Optical system for a scanning fluorometer

• US 6,316,774 B1
• Filed: 03/23/1999
• Issued: 11/13/2001
• Est. Priority Date: 08/18/1998
• Status: Expired due to Term

• Current Assignee: Molecular Devices
• Original Assignee: Molecular Devices
• Inventors: Kaye, Roger, Ogle, David G., Giebeler, Robert
• Primary Examiner(s): Hannaher, Constantine
• Assistant Examiner(s): Gabor, Otilia
• Application Number:
  US09/274,753
• Time in Patent Office: 966 Days
• Field of Search: 250/458.1, 250/461.1, 250/459.1, 250/461.2
• US Class Current: 250/458.1
• CPC Class Codes: G01N 2021/6421 Measuring at two or more wavelengths
• CPC Class Codes: G01N 2021/6471 Special filters, filter wheel
• CPC Class Codes: G01N 21/6452 Individual samples arranged in a regular 2D-array, e.g. multiwell plates

Abstract

A method and apparatus for determining the fluorescence, luminescence, or absorption of a sample is provided. The sample may either be contained within a cuvette or within one or more sample wells within a multi-assay plate. A combination of a broadband source, a monochromator, and a series of optical filters are used to tune the excitation wavelength to a predetermined value within a relatively wide wavelength band. A similar optical configuration is used to tune the detection wavelength. An optical scanning head assembly is used that includes mirrored optics for coupling the excitation source to the sample and the emitted light to the detector. An elliptical focussing mirror is used to magnify and focus the light projected from an optical fiber coupled to the source subassembly onto the sample. A portion of the source light is reflected by a beamsplitter onto a reference detector used to monitor the output of the source. The light from the elliptical mirror passes through an aperture in a second elliptical mirror prior to impinging upon the sample. The light emitted by the sample within the sample well is reflected by the second elliptical mirror and imaged onto the entrance aperture of an optical fiber coupled to the detector subassembly. The optical axes of both mirrors are slightly offset from the sample well normal. The mirror offset minimizes the amount of light reflected from the meniscus of the sample or the bottom surface of the sample well that enters the detection subassembly.

Claims

1. A fluorometer for characterizing at least one sample within at least one sample well of a sample plate, the fluorometer comprising:

a source of excitation light;

an optical head assembly coupled to said excitation light source, said optical head assembly comprising;

an optical element for directing said excitation light toward said at least one sample well along a first optical path, wherein a first optical path axis corresponding to said first optical path is offset by a first angle away from a normal to a bottom surface of said at least one sample well;

a first reflector for reflecting fluorescence emitted by said at least one sample within said at least one sample well; and

an aperture within said first reflector, wherein said excitation light directed by said optical element passes through said aperture prior to impinging on said at least one sample within said at least one sample well; and

a detector coupled to said optical head assembly, wherein said reflected emitted fluorescence is detected by said detector.

2. The fluorometer of claim 1, wherein said optical element is a second reflector.

3. The fluorometer of claim 2, wherein said second reflector focuses said excitation light.

4. The fluorometer of claim 2, wherein a portion of said fluorescence emitted by said at least one sample follows a second optical path and is reflected by said first reflector, and wherein a second optical path axis corresponding to said second optical path is offset by a second angle away from said normal to said bottom surface of said at least one sample well.

5. The fluorometer of claim 4, wherein said first optical axis coincides with said second optical axis.

6. The fluorometer of claim 4, wherein said first angle is equivalent to said second angle.

7. The fluorometer of claim 6, wherein said first angle and said second angle are approximately 10 degrees.

8. The fluorometer of claim 2, wherein a portion of said excitation light reflected by said second reflector is reflected by a beamsplitter to a reference detector.

9. The fluorometer of claim 2, wherein said first reflector and said second reflector are elliptical mirrors.

10. The fluorometer of claim 1, wherein said optical element further comprises a lens.

11. The fluorometer of claim 1, herein said excitation light impinges on a top surface of said at least one sample at a first location, said first location offset by a first distance from a center line of said at least one sample well.

12. The fluorometer of claim 11, wherein a portion of said excitation light reflects off of said top surface of said at least one sample into said detector, and wherein said first distance offset minimizes an intensity corresponding to said reflections from said top surface into said detector.

13. The fluorometer of claim 1, wherein said excitation source is coupled to said optical head assembly with a first optical fiber and said detector is coupled to said optical head assembly with a second optical fiber.

14. The fluorometer of claim 1, wherein said detector is a photomultiplier tube detector.

15. The fluorometer of claim 1, wherein said excitation source is a xenon lamp.

16. The fluorometer of claim 1, wherein said optical head assembly is coupled to a first carriage assembly, said first carriage assembly scanning along a first scanning axis, and wherein a sample plate holding fixture is coupled to a second carriage assembly, said second carriage assembly scanning along a second scanning axis, wherein said first scanning axis is substantially orthogonal to said second scanning axis.

17. The fluorometer of claim 16, further comprising a processor coupled to said first carriage assembly and to said second carriage assembly, wherein said processor controls scanning said first carriage assembly along said first scanning axis and controls scanning said second carriage assembly along said second scanning axis.

