High-resolution scanning microscopy

Patent metadata

Field	Value
Publication number	US-9632296-B2
Application number	US-201314373602-A
Country	US
Kind code	B2
Filing date	Feb 27, 2013
Priority date	Mar 15, 2012
Publication date	Apr 25, 2017
Grant date	Apr 25, 2017

Abstract

In a microscope for high resolution scanning microscopy of a sample, said microscope comprising—an illumination device for illuminating the sample, —an imaging device for scanning at least one point spot or line spot across the sample and for imaging the point spot or line spot into a diffraction-limited, stationary single image with magnification into a detection plane, —a detector device for detecting the single image in the detection plane for different scanning positions with a spatial resolution, which, taking into consideration the magnification, is at least twice as high as a full width at half maximum of the diffraction-limited single image, —an evaluation device for evaluating a diffraction pattern of the single image for the scanning positions from data of the detector device and for generating an image of the sample, said image having a resolution that is increased beyond the diffraction limit, provision is made for—the detector device to have a detector array, which has pixels and is larger than the single image, and —a non-imaging redistribution element, which is disposed upstream of the detector array and distributes the radiation from the detection plane in a non-imaging manner among the pixels of the detector array.

First claim

What is claimed is:

1. Microscope for high resolution scanning microscopy of a sample, said microscope comprising,
an illumination device for illuminating the sample,
an imaging device for scanning at least one point spot or line spot across the sample and for diffraction-limited imaging the point spot or line spot into a, stationary single image in a detection plane,
a detector device for detecting the single image in the detection plane for different scanning positions with a spatial resolution,
an evaluation device for evaluating a diffraction pattern of the single image for the scanning positions from data of the detector device and for generating an image of the sample, said image having a resolution that is increased beyond the diffraction limit,
wherein the detector device has a detector array, which has pixels and is larger than the single image, and a non-imaging redistribution element, which is disposed upstream of the detector array and distributes the radiation from the detection plane in a non-imaging manner among the pixels of the detector array,
said redistribution element comprising a bundle of optical fibers, said bundle having an input with fiber ends, which is arranged in the detection plane, and an output, at which the optical fibers terminate at the pixels of the detector array in a geometrical arrangement, which is different from that of said input, said input fiber ends defining individual image pixels and said pixels of the detector being larger than the image pixels.

2. Microscope, as claimed in claim 1 , wherein the optical fibers extend from the input to the output in such a way that the optical fibers that are adjacent to each other at the output are also adjacent to each other at the input, in order to minimize a radiation intensity-dependent cross talk of adjacent pixels.

3. Microscope, as claimed in claim 1 , wherein the redistribution element comprises a mirror having mirror elements with varying tilt, said mirror being one of a multi-faceted mirror, a DMD or an adaptive mirror, wherein said mirror deflects the radiation from the detection plane onto the pixels of the detector array, wherein the pixels of the detector array have a geometric arrangement that is different from that of the mirror elements.

4. Microscope, as claimed in claim 1 , wherein the imaging device has a zoom optical system, which is arranged upstream of the detection plane in the imaging device, in order to adjust the size of the single image to that of the detector device.

5. Microscope, as claimed in claim 4 , wherein the illumination device and the imaging device share a scanning device, so that the illumination device illuminates the sample with a diffraction-limited point spot or line spot, which is coincident with the spot, imaged by the imaging device, wherein the zoom optical system is arranged in such a way that it is also an integral part of the illumination device.

6. Microscope, as claimed in claim 1 , wherein the detector array is a detector line, being an avalanche photodiode or photo multiplier tube line.

7. Method for high resolution scanning microscopy of a sample, comprising
illuminating the sample,
guiding at least one point spot or line spot across the sample for scanning it and imaging it into a single image, wherein the spot is imaged in a diffraction limited manner into the single image, and the single image lies quiescent in a detection plane,
detecting the single image for different scanning positions with a spatial resolution, so that a diffraction pattern of the single image is detected,
evaluating the diffraction pattern of the single image for each scanning position, and generating an image of the sample that has a resolution that is increased beyond the diffraction limit, wherein—a detector array is provided that has pixels and is larger than the single image, and
redistributing radiation of the single image from the detection plane in a non-imaging manner among the pixels of the detector array said redistributing performed by a bundle of optical fibers, said bundle having an input with fiber ends, which is arranged in the detection plane, and an output, at which the optical fibers terminate at the pixels of the detector array in a geometrical arrangement, which is different from that of said input, said input fiber ends defining individual image pixels and said pixels of the detector being larger than the image pixels.

8. Method, as claimed in claim 7 , wherein the optical fibers are guided from the input to the output in such a way that the optical fibers, which are adjacent to each other at the output, are also adjacent to each other at the input, in order to minimize a radiation intensity-dependent cross talk of adjacent pixels.

9. Method, as claimed in claim 7 , wherein the bundle composed of optical fibers, and the detector array are calibrated by exposing each optical fiber individually to radiation, by detecting an interference signal in pixels, which are assigned to the optical fibers, which are adjacent to each other at the output, and by constructing a calibration matrix, with which during microscopy of the sample, a radiation intensity-dependent cross talk between adjacent pixels is corrected.

10. Method, as claimed in claim 7 , wherein the radiation of the single image is redistributed by means of a mirror having mirror elements with varying tilt, said mirror being a multi-faceted mirror, a DMD or an adaptive mirror, wherein said mirror directs the radiation from the detection plane onto the pixels of the detector array, and the pixels of the detector array have a geometric arrangement that is different from that of the mirror elements.

11. Method, as claimed in claim 7 , using a detector line, being an avalanche photodiode or photo multiplier tube line, as the detector array.

12. Method, as claimed in claim 7 , further comprising determining a direction of movement of the scanning of the point spot or the line spot by evaluating the signals of the individual pixels of the detector array by means of cross correlation.

13. Method, as claimed in claim 7 , further comprising detecting that variations in the sample by determining and evaluating a temporal variation of the diffraction-limited single image at the point spot or the line spot that is quiescent in the sample.

14. Method, as claimed in claim 1 , wherein said optical fibers are multi-mode optical fibers.

15. Method, as claimed in claim 7 , wherein said optical fibers are multi-mode optical fibers.

Assignees

• Zeiss Carl Microscopy Gmbh

Inventors

• Wolleschensky Ralf
• Kleppe Ingo
• Netz Ralf
• Nieten Christoph

Classifications

• G02B6/06

  the relative position of the fibres being the same at both ends, e.g. for transporting images · CPC title

• G02B21/0076

  arrangements using fluorescence or luminescence · CPC title

• G02B21/367

  providing an output produced by processing a plurality of individual source images, e.g. image tiling, montage, composite images, depth sectioning, image comparison · CPC title

• G02B21/06

  Means for illuminating specimens · CPC title

• G02B21/0072

  details concerning resolution or correction, including general design of CSOM objectives · CPC title