Method for fluorescence microscopy, and fluorescence microscope

• US 6,806,953 B2
• Filed: 10/09/2002
• Issued: 10/19/2004
• Est. Priority Date: 10/12/2001
• Status: Active Grant

• Current Assignee: Leica Microsystems Gmbh (Danaher Corporation)
• Original Assignee: Leica Microsystems Gmbh (Danaher Corporation)
• Inventors: Hoffmann, Juergen, Engelhardt, Johann
• Primary Examiner(s): Evans, F. L.
• Assistant Examiner(s): GEISEL, KARA E
• Application Number:
  US10/268,159
• Publication Number:
  US 20030071226A1
• Time in Patent Office: 741 Days
• Field of Search: 356/317-318, 356/417, 250/458--46
• US Class Current: 356/317
• CPC Class Codes: G01N 21/6458 Fluorescence microscopy fluorescence microscopes per se G02B21/0076 and G02B21/16

Abstract

The invention discloses a fluorescence microscope comprising a light source that emits excitation light for illumination of a specimen, means for defining a two-dimensional search region for the excitation and detection wavelengths, means for selecting a subregion from the search region, at least one detector that detects detected light proceeding from the specimen, and a display for displaying an image of at least a portion of the specimen. Furthermore the invention discloses a method for fluorescence microscopy.

Claims

1. A method for fluorescence microscopy, wherein a specimen is illuminated with excitation light, wherein detected light proceeding from the specimen is detected, and wherein an image of at least a portion of the specimen is generated, comprising the steps of:

defining a two-dimensional search region for excitation and detection wavelengths;

determining, from the image of the specimen, quality features for subregions of the search region, the quality features being at least one of a brightness, a contrast, a resolution, a sharpness and a lifetime of the image;

selecting a subregion on the basis of the quality features that have been determined;

illuminating the specimen with excitation light of the excitation wavelengths of the selected subregion; and

detecting the detected light of the detection wavelengths of the selected subregion.

2. The method as defined in claim 1, further comprising the step of:

storing parameters characterizing the subregion.

3. The method as defined in claim 1, further comprising the steps of:

acquiring an overview image; and

selecting an image region from the overview image.

4. The method as defined in claim 1, wherein the search region is varied during the determination of quality features.

5. A fluorescence microscope comprising:

a light source that emits excitation light for illumination of a specimen,

means for defining a two-dimensional search region for the excitation and detection wavelengths,

means for selecting a subregion from the search region based on quality features determined from the image of the specimen, the quality features being at least one of a brightness, a contrast, a resolution, a sharpness and a lifetime of the image,

at least one detector that detects detected light proceeding from the specimen, and

a display for displaying an image of at least a portion of the specimen.

6. The fluorescence microscope as defined in claim 5, further comprising means for determining the quality features from the image of the specimen.

7. The fluorescence microscope as defined in claim 5, further comprising a display apparatus for displaying the quality features.

8. The fluorescence microscope as defined in claim 5, wherein the means for selecting a subregion includes an apparatus for automatically selecting suitable subregions.

9. The fluorescence microscope as defined in claim 8, wherein the apparatus for automatic selection of the subregion contains a computer that pursues a definable search strategy on the basis of a definable algorithm.

10. The fluorescence microscope as defined in claim 5, wherein the excitation light comprises the excitation wavelengths of the selected subregion.

11. The fluorescence microscope as defined in claim 10, wherein the excitation light comprises a wavelength band.

12. The fluorescence microscope as defined in claim 5, wherein the light source contains photonic band gap material.

13. The fluorescence microscope as defined in claim 5, wherein the light source contains a microstructured light-guiding fiber.

14. The fluorescence microscope as defined in claim 5, wherein the light source contains a selection means for selecting the excitation wavelengths.

15. The fluorescence microscope as defined in claim 14, wherein the selection means consists essentially of an acoustooptical element or an interference filter or a prism or a liquid-crystal filter or a polarization filter or a Fabry-Perot filter or a Sagnac filter or a color beam splitter.

16. The fluorescence microscope as defined in claim 5, further comprising a memory, in which data regarding the subregion and the quality features can be stored.

17. The fluorescence microscope as defined in claim 5, wherein the detected light comprises detection wavelengths of the subregion.

18. The fluorescence microscope as defined in claim 17, wherein the detected light comprises at least one wavelength band.

19. The fluorescence microscope as defined in claim 5, wherein the detector is a multiband detector.

20. The fluorescence microscope as defined in claim 5, wherein the detector contains a means for spatial splitting of the detected light.

21. The fluorescence microscope as defined in claim 5, wherein the detector contains a selection means for selecting the detection wavelengths.

22. The fluorescence microscope as defined in claim 21, wherein the selection means consists essentially of an acoustooptical element or an interference filter or a prism or a liquid-crystal filter or a polarization filter or a Fabry-Perot filter or a Sagnac filter or a color beam splitter.

23. The fluorescence microscope as defined in claim 5, wherein the fluorescence microscope is a confocal scanning microscope.