Apparatus and method for differentiating multiple fluorescence signals by excitation wavelength

• US 20040014202A1
• Filed: 05/19/2003
• Published: 01/22/2004
• Est. Priority Date: 11/29/2001
• Status: Active Grant

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Inventors: Oldham, Mark F., Young, Eugene F., Boege, Steven J., King, Howard G.
• Granted Patent: US 7,635,588 B2
• Time in Patent Office: Days
• US Class Current: 435/287.2
• 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 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 21/6452 Individual samples arranged in a regular 2D-array, e.g. multiwell plates
• CPC Class Codes: G01N 2201/0612 Laser diodes
• CPC Class Codes: G01N 2201/062 LED's
• CPC Class Codes: G01N 2201/0627 Use of several LED's for spectral resolution
• CPC Class Codes: Y10S 435/808 Optical sensing apparatus
• CPC Class Codes: Y10S 435/809 Incubators or racks or holders for culture plates or containers

Abstract

An apparatus and method are provided for differentiating multiple detectable signals by excitation wavelength. The apparatus can include a light source that can emit respective excitation light wavelengths or wavelength ranges towards a sample in a sample retaining region, for example, in a well. The sample can contain two or more detectable markers, for example, fluorescent dyes, each of which can be capable of generating increased detectable emissions when excited in the presence of a target component. The detectable markers can have excitation wavelength ranges and/or emission wavelength ranges that overlap with the ranges of the other detectable markers. A detector can be arranged for detecting an emission wavelength or wavelength range emitted from a first marker within the overlapping wavelength range of at least one of the other markers.

Claims

1. An apparatus comprising:

at least one reaction region;

an excitation light source capable of providing a plurality of separate excitation beams, each excitation beam including a wavelength range that is not provided by the other excitation beams of the plurality of separate excitation beams, the excitation light source being capable of directing each excitation beam along a beam path toward the at least one reaction region;

a control unit capable of individually activating each of the plurality of excitation beams independently from activating the other ones of the plurality of excitation beams; and

a detector capable of detecting emission beams emitted from the at least one reaction region.

2. The apparatus of claim 1, wherein the excitation light source comprises a plurality of excitation wavelength sources each capable of forming a respective excitation beam having a wavelength range, wherein each excitation beam wavelength range is exclusive of any other excitation beam wavelength range.

3. The apparatus of claim 1, wherein the detector is capable of comparing the excitation beam with a detected signal to determine the presence or absence of a component.

4. The apparatus of claim 1, wherein the control unit is capable of storing in memory each of the respective excitation beams and respective detection signals resulting from illuminating a sample in the at least one reaction region with the respective excitation beam.

5. The apparatus of claim 1, wherein the control unit is capable of comparing a detection signal with a preset value and, based on that comparison, determining a presence or an absence of a component in a sample.

6. The apparatus of claim 1, wherein the excitation light source comprises at least one of a light emitting diode, an organic light emitting diode, a semiconductor laser, a solid state laser, and a micro-wire laser.

7. An instrument comprising:

a sample well;

a sample arranged in the sample well, the sample comprising a plurality of components and a plurality of detectable markers, each of the plurality of detectable markers being capable of generating increased emission due to a presence of a target component in the sample, each of the plurality of detectable markers having a respective excitation wavelength range and a respective emission wavelength range;

a light source capable of emitting a plurality of excitation wavelength ranges toward the sample to respectively excite each of the detectable markers, each of the plurality of excitation wavelength ranges corresponding to a respective excitation wavelength range of a respective one of the plurality of detectable markers; and

a detector capable of detecting an emission wavelength range emitted from each of the plurality of detectable markers.

8. The instrument of claim 7, wherein the light source is capable of sequentially emitting a plurality of excitation wavelength ranges toward the sample and the detector is capable of sequentially detecting whether a corresponding emission wavelength range is emitted from the sample.

9. The instrument of claim 7, further comprising a lens arranged to receive the plurality of excitation wavelengths emitted from the light source and emission wavelengths emitted from the sample.

10. The instrument of claim 9, wherein the lens is a focusing lens.

11. The instrument of claim 9, wherein the lens is a Fresnel lens or a prism.

12. The instrument of claim 9, wherein the light source comprises, a light emitting diode, a organic light emitting diode, a laser, a solid state laser, a micro-wire laser, or a combination thereof.

13. The instrument of claim 7, wherein the light source comprises an array of light generating sources.

14. The instrument of claim 13, wherein the array of light generating sources includes an array of light emitting diodes.

15. The instrument of claim 13, wherein the array of light generating sources includes an organic light emitting diode layout.

16. The instrument of claim 13, wherein the array of light generating sources comprises one or more light emitting diodes.

17. The instrument of claim 13, wherein the array of light generating sources includes one or more lasers.

18. The instrument of claim 13, wherein the array of light generating sources includes a solid state laser.

19. The instrument of claim 7, further comprising a lightpipe arranged between the light source and the sample.

20. The instrument of claim 7, further comprising a lightpipe arranged between the detector and the sample.

21. The instrument of claim 7, further comprising an excitation wavelength-excluding device arranged between the sample and the detector.

22. The instrument of claim 21, wherein the excitation wavelength-excluding device comprises a longpass filter, a multiple notch filter, a bandpass filter, or a combination thereof.

23. The instrument of claim 7, further comprising an excitation wavelength-excluding device arranged between the light source and the sample.

24. The instrument of claim 23, wherein the excitation wavelength-excluding device comprises a longpass filter, a multiple notch filter, a bandpass filter, or a combination thereof.

25. The instrument of claim 7, further comprising a mirror arranged between both the light source and the sample, and the detector and the sample.

26. A method comprising:

providing a sample that includes at least two detectable markers, wherein each detectable marker emits a respective detectable emission beam when illuminated by an excitation beam that includes light of a respective wavelength,

illuminating the sample with a first excitation beam that includes a first wavelength;

detecting emission beams emitted from the sample in response to illuminating the sample with the first excitation beam;

then illuminating the sample with a second excitation beam that includes a second wavelength that differs from the first wavelength; and

detecting emission beams emitted from the sample in response to illuminating the sample with the second excitation beam.

27. The method of claim 26, wherein the at least two detectable markers comprise fluorescent dyes.

28. The method of claim 26, wherein the sample includes at least four detectable markers and the method comprises illuminating with at least four different excitation beams.

29. A method of differentiating multiple fluorescent signals by excitation wavelength, comprising:

(a) providing a sample in a sample well, the sample comprising a plurality of components;

(b) adding a plurality of fluorescent dyes to the sample, each of the plurality of fluorescent dyes being capable of generating increased emission due to a presence of a respective target component in the sample, each of the plurality of fluorescent dyes possessing a respective excitation wavelength range and a respective emission wavelength range;

(c) directing an excitation wavelength range from a light source toward the sample to cause a first one of the fluorescent dyes to emit a detectable emission;

(d) detecting emission wavelengths emitted from the sample;

(e) determining whether a respective target component is present in the sample by analyzing the detected emission wavelengths; and

repeating (c) to (e) for each of the plurality of fluorescent dyes in the sample.

30. The method of claim 29, further comprising passing at least one of the excitation wavelength and the emission wavelength through a lens.

31. The method of claim 29, further comprising passing the emission wavelength through a filter before detecting.

32. The method of claim 30, wherein the method comprises includes subjecting the sample to nucleic acid sequence amplification.

33. The method of claim 30, wherein the nucleic acid sequence amplification comprises polymerase chain reaction.