US-12625354-B2 - Light sheet microscope

US12625354B2US 12625354 B2US12625354 B2US 12625354B2US-12625354-B2

Abstract

A light sheet microscope for illuminating a sample arranged in an object plane on a sample carrier, comprising: an illumination device having an objective for illuminating the sample via an illumination beam path with a first light sheet, which intersects the sample volume in a first light sheet plane, the first light sheet plane intersecting the object plane at a skew first illumination angle (α), and a detection device for imaging of light coming from the sample, having an objective, the focal plane lying in the sample volume parallel to or in the object plane, and an area detector having a detector plane. The area detector includes a first slit stop in the detection beam path upstream of the area detector so as to mask out such regions of the sample volume which are illuminated by the first light sheet but lie outside the focal plane of the detection objective.

Inventors

Richard Hollinger
Jörg Siebenmorgen
Helmut Lippert
Ralf Wolleschensky

Assignees

CARL ZEISS MICROSCOPY GMBH

Dates

Publication Date: 20260512
Application Date: 20230928
Priority Date: 20220929

Claims (20)

1 . A light sheet microscope for illuminating a sample having a sample volume, said sample being arranged in an object plane on a sample carrier, the light sheet microscope comprising: an illumination device having an illumination objective for illuminating the sample via an illumination beam path with a first light sheet, which intersects the sample volume in a first light sheet plane, the first light sheet plane intersecting the object plane at a skew first illumination angle (α); and a detection device for the imaging of light coming from the sample, having a detection objective, the focal plane of which lies in the sample volume parallel to or in the object plane, and an area detector having a detector plane; wherein the area detector comprises a first slit stop arranged in the detection beam path upstream of the area detector in such a way as to mask out such regions of the sample volume which are illuminated by the first light sheet but lie outside the focal plane of the detection objective.
2 . The light sheet microscope as claimed in claim 1 , wherein: the first slit stop is arranged in the detection beam path in a displaceable fashion parallel to the detector plane in order to detect different regions of the focal plane; the detection device comprises an adaptive optical detection element, which is arranged in the detection beam path or is introducible into the latter and is controlled by a control device and by which the focal plane of the detection objective can be displaced in the sample volume; and the control device controls the first slit stop in such a way that in the event of a displacement of the focal plane, the position of the opening of the first slit stop corresponds to that region of the focal plane which is intersected by the first light sheet, such that those regions of the sample volume which are illuminated by the first light sheet and lie outside the focal plane of the detection objective are masked out.
3 . The light sheet microscope as claimed in claim 2 , wherein the adaptive optical detection element comprises an Alvarez manipulator, a deformable mirror, a spatial light modulator, a microelectromechanical system (MEMS), a micromirror array lens system (MALS), an adjustable or adaptive lens and/or an adaptive lens system.
4 . The light sheet microscope as claimed in claim 1 , wherein the control device is connected to the illumination device, the sample carrier or both for the control thereof in order to change the position of the first light sheet plane relative to the object plane.
5 . The light sheet microscope as claimed in claim 1 , wherein the illumination device having the illumination objective is designed for illuminating the sample with a second light sheet, which intersects the sample volume in a second light sheet plane, the second light sheet plane intersecting the object plane at a skew second illumination angle (β), and the area detector comprises a second slit stop arranged in the detection beam path upstream of the area detector in such a way as to mask out such regions of the sample volume which are illuminated by the second light sheet but lie outside the focal plane of the detection objective.
6 . The light sheet microscope as claimed in claim 5 , wherein the first light sheet plane and the second light sheet plane form an intersection angle (γ) with one another which is different than zero.
7 . The light sheet microscope as claimed in claim 5 , the detection device comprising an adaptive optical detection element, which is arranged in the detection beam path or is introducible into the detection beam path and is controlled by a control device and by which the focal plane of the detection objective can be displaced in the sample volume; wherein: the second slit stop is arranged in the detection beam path in a displaceable fashion parallel to the detector plane in order to detect different regions of the focal plane, and the control device controls the second slit stop in such a way that in an event of a displacement of the focal plane, the position of the opening of the second slit stop corresponds to that region of the focal plane which is intersected by the second light sheet, such that those regions of the sample volume which are illuminated by the second light sheet and lie outside the focal plane of the detection objective are masked out.
