Light-microscopic method of localization microscopy for localizing point objects

Patent metadata

Field	Value
Publication number	US-10234672-B2
Application number	US-201414901740-A
Country	US
Kind code	B2
Filing date	Jul 1, 2014
Priority date	Jul 1, 2013
Publication date	Mar 19, 2019
Grant date	Mar 19, 2019

Abstract

A light-microscopic method of localization microscopy for localizing point objects in a sample arranged in an object space includes imaging, by an imaging optical unit having a depth of field range of predetermined axial z-extension along its optical axis in the object space, the sample onto a detector; localizing the point objects in the sample within the depth of field range in that, on the basis of a sample image, lateral x/y-positions of the point objects in a direction perpendicular to the optical axis are ascertained; displacing, in the object space relative to the sample the depth of field range within which the point objects are localized in the object space relative to the sample along the optical axis at least once by a predetermined axial z-travel distance; and imaging, by the imaging optical unit in the event of an axially displaced depth of field range.

First claim

The invention claimed is:

1. A light-microscopic method of localization microscopy for localizing point objects in a sample disposed in an object space, the method comprising:
imaging, by an imaging optical unit having a depth of field range of predetermined axial z-extension along its optical axis in the object space, the sample onto a detector;
localizing the point objects in the sample within the depth of field range on the basis of a sample image generated by imaging the sample onto the detector to determine lateral x/y-positions of the point objects in a direction perpendicular to the optical axis;
displacing, in the object space relative to the sample, the depth of field range within which the point objects are localized along the optical axis at least once by a predetermined axial z-travel distance which is smaller than the axial z-extension of the depth of field range;
imaging, by the imaging optical unit in the event of an axially displaced depth of field range, the sample anew onto the detector to generate at least one further sample image;
ascertaining the lateral x/y-positions of the point objects anew on the basis of the at least one further sample image;
ascertaining lateral x/y-position deviations between the lateral x/y-positions of the same point objects in the sample image and the at least one further sample image; and
using the ascertained lateral x/v-position deviations to generate correction information which, as a function of a distance of the point objects from a reference plane within the depth of field range, corrects lateral field distortion.

2. The light-microscopic method according to claim 1 , wherein the reference plane lies perpendicularly to the optical axis and remains stationary relative to the depth of field range when the depth of field range is displaced;
wherein one of the sample images is set as a reference image and a comparison structure which represents at least one of the point objects arranged in the reference plane of the depth of field range when taking the reference image is defined on the basis of the reference image;
wherein the comparison structure is identified in the at least one other sample image;
wherein on the basis of the various sample images, the lateral x/y-position of the comparison structure is ascertained in each case;
wherein the lateral x/y-position deviation between the lateral x/y-positions of the comparison structure which are ascertained on the basis of the various sample images is determined; and
wherein the correction information is generated as a function of the lateral x/y-position deviation ascertained for the comparison structure.

3. The light-microscopic method according to claim 2 , wherein the depth of field range is axially displaced in a plurality of steps,
wherein in each of these steps, the lateral x/y-position deviation of the comparison structure ascertained on the basis of the associated sample image is ascertained relative to the lateral x/y-position of the comparison structure ascertained on the basis of the reference image, and
wherein an association function is generated as correction information, the function values of which in each case indicate the lateral x/y-position deviation of the associated comparison structure ascertained in the respective step, depending on the axial z-position thereof along the optical axis.

4. The light-microscopic method according to claim 3 , wherein values of the association function which lie between the function values ascertained by step-wise displacement of the depth of field range are determined by interpolation.

5. The light-microscopic method according to claim 3 , wherein the lateral x/y-positions of the point objects are corrected by image processing directly in the associated sample image.

6. The light-microscopic method according to claim 3 , wherein the lateral x/y-positions of the point objects are corrected in a data set obtained from the associated sample image, and
wherein a corrected sample image is generated on the basis of the corrected data set.

7. The light-microscopic method according to claim 2 , wherein the comparison structure in the at least one other sample image is identified as a function of image brightness detected on the detector.

8. The light-microscopic method according to claim 1 , wherein the lateral x/y-position deviations are ascertained using a correlation method.

9. The light-microscopic method according to claim 1 , wherein the sum of the individual axial z-travel distances is substantially identical to the axial z-extension of the depth of field range.

10. The light-microscopic method according to claim 1 , wherein the axial z-travel distance is detected by a sensor.

11. The light-microscopic method according to claim 1 , wherein the depth of field range is displaced in the object space relative to the sample along the optical axis by the axial z-travel distance, in that the sample is displaced along the optical axis relative to the imaging optical unit or in that the imaging optical unit is displaced along the optical axis relative to the sample.

12. The light-microscopic method according to claim 1 , wherein the z-position of a respective point object along the optical axis is ascertained in that a parameter of a light spot which represents the respective point object in the sample image is ascertained and the z-position is associated with the parameter using predetermined association information.

13. The light-microscopic method according to claim 12 , wherein the z-positions of the point objects ascertained in the sample image are compared with the z-positions of the same point objects ascertained in the further sample image as a function of the predetermined axial z-travel distance; and
wherein z-correction information is generated as a function of this comparison using which the z-positions of the point objects which are ascertained as a function of the association information are corrected.

14. A light-microscopic apparatus for localizing point objects in a sample, the apparatus comprising:
an imaging optical unit having, in an object space and along its optical axis, a depth of field range of predetermined axial z-extension;
a detector onto which the imaging optical unit is operable to image a sample disposed in the object space;
a controller operable to localize point objects contained in the sample within the depth of field range to ascertain, on the basis of a sample image which the imaging optical unit generates on the detector, lateral x/y-positions of the point objects in a direction perpendicular to the optical axis; and
an adjustment unit, triggered by the control unit, operable to displace the depth of field range, within which the point objects are localized, in the object space relative to the sample along the optical axis at least once by a predetermined axial z-travel distance which is smaller than the axial extension of the depth of field range;
wherein, in the event of an axially displaced depth of field range, the imaging optical unit is operable to image the sample anew onto the detector and generate a further sample image;
wherein the control unit is operable to ascertain the lateral x/y-positions of the point objects anew on the basis of the further sample image;
wherein the control unit is operable to ascertain lateral x/y-position deviations between the lateral x/y-positions of the same point objects in each case ascertained on the basis of the sample images; and
wherein the control unit is operable to use the ascertained later x/y-position deviations to generate correction information which, as a function of a distance of the

Assignees

• Leica Microsystems

Inventors

• Foelling Jonas

Classifications

• G01N21/6458

  Fluorescence microscopy (fluorescence microscopes per se G02B21/0076 and G02B21/16) · CPC title

• G02B21/16

  adapted for ultraviolet illumination {; Fluorescence microscopes (G02B21/0076 takes precedence)} · CPC title

• G02B27/0025

  for optical correction, e.g. distorsion, aberration · CPC title

• G02B27/58

  Optics for apodization or superresolution; Optical synthetic aperture systems · CPC title

• G02B21/367 Primary

  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