Quantified fluorescence microscopy

• US 6,984,828 B2
• Filed: 08/07/2002
• Issued: 01/10/2006
• Est. Priority Date: 02/09/2000
• Status: Expired due to Term

• Current Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Original Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Inventors: Montagu, Jean I.
• Primary Examiner(s): Gabor, Otilia
• Assistant Examiner(s): Sung, Christine
• Application Number:
  US10/214,221
• Publication Number:
  US 20030015668A1
• Time in Patent Office: 1,252 Days
• Field of Search: 250/458.1, 378/44, 356/213, 600/477
• US Class Current: 250/458.1
• CPC Class Codes: B01J 2219/00364 Pipettes
• CPC Class Codes: B01J 2219/00387 Applications using probes
• CPC Class Codes: B01J 2219/00527 Sheets
• CPC Class Codes: B01J 2219/00585 Parallel processes
• CPC Class Codes: B01J 2219/0059 Sequential processes
• CPC Class Codes: B01J 2219/00596 Solid-phase processes
• CPC Class Codes: B01J 2219/00605 the compounds being directly bound or immobilised to solid supports
• CPC Class Codes: B01J 2219/00612 the surface being inorganic
• CPC Class Codes: B01J 2219/00659 Two-dimensional arrays
• CPC Class Codes: B01J 2219/00677 Ex-situ synthesis followed by deposition on the substrate
• CPC Class Codes: B01J 2219/00691 using robots
• CPC Class Codes: B01L 3/0244 using pins
• CPC Class Codes: B01L 3/0251 Pin and ring type or pin in tube type dispenser
• CPC Class Codes: B82Y 30/00 Nanotechnology for materials or surface science, e.g. nanocomposites
• CPC Class Codes: C40B 60/14 for creating libraries
• CPC Class Codes: G01N 2035/1034 Transferring microquantities of liquid
• CPC Class Codes: G01N 2035/1037 Using surface tension, e.g. pins or wires
• CPC Class Codes: G01N 21/274 Calibration, base line adjustment, drift correction
• CPC Class Codes: G01N 21/278 Constitution of standards
• CPC Class Codes: G01N 21/64 Fluorescence; Phosphorescence
• CPC Class Codes: G01N 21/6452 : Individual samples arranged in a regular 2D-array, e.g. multiwell plates
• CPC Class Codes: G01N 21/6458 : Fluorescence microscopy fluorescence microscopes per se G02B21/0076 and G02B21/16
• CPC Class Codes: G01N 35/1065 : Multiple transfer devices
• CPC Class Codes: G02B 21/16 : adapted for ultraviolet illumination ; Fluorescence microscopes G02B21/0076 takes precedence
• CPC Class Codes: G02B 21/34 : Microscope slides, e.g. mounting specimens on microscope slides
• CPC Class Codes: Y10T 428/24355 : Continuous and nonuniform or irregular surface on layer or component [e.g., roofing, etc.]

Abstract

The present invention relates to calibration apparatuses, methods or tools used in microscopy. A calibration tool for fluorescent microscopy includes a support, a solid surface layer including a fluorescent material, and a thin opaque mask of non-fluorescent material defining reference feature openings having selected dimensions exposing portions of the surface layer. A first type of the calibration tool may include an adhesion promoter facilitating contact between the surface of the support and the solid surface layer including the fluorescent material, which is in contact with the thin opaque mask. A second type of the calibration tool may include the thin opaque mask fabricated (with or without an adhesion promoter) onto the support, and the solid surface layer including the fluorescent material located on the thin opaque mask.

Claims

1. A calibration tool for fluorescent microscopy comprising a support, a solid surface layer including a fluorescent material, and a thin opaque mask of non-fluorescent material defining reference feature openings having selected dimensions exposing portions of said surface layer.

2. The calibration tool of claim 1 including an adhesion promoter facilitating contact between a surface of said support and said solid surface layer including said fluorescent material, said solid surface layer being in contact with said thin opaque mask.

3. The calibration tool of claim 1, wherein said thin opaque mask is fabricated onto said support, and wherein said solid surface layer includes the fluorescent material is deposited on said thin opaque mask.

4. The calibration tool of claim 3, wherein said thin opaque mask is fabricated onto said support using an adhesion promoter.

5. The calibration tool of claim 2 or 3 wherein said support is flat and rigid.

6. The calibration tool of claim 2 or 3 wherein said support includes fused quartz.

7. The calibration tool of claim 2 or 3 wherein said mask comprises a thin metal film.

8. The calibration tool of claim 3 wherein said support forms an optical window of a cassette.

9. The calibration tool of claim 3 wherein the fluorescent material includes fluorophores that are excited by optical radiation passing through said support.

