Method for preparing for and carrying out the acquisition of image stacks of a sample from various orientation angles

Patent metadata

Field	Value
Publication number	US-10001634-B2
Application number	US-201314412508-A
Country	US
Kind code	B2
Filing date	Jun 24, 2013
Priority date	Jul 3, 2012
Publication date	Jun 19, 2018
Grant date	Jun 19, 2018

Abstract

A method for preparing for and carrying out the microscopic recording of image stacks of a sample from various orientation angles, wherein the sample is retained in a sample retainer defining a sample coordinate system. The sample retainer can be translated and rotated in a space spanned by a detection coordinate system. In a first embodiment, for at least two different orientation angles, a projection image is recorded in the detection coordinate system and a first sample volume and a second sample volume are determined in the sample coordinate system. Then the intersection volume of the two sample volumes is determined in the sample coordinate system. Parameters necessary for image stack recording are determined for all other orientation angles based on the intersection volume.

First claim

The invention claimed is:

1. A method for preparing for and carrying out the microscopic acquisition of image stacks of a sample from various orientation angles (ai), where i=1, . . . , N, and where N is a natural number, the method comprising:
prior to acquisition of the image stacks the following preparation steps for determining a section volume:
placing the sample in a sample holder, defining a sample coordinate system (X p , Yp, Zp) in the sample holder, wherein the sample holder can be translated in a space spanned by a corresponding detection coordinate system (X D , Y D , Z D ) and can be rotated about at least one axis of rotation parallel to the axis (Yp) of the sample coordinate system, and wherein a detection direction lies parallel to the axis (Z D ) and the position of the two coordinate systems relative to each other is known;
acquiring a projection image for at least two orientation angles (al, a2) in the detection coordinate system along the detection direction, the projection image being generated by illuminating the sample, and as the first orientation angle (al) is set;
a) selecting a sample region to be examined, wherein the size of an image field is chosen such that the sample region to be examined is located, in a projection along the detection direction, at least partially in the region of the image field;
b) at the first orientation angle (al) defining a first region of interest which overlaps at least partially with the image field;
c) determining the position of the image field centre in the detection coordinate system in a plane perpendicular to the detection direction;
d) determining a set of coordinates in the detection coordinate system depending on the shape of the first region of interest, wherein the set of coordinates is sufficient to clearly define the position and the dimensions of the first region of interest in the image field and thus in the detection coordinate system;
e) determining a first sample volume in the sample coordinate system from the position of an image field centre of the projection image, from the set of coordinates, and from coordinates in the detection direction perpendicular to the first region of interest;

setting the second orientation angle (a2) and rotating the sample about the at least one axis of rotation by the angular difference between the two orientation angles, (a2−al), and wherein the sample is optionally translated in the detection coordinate system so that the sample region to be examined is located at least partially in the region of the image field;
performing steps a) to d) at the second orientation angle (a2), defining a second region of interest, and determining the position of the image field centre, a further set of coordinates in the detection coordinate system and a second sample volume;
determining a section volume in which the two sample volumes overlap; and
after the section volume has been determined,
determining the parameters in the sample coordinate system for image stack acquisition for the orientation angles (ai) with reference to the section volume, including initial and final positions for the stack acquisition along the detection direction for each orientation angle (ai) and the position of the section volume relative to the axis of rotation;
positioning the sample correspondingly in the detection coordinate system, and carrying out the image stack acquisition for each orientation angle (ai).

2. The method of claim 1 , wherein the regions of interest, are rectangular, and coordinates (x1 b , y1 b ), (x2 b , y2 b ) in each case specify the centre point of the rectangle in the detection coordinate system, and in each case the dimensions of the rectangles are stored in coordinates (x1 c , y1 c ), (x2 c , y2 c ).

3. The method of claim 1 , wherein the difference between the orientation angles, is 90°.

4. A method for preparing for and carrying out the microscopic acquisition of image stacks of a sample from various orientation angles (αi), where i=1, . . . , N, and where N is a natural number, the method comprising:
prior to acquisition of the image stacks the following preparation steps for determining a first sample volume:
placing the sample in a sample holder, defining a sample coordinate system (X P , Y P , Z P ) in the sample holder, wherein the sample holder can be translated in a space spanned by a corresponding detection coordinate system (X D , Y D , Z D ) and can be rotated about at least one axis of rotation parallel to the axis (Y P ) of the sample coordinate system, and wherein a detection direction lies parallel to the axis (Z D ) and the position of the two coordinate systems relative to each other is known;
acquiring a projection image for at least one orientation angle (α1) in the detection coordinate system along the detection direction, the projection image being generated by illuminating the sample, and as the first orientation angle (α1) is set;
a) selecting a sample region to be examined, wherein the size of an image field is chosen such that the sample region to be examined is located, in a projection along the detection direction, at least partially in the region of the image field;
b) at the first orientation angle (α1) defining a first region of interest which overlaps at least partially with the image field;
c) determining the position of the image field centre in the detection coordinate system in a plane perpendicular to the detection direction;
d) determining a set of coordinates in the detection coordinate system depending on the shape of the first region of interest, wherein the set of coordinates is sufficient to clearly define the position and the dimensions of the first region of interest in the image field and thus in the detection coordinate system;
e) determining the initial and final parameters of a first image stack acquisition at the first orientation angle (α1);
f) determining a first sample volume in the sample coordinate system from the position of the image field centre of the projection image, from the set of coordinates and from the initial and final parameters of the first image stack acquisition and

after the first sample volume has been determined,
determining parameters necessary for the acquisition of further image stacks in the sample coordinate system for the further orientation angles (αi) with reference to the sample volume, including initial and final positions for the stack acquisition along the detection direction for each further orientation angle (αi), positioning the sample correspondingly in the detection coordinate system, and carrying out the image stack acquisitions for each orientation angle (αi).

5. The method of claim 4 , wherein the region of interest is rectangular, and coordinates (x1 b , y1 b ) specify the centre point of the rectangle in the detection coordinate system, and the dimensions of the rectangle are stored in coordinates (x1 c , y1 c ).

6. The method of claim 1 , wherein the plane in the image field corresponds to the focal plane.

7. The method of claim 1 , wherein the position of the axis of rotation relative to the detection coordinate system is determined in a calibration step preceding the acquisition.

8. The method of claim 1 , wherein the acquisition of image stacks takes place within the framework of a microscopic sample analysis, during which the sample is illuminated with a light sheet, an expansion plane of which lies perpendicular to the detection direction.

9. The method of claim 1 , wherein the determination of the section volume takes place automated.

10. The method according to claim 4 , wherein the plane in the image field corresponds to the focal plane.

11. The method of claim 4 , wherei

Assignees

• Zeiss Carl Microscopy Gmbh

Inventors

• Lippert Helmut
• Selchow Olaf
• Tomancak Pavel

Classifications

• 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