Method and device for light-microscopic imaging of a sample structure

Patent metadata

Field	Value
Publication number	US-9772485-B2
Application number	US-201113806047-A
Country	US
Kind code	B2
Filing date	Jun 27, 2011
Priority date	Jun 29, 2010
Publication date	Sep 26, 2017
Grant date	Sep 26, 2017

Abstract

A method for light-microscopy imaging of a sample structure ( 2, 34 ) is described, having the following steps:
preparing the sample structure ( 2, 34 ) with markers that are transferrable into a state imageable by light microscopy, generating a sequence of individual-image data sets by sequential imaging of the sample structure ( 2, 34 ), in such a way that for each image, only a subset of the totality of the markers is in each case transferred into the state imageable by light microscopy, the markers of the respective subset having an average spacing from one another which is greater than the resolution limit of light-microscopy imaging which determines the extent of a light distribution representing one of the respectively imaged markers, generating at least two data blocks in which multiple successive individual-image data sets are respectively combined, superposing the individual-image data sets contained in the respective data block to yield a superposed-image data set, identifying an image offset between the superposed-image data sets, correcting the individual-image data sets that are contained in at least one of the superposed-image data sets on the basis of the identified image offset, determining center point positions of the light distributions representing the imaged markers, and assembling the center point positions into an offset-corrected overall image.

First claim

The invention claimed is:

1. A method for localization light-microscopy imaging of a sample structure, wherein said light microscopy imaging has a resolution limit; said method comprising:
preparing the sample structure with markers that are transferrable into a state imageable by light microscopy,
generating a sequence of individual-image raw-data image sets by sequential imaging of the sample structure in such a way that for each image, only a subset of the totality of the markers is in each case transferred into the state imageable by light microscopy, the markers of the respective subset having an average spacing from one another which is greater than the resolution limit of light-microscopy imaging which determines the extent of a light distribution representing one of the respectively imaged markers,
generating at least two data blocks in which multiple successive individual-image raw-data sets are respectively combined,
superposing for each data block the individual-image raw-data sets contained in the respective data block to yield a corresponding superposed-image data set,
identifying a drift-based image offset between the superposed-image data sets of the at least two data blocks, wherein identification of the drift-based image offset is based on the imaged sample structure itself,
correcting the individual-image data sets that are contained in at least one of the superposed-image data sets of one of the data blocks on the basis of the identified image offset,
determining center point positions of the corrected individual-image data sets of the light distributions representing the imaged markers, and
assembling the center point positions into an offset-corrected overall localization microscopy image, wherein the step of determining center point positions is performed after the step of correcting the individual-image data sets, wherein
the individual-image raw-data sets contained in the at least one superposed-image data set first are corrected based on the identified image offset, and
the center point positions of the light distributions representing the imaged markers then are determined.

2. The method according to claim 1 , wherein the number of successive individual-image data sets that are combined into the respective data blocks is defined in such a way that a correlation coefficient that is created by cross-correlation between the data blocks is greater than a predetermined threshold value.

3. The method according to claim 1 , wherein
more than two image data sets are superposed, and one of the superposed-image data sets is determined as a reference data set, and
the image offset of each of the other superposed-image data sets is determined relative to the reference data set.

4. The method according to claim 1 , wherein more than two image data sets are superposed, and the image offset is determined in each case between two immediately successive superposed-image data sets.

5. The method according to claim 1 , wherein
an interpolation function is determined based on the identified image offset;
said interpolation function defining values constituting individual image offset values, and
the individual-image data sets within the respective superposed-image data set are corrected based on said individual image offset values.

6. The method according to claim 1 , wherein
the center point positions of the light distributions representing the imaged markers first are determined in the individual-image data sets, and
the center point positions of the individual-image data sets contained in the at least one superposed-image data set then are corrected based on the identified image offset.

7. The method according to claim 1 , wherein the image offset is identified in accordance with an ICP algorithm.

8. The method according to claim 1 , wherein the image offset is identified by detecting a common substructure in the superposed-image data sets.

9. The method according to claim 1 , wherein a continuous time course of an image drift is identified by repeatedly identifying the image offset in multiple cycles and by modifying from one cycle to the next the number of individual-image data sets combined into the data blocks.

10. The method according to claim 1 , wherein
a quality parameter is determined during the identification of the image offset, and
a variable that indicates the resolution of the overall image is identified based on said quality parameter.

11. The method according to claim 1 , wherein the center point positions are determined in three spatial dimensions.

12. The method according to claim 1 , further comprising moving a component of a light microscope in accordance with the identified image offset in order to compensate for a drift motion of the sample structure relative to an image-producing system of the light microscope.

13. A light microscope comprising means for carrying out the method according to claim 1 .

14. The microscope according to claim 13 , wherein said means include a graphics processor.

Assignees

• Dyba Marcus
• Seyfried Volker
• Leica Microsystems

Inventors

• Dyba Marcus
• Seyfried Volker

Classifications

• G02B21/16

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

• G02B21/365 Primary

  Control or image processing arrangements for digital or video microscopes (G02B21/361, G02B21/362 take precedence) · CPC title

• G01N21/6458

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

• G01N21/6428 Primary

  Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" (in vivo A61B5/00; immunoassay G01N33/53) · CPC title