High-resolution scanning microscopy

Patent metadata

Field	Value
Publication number	US-9470883-B2
Application number	US-201414490806-A
Country	US
Kind code	B2
Filing date	Sep 19, 2014
Priority date	Sep 19, 2013
Publication date	Oct 18, 2016
Grant date	Oct 18, 2016

Abstract

A microscope for high resolution scanning microscopy of a sample, having:
an illumination device for the purpose of illuminating the sample, an imaging device for the purpose of scanning at least one point or linear spot over the sample and of imaging the point or linear spot into a diffraction-limited, static single image below am imaging scale in a detection plane. A detector device for detecting the single image in the detection plane for various scan positions, with a spatial resolution which, taking into account the imaging scale in at least one dimension/measurement, is at least twice as high as a full width at half maximum of the diffraction-limited single image. The amplitude and/or phase of a wavefront influenced by the sample is detected with spatial resolution by means for wavefront detection, and wherein the influence of the sample on the phase is determined by means of a wavefront sensor.

First claim

The invention claimed is:

1. A microscope for high resolution scanning microscopy of a sample, comprising
an illumination device for the purpose of illuminating the sample,
an imaging device for the purpose of scanning at least one point or linear spot over the sample and of imaging the point or linear spot into a diffraction-limited, static single image, with an imaging scale in a detection plane,
a detector device for the purpose of detecting the single image in the detection plane for various scan positions with a spatial resolution;
an evaluation device for the purpose of evaluating a diffraction structure of the single image for the scan positions, using data from the detector device, and for the purpose of generating an image of the sample which has a resolution which is enhanced beyond the diffraction limit, said detector device having a detector array which has pixels and which is larger than the single image,
a non-imaging redistribution element which is arranged in front of the detector array and which distributes the radiation from the detection plane onto the pixels of the detector array in a non-imaging manner, and
means for wavefront detection wherein an amplitude and/or phase of a wavefront influenced by the sample is detected with spatial resolution through said means for wavefront detection.

2. The microscope according to claim 1 , wherein said redistribution element comprises a bundle of optical fibers, preferably of multi-mode optical fibers, which has an input arranged in the detection plane, and an output where the optical fibers end at the pixels of the detector array in a geometric arrangement which differs from that of the input.

3. The microscope according to claim 2 , wherein said optical fibers run from the input to the output-in such a manner that optical fibers which are adjacent the output are also adjacent the input in order to minimize a radiation intensity-dependent crosstalk between adjacent pixels.

4. The microscope according to claim 1 , wherein said redistribution element has a mirror with differently inclined mirror elements, particularly a multi-facet mirror, a DMD, or an adaptive mirror, which deflects radiation from the detection plane onto the pixels of the detector array, whereby the pixels of the detector array have a geometric arrangement which differs from that of the mirror elements.

5. The microscope according to claim 1 , wherein said imaging device has a zoom lens arranged in front of the detection plane in the imaging direction, for the purpose of matching the size of the single image to that of the detector device.

6. The microscope according to claim 5 , wherein said illumination device and the imaging device share a scanning device such that the illumination device illuminates the sample with a diffraction-limited point or linear spot which coincides with the spot imaged by the imaging device, whereby the zoom lens is arranged in such a manner that it is also a component of the illumination device.

7. The microscope according to claim 1 , wherein said detector array is a detector row.

8. The microscope according to claim 7 , wherein said detector row is an APD row.

9. The microscope according to claim 7 , wherein said detector row is an PMT row.

10. The microscope according to claim 1 wherein the influence of the sample on the phase is determined by means of a wavefront sensor.

11. The microscope according to claim 10 , wherein the wavefront sensor is a Shack-Hartmann sensor.

12. The microscope according to claim 10 , wherein the wavefront sensor is a WIS sensor.

13. The microscope according to claim 10 , wherein the wavefront sensor is a PAW wavefront sensor or a pyramid sensor.

14. The microscope according to claim 1 , wherein determination of the wavefront is performed using interferometric or holographic methods.

15. The microscope according to claim 1 , wherein determination of the wavefront is performed using a phase retrieval method.

16. The microscope according to claim 1 , further comprising a lens array arranged in front of light input surfaces of the redistribution element, said lens array directing light from each lens to light input surfaces.

17. The microscope according to claim 16 , wherein the position of each lens spot is determined by means of the receivers which are functionally assigned to the light input surfaces via the intensity measurement of the light input surfaces to which light is directed.

18. The microscope according to claim 16 , wherein a wavefront deformation is determined by a local displacement of the lens spot.

19. The microscope according to claim 16 , wherein calibration measurement of the position of the lens spot is performed without influencing the sample, via a flat mirror in the sample plane.

20. The microscope according to claim 1 , wherein wavefront deformations are determined by means of multiple images being captured and/or by means of detection of the illumination spot with the position of the detection being shifted in the direction of the optical axis.

21. The microscope according to claim 1 , further comprising at least one beam splitter for splitting the detection light into at least two partial beam paths, in which one redistribution element is positioned in each partial beam path, the same having different path lengths with respect to each other.

22. The microscope according to claim 1 , wherein, for the purpose of determining the wavefront and phase shift, images or illumination spots from different axial planes are compared to each other, the same being captured by a displacement of the sample and/or the detection device in the axial direction.

23. The microscope according to claim 1 , further comprising a variable lens in the detection beam path and/or the shared illumination/detection beam path.

24. A method for high resolution scanning microscopy of a sample, comprising
illuminating said sample;
guiding at least one point or linear spot over the sample in a scanning manner so that it is imaged into a single image, wherein the spot is imaged into the single image, with an imaging scale, and diffraction-limited, and the single image is static in a detection plane;
detecting the single image for various scan positions with a location accuracy, such that a diffraction structure of the single image is detected;
evaluating the diffraction structure of the single image for each scan position, and generating an image of the sample which has a resolution which is enhanced beyond the diffraction limit;
a detector array being included which comprises the pixels and is larger than the single image;
radiation of the single image from the detection plane being redistributed on the pixels of the detector array in a non-imaging manner; and
detecting the amplitude and/or phase of a wavefront influenced by the sample with spatial resolution through means for the detection of said wavefront.

25. The method according to claim 24 , wherein said radiation of the single image is redistributed by means of a bundle of multi-mode optical fibers, which has an input arranged in the detection plane, and an output where the optical fibers end at the pixels of the detector array in a geometric arrangement which differs from that of the input.

26. The method according to claim 25 , wherein said optical fibers run from the input to the output in such a manner that optical fibers which are adjacent at the output ar

Assignees

• Zeiss Carl Microscopy Gmbh

Inventors

• Kalkbrenner Thomas
• Kleppe Ingo
• Lippert Helmut
• Netz Ralf

Classifications

• G02B21/0084

  time-scale detection, e.g. strobed, ultra-fast, heterodyne detection · CPC title

• G02B21/008 Primary

  Details of detection or image processing, including general computer control · CPC title

• G02B21/0032

  Optical details of illumination, e.g. light-sources, pinholes, beam splitters, slits, fibers (G02B21/0036 - G02B21/008; means for illumination of specimens in general G02B21/06) · CPC title

• G02B26/0816

  by means of one or more reflecting elements · CPC title

• G01J9/02

  by interferometric methods (using interferometers for measuring optically the linear dimensions of objects G01B9/02) · CPC title