18. The fluorometer of claim 1, said excitation light source further comprising:

a lamp;

a scanning monochromator; and

a plurality of optical filters.

19. The fluorometer of claim 18, wherein said lamp is a xenon lamp.

20. The fluorometer of claim 18, further comprising a processor coupled to said excitation light source, wherein said processor controls an output wavelength of said excitation light source by controlling said lamp, said scanning monochromator, and said plurality of optical filters.

21. The fluorometer of claim 1, said detector further comprising:

a scanning monochromator; and

a plurality of optical filters.

22. The fluorometer of claim 21, wherein said detector is a photomultiplier tube detector.

23. The fluorometer of claim 22, further comprising a processor coupled to said detector, wherein said processor controls a detection wavelength of said detector by controlling said scanning monochromator, said plurality of optical filters, and an output voltage of a high voltage source coupled to said photomultipler tube detector.

24. The fluorometer of claim 1, wherein said sample plate is a multi-assay plate.

25. The fluorometer of claim 24, further comprising:

a heater;

a temperature monitor,

an air circulation system; and

an enclosure substantially surrounding a sample plate holding fixture for use with said multi-assay plate, wherein a humidity proximate to said multi-assay plate is in excess of 90 percent.

26. The fluorometer of claim 25, wherein a temperature corresponding to each of said plurality of sample wells varies by less than 0.2°

C.

27. A fluorometer, comprising:

a source of excitation light;

an optical head assembly coupled to said excitation light source, said optical head assembly comprising;

a first elliptical mirror for reflecting said excitation light toward a sample well along a first optical path,-wherein a first optical path axis corresponding to said first optical path is offset by a first angle away from a normal to a bottom surface of said sample well;

a second elliptical mirror for reflecting fluorescence emitted by a sample within said sample well, wherein a portion of said fluorescence emitted by said at least one sample follows a second optical path and is reflected by said second elliptical mirror, wherein a second optical path axis corresponding to said second optical path is offset by a second angle away from said normal to said bottom surface; and

an aperture within said second elliptical mirror, wherein said excitation light reflected by said first elliptical mirror passes through said aperture prior to impinging on said sample well; and

a detector coupled to said optical head assembly, wherein said reflected emitted fluorescence is detected by said detector.

28. A fluorometer, comprising:

a source of excitation light;

an optical head assembly coupled to said excitation light source, said optical head assembly comprising;

a first mirror for reflecting said excitation light toward a sample well along a first optical path, wherein a first optical path axis corresponding to said first optical path is offset by a first angle away from a normal to a bottom surface of said sample well;

a second mirror for reflecting fluorescence emitted by a sample within said sample well, wherein a portion of said fluorescence emitted by said at least one sample follows a second optical path and is reflected by said second mirror, wherein a second optical path axis corresponding to said second optical path coincides with said first optical path axis; and

an aperture within said second mirror, wherein said excitation light reflected by said first mirror passes through said aperture prior to impinging on said sample well; and

a first carriage assembly coupled to said optical head assembly, said first carriage assembly scanning along a first scanning axis;

a second carriage assembly coupled to said sample well, said second carriage assembly scanning along a second scanning axis, wherein said first scanning axis is substantially orthogonal to said second scanning axis; and

a detector coupled to said optical head assembly, wherein said reflected emitted fluorescence is detected by said detector.

29. A fluorometer for characterizing at least one sample within at least one sample well of a sample plate, the fluorometer comprising:

a source of excitation light;

an optical head assembly coupled to said excitation light source, said optical head assembly comprising;

an optical element for directing said excitation light toward said at least one sample well, wherein said excitation light impinges on a top surface of said at least one sample at a first location, said first location offset by a first distance from a center line of said at least one sample well;

a first reflector for reflecting fluorescence emitted by said at least one sample within said at least one sample well; and

an aperture within said first reflector, wherein said excitation light directed by said optical element passes through said aperture prior to impinging on said at least one sample within said at least one sample well; and

a detector coupled to said optical head assembly, wherein said reflected emitted fluorescence is detected by said detector.

30. The fluorometer of claim 29, wherein a portion of said excitation light reflects off of said top surface of said at least one sample into said detector, and wherein said first distance offset minimizes an intensity corresponding to said reflections from said top surface into said detector.

31. A fluorometer for characterizing at least one sample within at least one sample well of a multi-assay sample plate, the fluorometer comprising:

a source of excitation light;

an optical head assembly coupled to said excitation light source, said optical head assembly comprising;

an optical element for directing said excitation light toward said at least one sample well;

a first reflector for reflecting fluorescence emitted by said at least one sample within said at least one sample well; and

an aperture within said first reflector, wherein said excitation light directed by said optical element passes through said aperture prior to impinging on said at least one sample within said at least one sample well;

a detector coupled to said optical head assembly, wherein said reflected emitted fluorescence is detected by said detector

a heater;

a temperature monitor;

an air circulation system; and

an enclosure substantially surrounding a sample plate holding fixture for use with said multi-assay plate, wherein a humidity proximate to said multi-assay sample plate is in excess of 90 percent.

32. The fluorometer of claim 31, wherein a temperature corresponding to each sample well of said multi-assay sample plate varies by less than 0.2°

C.