Pulsed-multiline excitation for color-blind fluorescence detection
View Patent ↗The present invention provides a technology called Pulse-Multiline Excitation or PME. This technology provides a novel approach to fluorescence detection with application for high-throughput identification of informative SNPs, which could lead to more accurate diagnosis of inherited disease, better prognosis of risk susceptibilities, or identification of sporadic mutations. The PME technology has two main advantages that significantly increase fluorescence sensitivity: (1) optimal excitation of all fluorophores in the genomic assay and (2) “color-blind” detection, which collects considerably more light than standard wavelength resolved detection. Successful implementation of the PME technology will have broad application for routine usage in clinical diagnostics, forensics, and general sequencing methodologies and will have the capability, flexibility, and portability of targeted sequence variation assays for a large majority of the population.
1. A pulse-multiline excitation apparatus for analyzing a sample containing one or more fluorescent species, comprising:
one or more lasers configured to emit two or more excitation lines, each excitation line having a different wavelength;
a timing circuit coupled to the one or more lasers and configured to generate the two or more excitation lines sequentially according to a timing program that pulses the one or more lasers on and off in a firing sequence to produce time-correlated fluorescence emission signals from the sample;
a non-dispersive detector positioned to collect the time-correlated fluorescence emission signals emanating from the sample; and
an analyzer coupled to the detector and configured to associate the time-correlated fluorescence emission signals with the timing program to identify constituents of the sample.
2. The apparatus of claim 1 , wherein the detector and the analyzer are integral.
3. The apparatus of claim 1 , wherein the two or more excitation lines intersect at the sample.
4. The apparatus of claim 1 , wherein the two or more excitation lines are configured so that the two or more excitation lines do not intersect in the sample.
5. The apparatus of claim 1 , wherein the two or more excitation lines are configured so that the two or more excitation lines are coaxial.
6. The apparatus of claim 1 , further comprising an assembly of one or more prisms in operative relation with the one or more lasers and configured to render radiation of the two or more excitation lines substantially colinear.
7. The apparatus of claim 1 , further comprising at least four excitation lines having four excitation wavelengths.
8. The apparatus of claim 7 , further comprising at least eight excitation lines having eight excitation wavelengths.
9. The apparatus of claim 8 , further comprising at least sixteen excitation lines having sixteen excitation wavelengths.
10. The apparatus of claim 1 , wherein said sample is comprised in at least one capillary.
11. The apparatus of claim 1 , wherein said sample is comprised in at least 4 capillaries.
12. The apparatus of claim 11 , wherein said sample is comprised in at least 8 capillaries.
13. The apparatus of claim 12 , wherein said sample is comprised in at least 16 capillaries.
14. The apparatus of claim 13 , wherein said sample is comprised in at least 48 capillaries.
15. The apparatus of claim 14 , wherein said sample is comprised in at least 96 capillaries.
16. The apparatus of claim 15 , wherein said sample is comprised in at least 384 capillaries.
17. The apparatus of claim 1 , further comprising a sheath flow cuvette.
18. The apparatus of claim 1 , wherein the timing program comprises a delay between the firing of each laser of between about 10 femtosecond and about 5 seconds.
19. The apparatus of claim 18 , wherein the timing program comprises a delay between the firing of each laser of between about 1 millisecond and about 100 milliseconds.
20. The apparatus of claim 18 , wherein the timing program comprises a delay between the firing of each laser of between about 50 ps and about 500 ps.
21. The apparatus of claim 1 , wherein at least one or more of the excitation lines is pulsed.
22. The apparatus of claim 21 , wherein said pulsed excitation line is controlled by transistor-transistor logic (TTL).
23. The apparatus of claim 21 , wherein said pulsed excitation line is controlled by mechanical or electronic means.
24. The apparatus of claim 22 , wherein said apparatus generates a sequence of discrete excitation lines that are time-correlated with the fluorescence emission signals from the sample.
25. The apparatus of claim 1 , wherein at least one of the lasers comprises a diode laser.
26. The apparatus of claim 1 , wherein at least one of the lasers comprises a semiconductor laser.
27. The apparatus of claim 1 , wherein at least one of the lasers comprises a gas laser.
28. The apparatus of claim 1 , wherein at least one of the lasers comprises a diode pumped solid state laser.
29. The apparatus of claim 28 , wherein at least one of the solid state lasers comprises a Neodymium laser.
30. The apparatus of claim 1 , further comprising a Raman shifter in operable relation with at least one laser beam.
31. The apparatus of claim 1 , wherein the excitation wavelength provided by each laser is optically matched to the absorption wavelength of each fluorophore.
32. The apparatus of claim 1 , wherein the detector comprises a charged couple device.
33. The apparatus of claim 1 , wherein the detector comprises a photomultiplier tube.
34. The apparatus of claim 1 , wherein the detector comprises a silicon avalanche photodiode.
35. The apparatus of claim 1 , wherein the detector comprises a silicon PIN detector.
36. The apparatus of claim 1 , wherein the footprint of said device is less than 4 ft×4 ft×2 ft.
37. The apparatus of claim 36 , wherein the footprint of said device is less than 1 ft×1 ft×2 ft.
38. The apparatus of claim 36 , wherein the footprint of said device is less than 1-in×3-in×6-in.