Instrument for monitoring nucleic acid sequence amplification reaction
View Patent ↗An instrument is provided that can monitor nucleic acid sequence amplifications reactions, for example, PCR amplification of DNA and DNA fragments. The instrument includes a multi-notch filter disposed along one or both of an excitation beam path and an emission beam path. Methods are also provided for monitoring nucleic acid sequence amplifications using an instrument that includes a multi-notch filter disposed along a beam path.
1. An optical instrument comprising:
an excitation light source that provides a plurality of different excitation wavelength ranges;
at least one reaction region retaining at least one respective sample comprising a plurality of different dyes, each dye having a different respective excitation wavelength range relative to the other dyes of the plurality, the plurality of different dyes being capable of emitting emission beams of different respective wavelength ranges along an emission beam path, wherein the different excitation wavelength ranges provided by the excitation light source correspond to the different respective excitation wavelength ranges of the plurality of different dyes;
a multi-notch beam splitter disposed along an excitation beam path between the excitation light source and the at least one reaction region, and disposed along the emission beam path, wherein the multi-notch beam splitter is configured to reflect one of (1) the plurality of different excitation wavelength ranges provided by the excitation light source and (2) the plurality of emission beams of different respective wavelength ranges, and to the pass the other of (1) and (2);
a detector arranged along the emission beam path to detect the emission beams of the different respective wavelength ranges and generate a data signal corresponding to the emission beams;
a processing device, operatively connected to the detector, the processing device adjusting exposure conditions of the detector to maintain the data signal within a predetermined operating range; and
a controller for controlling the temperature of a sample in the at least one reaction region.
2. The instrument of claim 1 , wherein the at least one reaction region comprises a plurality of reaction regions.
3. The instrument of claim 1 , further comprising a Fresnel lens disposed along an excitation beam path between the excitation light source and the at least one reaction region.
4. The instrument of claim 1 , further comprising a fold mirror disposed along an excitation beam path between the excitation light source and the at least one reaction region.
5. The instrument of claim 1 , further comprising a dichroic mirror disposed along an excitation beam path between the excitation light source and the at least one reaction region.
6. The instrument of claim 1 , wherein the multi-notch bean splitter is a Rugate filter.
7. A method comprising:
generating excitation beams that provide a plurality of different excitation wavelength ranges;
passing the excitation beams through a multi-notch beam splitter to form filtered excitation beams;
directing the filtered excitation beams at, at least one reaction region retaining at least one respective sample comprising a plurality of different dyes, each dye having a different respective excitation wavelength range relative to the other dyes of the plurality, the plurality of different dyes being capable of emitting emission beams of different respective wavelength ranges;
causing the at least one respective sample retained in the at least one reaction region to emit emission beams of different respective wavelength ranges wherein the different excitation wavelength ranges provided by the excitation beams correspond to the different respective excitation wavelength ranges of the plurality of different dyes;
directing the emission beams through the multi-notch beam splitter and toward a detector;
detecting the emission beams of different respective wavelengths emitted from the at least one respective sample;
generating a data signal corresponding to the detected emission beams;
adjusting exposure conditions of the detector to maintain the data signal within a predetermined operating range; and
controlling the temperature of the at least one respective sample, during the detecting.
8. The method of claim 7 , wherein the at least one reaction region comprises a plurality of reactions regions, the at least one respective sample comprises a plurality of respective samples, and each of the plurality of reaction regions retains a respective one of the plurality of respective samples.
9. The method of claim 7 , further comprising passing at least one of the excitation beams and the filtered excitation beams through a Fresnel lens.
10. The method of claim 7 , further comprising passing the emission beams through a Fresnel lens.
11. The method of claim 7 , further comprising reflecting at least one of the excitation beams and the filtered excitation beams with a fold mirror.
12. The method of claim 7 , wherein the reaction region is a well.
13. The method of claim 7 , further comprising directing at least one of the excitation beams and the filtered excitation beams toward a dichroic mirror.
