Method and arrangement for light sheet microscopy

Patent metadata

Field	Value
Publication number	US-10031325-B2
Application number	US-201515120162-A
Country	US
Kind code	B2
Filing date	Feb 19, 2015
Priority date	Feb 20, 2014
Publication date	Jul 24, 2018
Grant date	Jul 24, 2018

Abstract

An arrangement for light sheet microscopy that includes a means for scanning a sample volume with a light sheet, which includes an angle δ≠90° with the optical axis of an objective. The light sheet passes through the entire sample volume in the propagation direction, and the depth of field S obj of the objective is less than the optical-axis depth T of this sample volume. An optical device, disposed downstream of the objective, increases the depth of field S obj to a depth of field S eff ≥the depth T of this sample volume. The arrangement also includes a means for positioning the sample volume within the region of the depth of field S eff . A spatially resolving optoelectronic area sensor is disposed downstream of the optical device, and hardware and software are provided to generate sample-volume images from the electronic image signals output by the area sensor.

First claim

The invention claimed is:

1. A method for light sheet microscopy, comprising the following steps:
scanning a sample volume with a light sheet which is formed by illumination light and has an angle δ≠90° with an optical axis of an objective, wherein:
the light sheet is directed to the entire sample volume to be imaged in a propagation direction; and
the objective directs both the illumination light to the sample volume and detection light from the sample volume; and
the illumination light is shined decentrally into an entrance pupil of the objective and in parallel with the optical axis of the objective;
a depth of field S obj of the objective is less than a depth T of the sample volume in a direction of the optical axis;

increasing the depth of field S obj in the detection beam path to a depth of field S eff that is greater than or equal to the depth T of the sample volume, and positioning the sample volume within the depth of field S eff ;
converting the optical image signals obtained with the increased depth of field S eff into electronic image signals with assignment to the regions of origin thereof in the sample volume; and
generating images of the scanned sample volume from the electronic image signals.

2. The method for light sheet microscopy as claimed in claim 1 ;
wherein, in a manner dependent on the sample volume to be scanned:
a position of the light sheet in relation to the objective is varied in the x, y or z-direction;
the angle δ is varied;
a spatial extent of the light sheet within the sample volume is varied by rotation about the optical axis; or
any combination of the above is performed.

3. The method for light sheet microscopy as claimed in claim 1 ;
wherein the sample volume is scanned using a reflected-light method.

4. The method for light sheet microscopy as claimed in claim 1 ;
wherein two-dimensional or three-dimensional images of the scanned sample volume are generated according to an extended depth of field technique or a light-field technique with an insertion of diffractive optical elements into the detection beam path.

5. The method for light sheet microscopy as claimed in claim 4 ;
wherein the two- or three-dimensional images of the scanned sample volume are generated according to the extended depth of field technique or light-field technique with the insertion of diffractive optical elements (DOE) into the detection beam path.

6. An arrangement for light sheet microscopy, comprising:
a scanning means configured to scan a sample volume to be imaged with a light sheet which is formed by illumination light and has an angle δ≠90° with an optical axis of an objective,
wherein:
the objective directs both the illumination light to the sample volume and detection light from the sample volume, the illumination light being shined to an entrance pupil of the object at a location offset in parallel to the optical axis of the objective; and
the light sheet is directed into a sample volume to be imaged in a propagation direction; and
a depth of field S obj of the objective is less than a depth T of the sample volume in a direction of the optical axis;

a device configured to vary a direction for shining the illumination light into the entrance pupil; and
an optical device, disposed downstream of the objective, configured to increase the depth of field S obj to a depth of field S eff that is equal to or greater than the depth T of the sample volume;
a positioning means configured to position the sample volume within a region of the depth of field S eff ;
a spatially resolving optoelectronic area sensor disposed downstream of the optical device; and
hardware and software, which are configured to generate images of the sample volume from the electronic image signals output by the area sensor.

7. The arrangement for light sheet microscopy as claimed in claim 6 , further comprising:
an optical means configured to:
generate a light sheet scanned into the sample volume; and
vary at least one of:
a position of the light sheet in relation to the objective in the x, y, or z-direction;
the angle δ; and
a spatial extent of the light sheet by rotation about the optical axis or about an axis parallel to the optical axis.

8. The arrangement for light sheet microscopy as claimed in claim 6 ;
wherein the optical device comprises:
a cubic phase mask; or
a microlens array.

9. The arrangement for light sheet microscopy as claimed in claim 6 ;
wherein a CCD sensor with a reception area aligned at right angles, at least substantially at right angles, to the detection beam path is provided as an area sensor.

10. The arrangement for light sheet microscopy as claimed in claim 6 , further comprising:
a means for changing the entrance location for shining illumination light into the entrance pupil of the objective, in a decentralized manner in relation to the optical axis thereof.

11. The arrangement for light sheet microscopy as claimed in claim 6 ;
wherein the hardware comprises:
a personal computer (“PC”); and
a means for electronic storage and visually perceivable reproduction of two-dimensional or three-dimensional images; and

wherein the software comprises a predetermined algorithm for signal evaluation and for generating the two-dimensional or three-dimensional images.

12. An arrangement for light sheet microscopy, comprising:
scanning means configured to scan a sample volume to be imaged with a light sheet which is formed by illumination light and has an angle δ≠90° with an optical axis of an objective,
wherein the light sheet is directed into a sample volume to be imaged in a propagation direction; and wherein a depth of field S obj of the objective is less than a depth T of the sample volume in a direction of the optical axis;
an optical device configured as an axicon, disposed downstream of the objective, and configured to increase the depth of field S obj to a depth of field S eff that is equal to or greater than the depth T of the sample volume;
positioning means configured to position the sample volume within a region of the depth of field S eff ;
a spatially resolving optoelectronic area sensor disposed downstream of the axicon; and
generating means configured to generate images of the sample volume from the electronic image signals output by the area sensor.

Assignees

• Zeiss Carl Microscopy Gmbh

Inventors

• Anhut Tiemo
• Kalkbrenner Thomas
• Schwedt Daniel
• Siebenmorgen Jörg
• Lippert Helmut

Classifications

• H04N25/71

  Charge-coupled device [CCD] sensors; Charge-transfer registers specially adapted for CCD sensors · 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

• H04N3/02

  by optical-mechanical means only (H04N3/36 takes precedence) · CPC title

• G02B21/365

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

• G02B21/008 Primary

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