Methods and systems for biological instrument calibration

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, wherein the ROI calibration minimizes at least one of the following groups of reaction site determination errors: dye saturation within the plurality of reaction sites, grid rotation, variation of magnification factors, and optical radial distortion;

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 a validation assay to validate that 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 validation assay comprises an assay of the RNase P gene.

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 validation assay 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 (Ct) values of amplification curves generated from the samples of the first quantity and a second set of Ct 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 Ct 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 non-transitory 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, wherein the ROI calibration minimizes at least one of the following groups of reaction site determination errors: dye saturation within the plurality of reaction sites, grid rotation, variation of magnification factors, and optical radial distortion;

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 a validation assay to validate the instrument is capable of distinguishing between two different quantities of sample.

9. The non-transitory 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 non-transitory computer readable storage medium of claim 8 , wherein the validation assay comprises an assay of the RNase P gene.

11. The non-transitory 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 non-transitory 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 non-transitory computer readable storage medium of claim 8 , wherein the instructions for the validation assay 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 (Ct) values of amplification curves generated from the samples of the first quantity and a second set of Ct 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 Ct values at each of the plurality of fluorescence thresholds.

14. The non-transitory 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, wherein the ROI calibration minimizes at least one of the following groups of reaction site determination errors: dye saturation within the plurality of reaction sites, grid rotation, variation of magnification factors, and optical radial distortion;

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;

a validator utilizing a known assay 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 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 (Ct) values of amplification curves generated from the samples of the first quantity and a second set of Ct 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 Ct 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.