METHODS AND SYSTEMS FOR ANALYSIS OF FLUORESCENT REACTIONS WITH MODULATED EXCITATION

• US 20090181396A1
• Filed: 01/09/2009
• Published: 07/16/2009
• Est. Priority Date: 01/10/2008
• Status: Active Grant

• Current Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Original Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Inventors: Turner, Stephen, Lyle, John, Lundquist, Paul, Luong, Khai, Dalal, Ravi
• Granted Patent: US 8,802,424 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12M 1/34 Measuring or testing with condition measuring or sensing means, e.g. colony counters
• CPC Class Codes: G01N 2021/6417 Spectrofluorimetric devices
• CPC Class Codes: G01N 2021/6419 Excitation at two or more wavelengths
• CPC Class Codes: G01N 2021/6421 Measuring at two or more wavelengths
• CPC Class Codes: G01N 2021/6441 with two or more labels
• CPC Class Codes: G01N 2021/7786 Fluorescence
• CPC Class Codes: G01N 21/6428 Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" in vivo A61B5/00; immunoassay G01N33/53
• CPC Class Codes: G01N 21/645 Specially adapted constructive features of fluorimeters
• CPC Class Codes: Y10T 436/2525 Stabilizing or preserving

Abstract

Methods, systems and their components for monitoring fluorescent signals and particularly transient fluorescent signals from reaction mixtures of interest, which methods and systems employ modulated excitation light sources to reduce impacts of excessive illumination on the reaction components or the data obtained therefrom.

Claims

1. A system for analysis of fluorescent materials, comprising:

a reaction region for containing a fluorescent reaction mixture;

an excitation light source;

a detector; and

an optical train for directing excitation light from the excitation light source to the reaction region and collecting fluorescent signals from the reaction region and directing the fluorescent signals to the detector, wherein at least one of the optical train and the excitation light source is configured to provide a modulated beam of excitation light to the reaction region.

2. The system of claim 1, comprising at least first and second excitation light sources, wherein the optical train is configured to provide first and second modulated beams of excitation light to the reaction region.

3. The system of claim 2, wherein the optical train is configured to provide the first and second modulated beams to ihe reaction region at different times.

4. The system of claim 1, wherein the optical train comprises a modulated excitation light source.

5. The system of claim 1, wherein the optical train comprises a beam modulating element that modulates an excitation beam from the excitation light source.

6. The system of claim 5, wherein the beam modulation component comprises a mechanical beam chopper.

7. The system of claim 5, wherein the beam modulation component comprises an acousto-optical modulator.

8. The system of claim 5, wherein the beam modulation component comprises a digital light processor.

9. The system of claim 1, wherein the optical train is configured to modulate the beam at a frequency of between about 20 Hz and about 1000 Hz.

10. The system of claim 9, wherein the optical train is configured to modulate the excitation beam at a frequency of greater than 50 Hz.

11. The system of claim 9, wherein the optical train is configured to modulate the excitation beam at a frequency of between about 100 Hz and about 500 Hz.

12. The system of claim 1, wherein the detector has a sampling rate that is greater than 1×

a frequency of modulation of the excitation beam.

13. The system of claim 1, wherein the detector has a sampling rate that is greater than 2×

a frequency of modulation of the excitation beam.

14. The system of claim 1, wherein the detector has a sampling rate that is greater than 4×

a frequency of modulation of the excitation beam.

15. The system of claim 1, wherein the detector has a sampling rate that is greater than 10×

a frequency of modulation of the excitation beam.

16. The system of claim 1, wherein the reaction region comprises an enzyme reagent and at least a first fluorescent reactant for the enzyme.

17. The system of claim 16, wherein the enzyme reagent comprises a nucleic acid polymerizing enzyme and the fluorescent reactant comprises at least a first fluorescent nucleotide analog.

18. The system of claim 17, wherein the fluorescent reactant comprises at least four different types of fluorescent nucleotide analogs.

19. The system of claim 17, wherein the polymerizing enzyme is immobilized upon a substrate in a single molecule configuration.

20. A system for detecting fluorescent reactants, comprising:

a reaction region containing at least first and second fluorescent reactants, the first and second fluorescent reactants being excited by first and second excitation beams having different wavelength spectra, respectively, and wherein simultaneous excitation of the first and second fluorescent reactants further excites at least one of the first and second fluorescent reactants to a triplet state;

a source of first and second excitation beams;

a detector; and

an optical train for directing the first and second excitation beams to the reaction region, modulating at least one of the first and second excitation beams, and directing fluorescent signals emitted by the first and second fluorescent reactants to the detector.

21. A method of detecting fluorescent reactants, comprising:

providing a reaction mixture containing at least first and second fluorescent reactants, the first and second fluorescent reactants being excited by first and second excitation beams having different wavelength spectra, respectively, and wherein simultaneous excitation of the first and second fluorescent reactants further excites at least one of the first and second fluorescent reactants to a triplet state;

directing the first and second excitation beams at the reaction region;

modulating at least one of the first and second excitation beams directed at the reaction region; and

detecting fluorescent signals emitted from the first and second fluorescent reactants.

22. A method for detecting fluorescent reactants from a reaction mixture that comprises at least first and second fluorescent, reactants that are excited by excitation beams of different wavelengths, where simultaneous excitation of the first and second fluorescent reactants excites at least one of the first and second fluorescent reactants to a triplet state, comprising:

modulating at least one of a first and second excitation beam directed at the reaction mixture.

23. A system for analysis of fluorescent materials, comprising:

a reaction region containing a reaction mixture that comprises at least first and second fluorescent reactants, the first and second fluorescent reactants having at least first and second distinct excitation spectra;

first and second excitation light sources configured to provide excitation light at the first and second excitation spectra, respectively; and

an optical train for directing excitation light from the first and second excitation light sources to the reaction region, and for modulating excitation beams from at least one of the first and second excitation light sources at a frequency of at least 50 Hz.

24. The system of claim 23, wherein the optical train modulates the excitation beams from the at least first and second excitation light sources, such that an excitation beam incident upon the substrate is substantially from only one of the at least first and second excitation light sources at a given time.

25. A method for analyzing fluorescent materials, comprising:

providing a reaction mixture comprising one or more of fluorescent reactants or products;

directing a modulated excitation illumination beam at the reaction mixture; and

detecting fluorescent signals produced by the reaction mixture in response to the modulated excitation beam.

26. The method of claim 25, comprising directing at least first and second modulated excitation illumination beams at the reaction mixture.

27. The method of claim 26, further comprising directing each of the first and second modulated excitation illumination beams at the reaction mixture at different times.

28. The method of claim 25, comprising directing at least first second and third modulated excitation beams at the reaction region.

29. The method of claim 27, further comprising identifying a type of fluorescent signal produced by the reaction mixture based, at least in part, upon which of the first or second excitation beam was directed at the reaction mixture at a time that the fluorescent signal was produced.