Single plane illumination microscope

Patent metadata

Field	Value
Publication number	US-11719922-B2
Application number	US-202117230613-A
Country	US
Kind code	B2
Filing date	Apr 14, 2021
Priority date	Oct 28, 2016
Publication date	Aug 8, 2023
Grant date	Aug 8, 2023

Abstract

A single plane illumination microscope having an illumination optical system for illuminating a sample located on a sample carrier in a medium, and which is parallel to a planar reference surface. The sample is illuminated by a light sheet via an illumination light path. A detection optical system has a detection beam path. The optical axes of the illumination and detection optical systems each define an angle that is not equal to zero degrees along with the normal to the reference surface. A barrier layer system includes at least one layer of a given material having a given thickness and separates the medium from the illumination and detection optical systems. A base area of the barrier layer system is in contact with the region that is accessible for illumination and detection activities, said base area running parallel to the reference surface. At least one corrective element in the illumination beam path and/or the detection beam path allows those aberrations to be reduced which are created when light to be detected or light for illuminating the sample penetrates interfaces of the barrier layer system at an angle. The microscope has means, which are independent of the generation of the light sheet, for applying, via at least one manipulation beam path, light intensity to the sample in substantially point-shaped regions of the light sheet plane or in a given volume that at least temporarily encompasses the light sheet plane.

First claim

What is claimed is:

1. A light sheet microscope, comprising
means for generating a light sheet,
an illumination optical unit for illuminating a sample which is located on a sample carrier in a medium, said sample carrier being oriented parallel to a planar reference surface, via an illumination beam path with said light sheet, the latter having a light sheet plane that encloses an illumination angle that differs from zero with the normal of the reference surface,
a detection optical unit having a detection beam path with an optical axis, which encloses a detection angle that differs from zero with the normal of the reference surface in the region of the sample carrier,
a separation layer system with at least one layer that is made of a specified material with a specified thickness and separates the medium from the illumination and detection optical units, wherein the separation layer system is in contact, at least in the region that is accessible for illumination and detection, with the medium by way of a base surface that is parallel to the reference surface,
at least one correction element in the illumination and/or in the detection beam path for reducing such aberrations that occur due to the oblique passage of light that is to be detected or of light for illuminating the sample through interfaces of the separation layer system,
means for optical manipulation of the sample by manipulation light by causing manipulation light intensity to impinge on the sample in substantially point-shaped areas in a specified volume which at least temporarily comprises the light sheet plane, via at least one manipulation beam path,
means for sequential switching between an illumination of the sample by manipulation light and an illumination of the sample with the light sheet.

2. The light sheet microscope as claimed in claim 1 , wherein the illumination optical unit includes an illumination objective and the detection optical unit includes a detection objective.

3. The light sheet microscope as claimed in claim 2 , wherein the illumination objective is identical to the detection objective.

4. The light sheet microscope as claimed in claim 2 , wherein a manipulation objective is positioned in the manipulation beam path.

5. The light sheet microscope as claimed in claim 4 , wherein the manipulation objective is identical to the illumination objective or to the detection objective.

6. The light sheet microscope as claimed in claim 4 , the means for optical manipulation further comprising a point scanner for temporally sequential impingement of light intensity on individual substantially point-shaped areas of the sample, or a multipoint scanner for the temporally sequential impingement on a plurality of substantially point-shaped areas of the sample, a respective quantity of which is able to be impinged on at the same time by light intensity.

7. The light sheet microscope as claimed in claim 6 , wherein the means for optical manipulation comprise a first spatial light modulator for causing a specified intensity distribution to impinge on the sample.

8. The light sheet microscope as claimed in claim 7 , wherein the means for optical manipulation comprise a second spatial light modulator for imparting a specified amplitude distribution to the specified intensity distribution.

9. The light sheet microscope as claimed in claim 7 , wherein the means for optical manipulation comprise a digital micromirror array for producing a specified intensity distribution in a plane, parallel to the light sheet plane, and particularly in the light sheet plane.

10. The light sheet microscope as claimed in claim 9 , further comprising a switching means for switching between the first spatial light modulator, the digital micromirror array, or a combination of both.

11. The light sheet microscope as claimed in claim 10 , wherein said switching means, is a switching mirror.

12. The light microscope as claimed in claim 7 , in which the manipulation objective is identical to the detection objective, and wherein a first region of a spatial light modulator is embodied for impingement of a specified intensity or amplitude distribution on the sample and as a correction element, and a second region of the spatial light modulator is embodied exclusively as a correction element in the detection beam path, wherein deflection means for steering the light to be detected onto the second region and the light that is used for the manipulation onto the first region are provided.

13. The light sheet microscope as claimed in claim 12 , wherein the deflection means comprise a dichroic element having active optical surfaces which are arranged parallel with respect to one another and preferably perpendicularly to the active optical surface of the first spatial light modulator, wherein the beam path is guided such that the light that is used for the manipulation and is to be detected is guided in each case twice onto the dichroic element, and the deflection onto the respective region is achieved by way of the thickness of the dichroic element.

14. The light sheet microscope as claimed in claim 4 , in which the manipulation objective is identical to the illumination objective, and wherein the illumination optical unit comprises a spatial light modulator, onto which a phase pattern for the light sheet forming is imprinted in a light sheet generation region and a phase pattern for causing a specified intensity distribution to impinge on the sample is imprinted in a manipulation region, and in that the illumination optical unit comprises means for deflecting the light for the manipulation exclusively on to the manipulation region and light for the light sheet generation exclusively onto the light sheet generation region.

15. The light sheet microscope as claimed in claim 14 , wherein the light sources, wavelengths, and intensities of the manipulation light is designed for exciting two-photon and multi-photon transitions.

16. The light sheet microscope as claimed in claim 15 , further comprising a mechanism for temporal focusing for axial localization of the intensity of the light that is used for manipulation along the axis of the manipulation beam path.

17. The light sheet microscope as claimed in claim 16 , wherein said mechanism for temporal focusing comprises a femtosecond pulsed laser with a controller for controlling group velocity dispersion for axial localization of the intensity of the light that is used for manipulation along the axis of the manipulation beam path.

18. The light sheet microscope as claimed in claim 1 , comprising a spatial light modulator comprising a manipulation region, onto which manipulation light is directed, and a light sheet generation region, onto which light for light sheet generation is directed, the light sheet generation region and the manipulation region overlapping at least partially.

19. The light sheet microscope as claimed in claim 1 , comprising a stimulation laser emitting manipulation light, and comprising an illumination laser emitting light for light sheet generation, wherein the emission wavelengths of both lasers are different from each other and/or switching between illumination with manipulation light and illumination with the light sheet is time-synchronized with a switching of the stimulation laser and the illumination laser.

Assignees

• Zeiss Carl Microscopy Gmbh

Inventors

• Kalkbrenner Thomas
• Netz Ralf
• Lippert Helmut
• Siebenmorgen Joerg

Classifications

• G02B21/0032 Primary

  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

• G02B21/006 Primary

  focusing arrangements; selection of the plane to be imaged · CPC title

• G02B21/32

  Micromanipulators structurally combined with microscopes · CPC title