Quantified fluorescence microscopy

• US 6,472,671 B1
• Filed: 02/09/2000
• Issued: 10/29/2002
• Est. Priority Date: 02/09/2000
• Status: Expired due to Term

• Current Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Original Assignee: Genetic MicroSystems, Inc. (Thermo Fisher Scientific Incorporated)
• Inventors: Montagu, Jean I.
• Primary Examiner(s): Hannaher, Constantine
• Assistant Examiner(s): Israel, Andrew
• Application Number:
  US09/500,626
• Time in Patent Office: 993 Days
• Field of Search: 250/458.1, 378/44, 356/213
• 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

A calibration tool for fluorescent microscopy comprises a support on which is carried a solid surface layer comprised of effective fluorophores, and a thin mask of non-fluorescent material defining reference feature openings of limited dimensions exposes portions of the fluorophore-comprising surface layer.

Claims

1. A calibration tool for fluorescent microscopy comprising a support on which is carried a solid surface layer comprised of effective fluorophores, and a thin mask of non-fluorescent material, wherein the fluorophore-comprising surface layer and the mask are deposited substantially in contact with each other defining reference feature openings of limited dimensions exposing portions of the fluorophore-comprising surface layer.

2. The calibration tool of claim 1 in which the support is flat and rigid.

3. The calibration tool of claim 1 in which the surface layer is opaque.

4. The calibration tool of claim 1 in which the mask comprises an etched thin metal film which is intimately engaged face-to-face with the solid surface layer comprised of effective fluorophores.

5. The calibration tool of claims 1, 2 or 3 in which the tool has a transparent non-fluorescing transparent layer overlying the mask and the solid surface layer is arranged so that the fluorophores are excited by radiation passing through the transparent, non-fluorescing layer.

6. The calibration tool of claim 1, 2 or 3 in which the fluorophore-containing surface layer is directly exposed through openings in the mask to exciting radiation.

7. The calibration tool of claim 1 in which the solid surface layer is a broad band fluorescence emitter.

8. The calibration tool of claim 7 in which either the thin mask or the solid surface layer comprised of fluorophores is directly deposited upon the other.

9. The calibration tool of claim 1, 2, or 3 combined with a confocal microscope having a restricted depth of field and a response, and the solid surface layer comprised of fluorophore has an effective depth of less than the depth of field of the confocal microscope.

10. The calibration tool of claim 9 in which the surface layer has an effective fluorescent emittance that can produce a full scale response of the microscope.

11. The calibration tool of claim 1, 2, or 3 in which the solid surface layer is comprised of fluorescent polyimide.

12. The calibration tool of claim 1, 2, or 3 in which the solid surface layer is comprised of a thin layer of fluorescent glass or glass-like material.

13. The calibration tool of claim 12 in which the solid surface layer is a congealed sol-gel coating layer, with fluorophores disposed in the coating layer.

14. The alignment tool of claim 1, 2 or 3 in which the fluorophores comprise a dye, such as Cy3, Cy5 or fluorescene, which is fluorescent at a desired wavelength.

15. The calibration tool of claim 1 including an adhesion promoter facilitating contact between a surface of the support and the solid surface layer including the fluorophores.

20. The calibration tool of claim 1, 2, or 3 in which the surface layer is comprised of polyimide which fluoresces in response to excitation over a wide band of wavelengths.

16. A process for producing the a calibration tool comprising:

providing a support,

creating in contact a solid surface layer, comprising effective fluorophores emitting surface fluorescence, and a thin mask of non-fluorescent material, wherein the mask is patterned to define reference feature openings of limited dimensions exposing portions of the fluorophore-comprising surface layer, the reference feature openings defining alignment features for fluorescent microscopy.

17. The process of claim 16 in which the support is flat and rigid.

18. The process of claim 16 in which a uniform metal film is deposited on the face of the surface layer to form the thin mask and subsequently etched to produce the pattern.

19. The process of claim 16 in which the surface layer containing effective fluorophores is deposited over the thin mask.

21. A method of quantified fluorescence microscopy comprising

providing a fluorescence detecting microscope,

providing a calibration tool including support arranged to carry a solid surface layer comprising effective fluorophores, and a thin mask of non-fluorescent material, wherein the fluorophore-comprising surface layer and the mask are deposited substantially in contact with each other defining reference feature openings of limited dimensions exposing portions of the fluorophore-comprising surface layer,

detecting surface fluorescence emitted from the fluorophore-comprising surface layer,

employing said calibration tool to calibrate the microscope, and

performing fluorescence microscopy of specimens employing the calibrated microscope.

22. The method of claim 21 in which the microscope is an on-axis flying objective microscope.

23. The method of claims 21 or 22 in which the microscope has a micro-lens objective carried upon an oscillating rotary arm.

24. 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 the surface layer, wherein said thin opaque mask is fabricated onto the support being optically transparent, and wherein solid surface layer including the fluorescent material is deposited on the thin opaque mask.

25. The calibration tool of claim 24, wherein the thin opaque mask is fabricated onto said support using an adhesion promoter.

26. The calibration tool of claim 24, wherein the support is made in form of a chip.