Method and device for focussing a microscope automatically

Patent metadata

Field	Value
Publication number	US-9696686-B2
Application number	US-201213672247-A
Country	US
Kind code	B2
Filing date	Nov 8, 2012
Priority date	Nov 9, 2011
Publication date	Jul 4, 2017
Grant date	Jul 4, 2017

Abstract

A method for automatic focusing of a microscope with a microscope objective on a selected area of a specimen, in which a digital hologram of the selected area of the specimen is generated in an off-axis mode and a microscope with which the method is implemented. The digital hologram is used to determine, on the optical axis of the microscope objective, a focus position to be set in which the selected area of the specimen is optimally in focus. Subsequently, a control system is used to set the microscope to the focus position determined and thus is focused on the area selected.

First claim

What is claimed is:

1. A method for automatic focusing of a microscope with a microscope objective on a selected area of a specimen, comprising:
producing a digital hologram of the selected area of the specimen in an off-axis mode prior to or during microscopic imaging, wherein the microscopic imaging is accomplished by an imaging method other than digital holography;
determining a focus position to be set on an optical axis of the microscope objective by analysis of the digital hologram prior to or during the microscopic imaging;
the focus position being, a position in which the selected area of the specimen is optimally in focus, and
setting a position, by application of a control system, of a stage, a position of an objective lens of positions of both the stage and the objective lens of the microscope to the focus position that is determined by the analysis of the digital hologram, and, thereby, focusing the microscope on the area selected.

2. The method as claimed in claim 1 , further comprising;
performing the determination of the focus position and subsequent focusing on several selected areas of the specimen sequentially;
recording a partial image of each area at the focus position determined and set; and
assembling the partial images to form a total image.

3. The method as claimed in claim 1 , further comprising assembling the partial images to form an image stack along the optical axis, an image in a plane normal to the optical axis or both.

4. The method as claimed in claim 1 , further comprising:
for generating the digital hologram, splitting focusing light of a coherent focusing light source with a focusing wavelength into an object beam and a reference beam;
directing the object beam onto the specimen and from the specimen onto a focusing sensor;
directing the reference beam onto the focusing sensor immediately;
detecting intensities of the reference beam and the object beam separately;
detecting an intensity of the superimposition of reference beam and object beam on the focusing sensor, and
in determining the best possible focus position, analyzing the amplitude of the intensity, integrated across the plane of the focusing sensor as a function of the position on the optical axis, with regard to the presence of extreme and as a function of the focus position to be set on the optical axis, and
in a case that the specimen is an amplitude object, selecting a position in which the integrated amplitude is a minimum, and
in a case that the specimen is a phase object, selecting a position in which the integrated amplitude is a maximum.

5. The method as claimed in claim 4 , further comprising directing the object beam onto the specimen and into a detection beam path of the microscope and, uncoupling the object beam that has passed through the microscope objective from the detection beam path and directing the object beam onto the focusing sensor.

6. The method as claimed in claim 5 , further comprising coupling the object beam into an illuminating beam path of the microscope, via which the specimen is illuminated with illuminating light of the illuminating wavelength or of the range of illuminating wavelengths, before the said object beam is directed onto the specimen.

7. The method as claimed in claim 1 , further comprising selecting a focusing wavelength and at least one illuminating wavelength or at least one range of illuminating wavelengths for illuminating the specimen to be different from each other.

8. The method as claimed in claim 7 , further comprising illuminating the selected area of the specimen simultaneously with light of the focusing wavelength and light of the illuminating wavelength or of the range of illuminating wavelengths, so that automatic focusing can be effected during observation.

9. An autofocusing transmitted light or reflected light microscope for automatic focusing of a microscope objective on a selected area of a specimen, comprising:
means for generating a digital hologram of the selected area in an off-axis mode prior to or during microscopic imaging, wherein the microscopic imaging is accomplished by an imaging method other than digital holography;
means for analyzing the digital hologram in such a way that a position of a best possible focus position in the selected area is determined along the optical axis of the microscope objective prior to or during the microscopic imaging; and
a controller that sets the previously determined best possible focus position of a stage, an objective lens or both the stage and the objective lens of the transmitted light or reflected light microscope based on analysis of the digital hologram that is performed by the means for analyzing the digital hologram.

10. The microscope as claimed in claim 9 , further comprising:
a focusing light source emitting a coherent focusing light of a focusing wavelength, and
at least one illuminating light source that illuminates the specimen with illuminating light of at least one illuminating wavelength or light of at least one range of illuminating wavelengths via an illuminating beam path.

11. The microscope as claimed in claim 10 , further comprising
means for splitting the focusing light into an object beam that illuminates the specimen with focusing light, and a reference beam,
a focusing sensor that detects intensities of an object beam, a reference beam and a superimposition of the object beam and the reference beam, and
means for uncoupling the object beam coming from the specimen from a detection beam path of the microscope after the object beam has passed through the microscope objective, and for directing the object beam onto the focusing sensor.

12. The microscope as claimed in claim 11 , wherein the means for uncoupling the object beam comprises a dichroic mirror or a polarizing beam splitter.

13. The microscope as claimed in claim 10 , further comprising means for coupling the object beam into the illuminating beam path of the microscope via which the specimen is illuminated also with illuminating light.

14. The microscope as claimed in claim 13 , wherein the means for coupling the object beam in, comprises a dichroic mirror or a polarizing beam splitter.

Assignees

• Zeiss Carl Microscopy Gmbh
• Zeiss Carl Ag

Inventors

• Sticker Markus
• Konradi Jakow
• Westphal Peter

Classifications

• G03H2001/0883

  Reconstruction aspect, e.g. numerical focusing · CPC title

• G03H1/0866

  Digital holographic imaging, i.e. synthesizing holobjects from holograms · CPC title

• G03H1/0443

  Digital holography, i.e. recording holograms with digital recording means (holobject computation G03H1/0866) · CPC title

• G03H2001/005

  in microscopy, e.g. digital holographic microscope [DHM] · CPC title

• G03H1/0005 Primary

  Adaptation of holography to specific applications (holographic optical element G02B5/32; holographic scanner G02B26/106; recognition using holographic mask G06V10/88; holographic memories G11B7/0065, G11C13/042) · CPC title