14. An instrument comprising:
an excitation light source that provides a plurality of different excitation wavelength ranges;
at least one reaction well retaining at least one respective sample comprising a plurality of different dyes, each dye having a different respective excitation wavelength range relative to the other dyes of the plurality, the plurality of different dyes being capable of emitting emission beams of different respective wavelength ranges along an emission beam path, wherein the different excitation wavelength ranges provided by the excitation light source correspond to the different respective excitation wavelength ranges of the plurality of different dyes;
a detector disposed along the emission beam path and capable of detecting emission beams of the different respective wavelength ranges emitted from the at least one reaction well and generating a data signal corresponding to the emission beams;
a processing device, operatively connected to the detector, the processing device adjusting exposure conditions of the detector to maintain the data signal within a predetermined operating range;
a multi-notch beam splitter spaced along the emission beam path between the at least one reaction well and the detector and configured to (1) reflect the plurality of emission beams of different respective wavelength ranges and pass the plurality of different respective excitation wavelength ranges, or (2) pass the plurality of emission beams of different respective wavelength ranges and reflect the plurality of different respective excitation wavelength ranges; and
a controller for controlling the temperature of a sample in the at least one reaction well.
15. The instrument of claim 14 , further comprising a Fresnel lens disposed along the emission beam path between the at least one reaction well and the detector.
16. The instrument of claim 14 , further comprising a dichroic mirror disposed along the emission beam path between the at least one reaction well and the detector.
17. The instrument of claim 14 , further comprising a fold mirror disposed along the emission beam path between the at least one reaction well and the detector.
18. The instrument of claim 14 , further comprising a focusing lens disposed along the emission beam path between the at least one reaction well and the detector.
19. The instrument of claim 14 , wherein the multi-notch beam splitter is a Rugate filter.
20. A method comprising:
generating excitation beams that provide a plurality of different excitation wavelength ranges;
directing the excitation beams through a multi-notch beam splitter and at, at least one reaction well retaining at least one respective sample comprising a plurality of different dyes, each dye having a different respective excitation wavelength range relative to the other dyes of the plurality, the plurality of different dyes being capable of emitting emission beams of different respective wavelength ranges;
causing the at least one respective sample to emit emission beams of different respective wavelength ranges, wherein the different excitation wavelength ranges provided by the excitation beams correspond to the different respective excitation wavelength ranges of the plurality of different dyes;
passing the emission beams of the different respective wavelength ranges through the multi-notch beam splitter to form filtered emission beams;
directing the filtered emission beams toward a detection region;
detecting the filtered emission beams emitted from the at least one respective sample;
generating a signal corresponding to the detected filtered emission beams;
adjusting exposure conditions of the detection region to maintain the data signal within a predetermined operating range; and
controlling the temperature of the at least one respective sample, during the detecting.
21. The method of claim 20 , further comprising reflecting the emission beams with a fold mirror.
22. The method of claim 20 , further comprising passing at least one of the emission beams and the filtered emission beams through a dichroic mirror.
23. The method of claim 20 , further comprising passing at least one of the emission beams and the filtered emission beams through a Fresnel lens.
24. A method comprising:
generating excitation beams that provide a plurality of different excitation wavelength ranges;
directing the excitation beams through a multi-notch beam splitter and at, at least one reaction region retaining at least one respective sample comprising a plurality of different dyes, each dye having a different respective excitation wavelength range relative to the other dyes of the plurality, the plurality of different dyes being capable of emitting emission beams of different respective wavelength ranges;
causing the at least one respective sample to undergo a nucleic acid amplification reaction:
causing the at least one sample to emit emission beams of different respective wavelength ranges, wherein the different excitation wavelength ranges provided by the excitation beams correspond to the different respective excitation wavelength ranges of the plurality of different dyes;
passing the emission beams of different respective wavelength ranges through the multi-notch beam splitter to form filtered emission beams;
directing the filtered emission beans toward a detection region;
detecting the filtered emission beams emitted from the at least one respective sample;
generating a signal corresponding to the detected filtered emission beams;
adjusting exposure conditions of the detection region to maintain the data signal within a predetermined operating range; and
controlling the temperature of the at least one respective sample, during the detecting.
25. The method of claim 24 , further comprising reflecting the emission beams with a fold mirror.
26. The method of claim 24 , further comprising passing at least one of the emission beams and the filtered emission beams through a dichroic mirror.
27. The method of claim 24 , further comprising passing at least one of the emission beams and the filtered emission beams through a Fresnel lens.
28. The method of claim 24 , wherein the at least one reaction region comprises a plurality of reaction regions.