10. The calibration tool of claim 3 wherein the fluorescent material includes fluorophores that are excited by optical radiation passing through said support and through said openings in said mask.

11. The calibration tool of claim 3 wherein the fluorescent material includes fluorophores that are excited by optical radiation passing through said support and through said openings in said mask and said support absorbs excited fluorescent radiation.

12. The calibration tool of claim 2 or 3 wherein said solid surface layer including said fluorescent material has a thickness in the range of about 2 μ

m to about 250 μ

m.

13. The calibration tool of claim 1, 2 or 3 wherein said solid surface layer is polyimide.

14. The calibration tool of claim 2 or 3 wherein said thin opaque mask has a thickness in the range of about 10 nm to about 10 μ

m.

15. The calibration tool of claim 14 wherein said thin opaque mask has a thickness in the range of about 10 nm to about 100 nm.

16. The calibration tool of claim 1 wherein said surface layer is opaque.

17. The calibration tool of claim 1 wherein said solid surface layer provides a broadband fluorescence emitter.

18. The calibration tool of claim 1 wherein said solid surface layer provides a fluorescence emitter active at at least two wavelengths and having emission characteristics similar to Cy3 and Cy5 fluorescent dyes.

19. The calibration tool of claim 1 wherein said solid surface layer provides a fluorescence emitter having effective fluorescent emittance that can produce a full scale response for microscope calibration.

20. A process for producing a calibration tool for fluorescent microscopy comprising:

providing a support;

providing a solid surface layer including a fluorescent material; and

fabricating a thin opaque mask of non-fluorescent material defining reference feature openings having selected dimensions exposing portions of said surface layer.

21. The process of claim 20 wherein said providing a solid surface layer includes depositing onto said support said solid surface layer including said fluorescent material.

22. The process of claim 21 including depositing an adhesion promoting layer onto said support for forming said solid surface layer including said fluorescent material.

23. The process of claim 21 wherein said depositing includes delivering vapor forming said solid surface layer including said fluorescent material.

24. The process of claim 23 wherein said delivering vapor includes evaporating material, onto said support, forming said solid surface layer including said fluorescent material.

25. The process of claim 23 wherein said delivering vapor includes sputtering material, onto said support, forming said solid surface layer including said fluorescent material.

26. The process of claim 21 wherein said depositing includes spin coating to form said solid surface layer including said fluorescent material.

27. The process of claim 20 wherein said fabricating said thin opaque mask includes depositing onto said support said non-fluorescent material and patterning said non-fluorescent material to form said reference feature openings.

28. The process of claim 27 wherein said providing a solid surface layer includes depositing said solid surface layer including said fluorescent material onto said thin opaque mask.

29. The process of claim 20, 21 or 27 wherein said support is fused quartz.

30. The process of claim 20, 21 or 27 wherein said thin opaque mask includes metal.

31. The process of claim 27 including using said support as an optical window for a cassette used in examination of biological material.

32. The process of claim 20, 21 or 27 wherein said solid surface layer provides a broadband fluorescence emitter.

33. The process of claim 20, 21 or 27 wherein said solid surface layer provides a fluorescence emitter active at at least two wavelengths and having emission characteristics similar to Cy3 and Cy5 fluorescent dyes.

34. The process of claim 20, 21 or 27 wherein said solid surface layer provides a fluorescence emitter having effective fluorescent emittance that can produce a full scale response for microscope calibration.

35. The process of claim 20, 21 or 27 wherein said solid surface layer including said fluorescent material has a thickness in the range of about 2 μ

m to about 250 μ

m.

36. The process of claim 20, 21 or 27 wherein said solid surface layer is polyimide.

37. The process of claim 20, 21 or 27 wherein said thin opaque mask has a thickness in the range of about 10 nm to about 10 μ

m.

38. The process of claim 37 wherein said thin opaque mask has a thickness in the range of about 10 nm to about 100 nm.

39. A method of calibrating a microscope comprising:

providing a scanning microscope,

providing a calibration tool comprising a support, a solid surface layer including a fluorescent material, and a thin opaque mask of non-fluorescent material defining reference feature openings having selected dimensions exposing portions of said surface layer,

bringing in focus an excitation beam emitted from an objective of said microscope,

examining at least some of said reference feature openings, and

calibrating detection intensity of said microscope.

40. The method of claim 39 wherein said bringing in focus includes adjusting a position of a sample table of scanning microscope where said calibration tool is located.

41. The method of claim 39 wherein said microscope is an on-axis flying objective microscope.

42. The method of claims 41 wherein said microscope has a micro-lens objective carried upon an oscillating rotary arm.