence threshold of the set, a first set of cycle threshold (C t ) values of amplification curves generated from the samples of the first quantity and a second set of C t  values of amplification curves generated from the samples of the second quantity; and   calculating if the first and second quantities are sufficiently distinguishable based on C t  values at each of the plurality of fluorescence thresholds.   
     
     
         14 . The computer readable storage medium of  claim 8 , further comprising instructions for:
 performing an auto-dye correction for real-time spectral calibration of the multi-component data;   performing a plate detection to determine whether there is a plate loading error;   performing an auto-background calibration to compensate for background changes; and   performing instrument normalization using a reflective material to detect any changes or variability in fluorescent emissions.   
     
     
         15 . A system for calibrating an instrument, wherein the instrument includes an optical system capable of imaging florescence emission from a plurality of reaction sites, the system comprising:
 a region-of-interest (ROI) calibrator configured to determine reaction site positions in an image;   a pure dye calibrator configured to determine the contribution of a fluorescent dye used in each reaction site by comparing a raw spectrum of the fluorescent dye to a pure spectrum calibration data of the fluorescent dye;   an instrument normalization calibrator configured to determine a filter normalization factor;   an RNase P validator configured to validate the instrument is capable of distinguishing between two different quantities of sample;   and a display engine configured to display calibration results.   
     
     
         16 . The system of  claim 15 , wherein the ROI calibrator is configured to:
 estimate initial region of interest (ROI) from fluorescence thresholds from each sample well;   estimate the center locations of each ROI;   estimate the size of each ROI;   determine the average size of the ROIs from the plurality of reaction sites;   derive global gridding models;   apply the global gridding models to the ROIs, wherein the application of the global gridding models improve the precision of the ROI center locations;   recover missing ROIs; and   adjust the radius of the ROIs, wherein the adjustment improves the signal-to-noise ratio of the optical system.   
     
     
         17 . The system of  claim 15 , wherein the pure dye calibrator is configured to:
 image a sample holder, loaded into the instrument, at more than one channel, the sample holder comprising a plurality of reaction sites and more than one dye type, each dye occupying more than one reaction site;   identify a peak channel for each dye on the sample holder;   normalize each channel to the peak channel for each dye; and   produce a dye matrix comprising a set of dye reference values.   
     
     
         18 . The system of  claim 15 , wherein the optical system comprises a plurality of excitation filters and a plurality of emission filters, and wherein the instrument normalization calibrator is configured to:
 determine a first correction factor for each of the excitation filters and emission filters;   calculate a second correction factor for a pair of filters, wherein each pair of filters comprises one excitation filter and one emission filter; and   apply the second correction factors to filter data.   
     
     
         19 . The system of  claim 15 , wherein the RNase P validator is configured to:
 receive amplification data from a validation plate to generate a plurality of amplification curves, wherein the validation plate includes a sample of a first quantity and a second quantity, and each amplification curve includes an exponential region;   determine a set of fluorescence thresholds based on the exponential regions of the plurality of amplification curves;   determine, for each fluorescence threshold of the set, a first set of cycle threshold (C t ) values of amplification curves generated from the samples of the first quantity and a second set of C t  values of amplification curves generated from the samples of the second quantity; and   calculate if the first and second quantities are sufficiently distinguishable based on C t  values at each of the plurality of fluorescence thresholds.   
     
     
         20 . The system of  claim 15 , further comprising:
 an auto-dye corrector configured to perform real-time spectral calibration of the multi-component data;   a plate detector configured to determine whether there is a plate loading error;   an auto-background calibrator configured to compensate for background changes; and   an instrument normalizer configured to use a reflective material to detect any changes or variability in fluorescent emissions.