Reducing effects of spectral nonuniformity
View Patent ↗Method and system providing calibration of light detected from biological samples with a correction factor including components for each of a plurality of spectrally distinguishable species and/or for each well and/or for each filter.
1. A method for calibrating detection of light from biological samples comprising:
providing a system adapted to excitation and detection of a plurality of spectrally distinguishable species, wherein the system comprises a plurality of filters;
providing a calibration plate comprising a plurality of wells, wherein each well comprises a sample with the plurality of spectrally distinguishable species;
detecting light from the plurality of spectrally distinguishable species for each well;
determining a correction factor for each spectrally distinguishable species distinguished by the plurality of filters for each well.
2. The method of claim 1 , wherein determining a correction factor further comprises determining a correction factor for each spectrally distinguishable species for each filter, for each well.
3. The method of claim 1 , wherein each spectrally distinguishable species is associated with at least one filter of the plurality of filters.
4. The method of claim 1 , wherein the plurality of spectrally distinguishable species comprises at least one dye chosen from FAM, SYBR Green, VIC, JOE, TAMRA, NED, CY-3, Texas Red, CY-5, and ROX.
5. The method of claim 1 , further comprising normalizing the light detected from each well.
6. The method of claim 5 , wherein the correction factor is determined prior to normalizing.
7. The method of claim 5 , wherein the correction factor is determined after normalizing.
8. The method of claim 1 , wherein the correction factor is determined from a plateau at the end of a run.
9. The method of claim 1 , wherein the correction factor is determined from an unquenched dye at the beginning of a run.
10. The method of claim 1 , wherein the correction factor is determined from log-log analysis of the run.
11. A method for calibrating detection of light from biological samples comprising:
providing a system adapted to excitation and detection of a plurality of spectrally distinguishable species, wherein the system comprises a plurality of filters;
providing a calibration plate comprising a plurality of wells, wherein each well comprises a sample with the plurality of spectrally distinguishable species;
detecting light from each filter for each well;
determining a correction factor for each filter for each well.
12. The method of claim 11 , wherein determining a correction factor further comprises determining a correction factor for each spectrally distinguishable species for each filter, for each well.
13. The method of claim 11 , wherein each spectrally distinguishable species is associated with at least one filter of the plurality of filters.
14. The method of claim 11 , wherein the plurality of spectrally distinguishable species comprises at least one dye chosen from FAM, SYBR Green, VIC, JOE, TAMRA, NED, CY-3, Texas Red, CY-5, and ROX.
15. The method of claim 11 , further comprising normalizing the light detected from each well.
16. The method of claim 15 , wherein the correction factor is determined prior to normalizing.
17. The method of claim 15 , wherein the correction factor is determined after normalizing.
18. The method of claim 11 , wherein the correction factor is determined from a plateau at the end of a run.
19. The method of claim 11 , wherein the correction factor is determined from an unquenched dye at the beginning of a run.
20. The method of claim 11 , wherein the correction factor is determined from log-log analysis of the run.