8 . The light sheet microscope as claimed in claim 5 , wherein a second length of the second slit stop corresponds to a second width of the second light sheet projected into the focal plane.
9 . The light sheet microscope as claimed in claim 1 , wherein the illumination objective and the detection objective are one and the same double objective.
10 . The light sheet microscope as claimed in claim 9 , wherein a dichroic mirror or a glass plate having a reflective coating for coupling the illumination beam path and the detection beam path into the double objective is introduced into the illumination beam path.
11 . The light sheet microscope as claimed in claim 1 , wherein the light sheet microscope is embodied as an inverse microscope.
12 . The light sheet microscope as claimed in claim 1 , wherein the detection device comprises an image field rotator, which is arranged in the detection beam path or is introducible into the detection beam path and by which the focal plane of the detection objective can be rotated in the sample volume.
13 . The light sheet microscope as claimed in claim 1 , wherein a first length of the first slit stop corresponds to a first width of the first light sheet projected into the focal plane.
14 . The light sheet microscope as claimed in claim 1 , wherein the first slit stop and/or the second slit stop are/is a rolling shutter of the area detector.
15 . A method for light sheet microscopy, comprising the following steps: (a) illuminating a sample having a sample volume, said sample being arranged in an object plane on a sample carrier, with a first light sheet, which intersects the sample volume in a first light sheet plane, the first light sheet plane intersecting the object plane at a skew first illumination angle (α); (b) setting a position of a focal plane of a detection objective in the sample volume, the focal plane of the detection objective lying parallel to or in the object plane in the sample volume; (c) displacing a first slit stop arranged upstream of an area detector into a position at which the opening of the first slit stop corresponds to that region of the focal plane which is intersected by the first light sheet and those regions of the sample volume which are illuminated by the first light sheet and lie outside the focal plane of the detection objective are masked out; and (d) imaging light coming from the sample onto the area detector via the detection objective; and repeating steps b) to d), the position of the focal plane being displaced perpendicularly to the object plane until a region of interest in the sample volume has been imaged.
16 . The method as claimed in claim 15 , wherein, in step a), the sample is additionally illuminated with a second light sheet, which intersects the sample volume in a second light sheet plane, the second light sheet plane forming a skew second illumination angle (β) with the object plane, and, in step c), a second slit stop arranged upstream of an area detector is additionally displaced such that the opening of the second slit stop corresponds to that region of the focal plane which is intersected by the second light sheet and those regions of the sample volume which are illuminated by the second light sheet and lie outside the focal plane of the detection objective are masked out.
17 . The method as claimed in claim 16 , wherein, in step d), the position of the second light sheet plane relative to the object plane is changed after the imaging.
18 . The method as claimed in claim 15 , wherein, in step d), the position of the first light sheet plane relative to the object plane is changed after the imaging.
19 . The method as claimed in claim 15 , wherein, in step d), the focal plane of the detection objective is rotated in the sample volume after the imaging.
20 . The method as claimed in claim 15 , wherein a rolling shutter of the area detector is used as the first slit stop and/or the second slit stop.

Description

PRIORITY CLAIM The present application claims priority to German Patent Application No. 10 2022 125 117.9, filed on Sep. 29, 2022, which said application is incorporated by reference in its entirety herein. FIELD OF THE INVENTION The invention relates to a light sheet microscope for illuminating a sample having a sample volume, said sample being arranged in an object plane on a sample carrier. The sample carrier can be embodied for example as a multiwell plate, a Petri dish, an object carrier or as any desired sample carrier that is commonplace in the prior art for light sheet microscopy. The sample volume can be a volume of one sample, or a plurality of samples can be arranged in the sample volume. The light sheet microscope comprises an illumination device having an illumination objective for illuminating the sample via an illumination beam path with a first light sheet, which intersects the sample volume in a first light sheet plane. The first light sheet plane intersects the object plane at a skew first illumination angle. Skew is any angle which is greater than 0° and less than 90°. The light sheet microscope furthermore comprises a detection device for the imaging of light coming from the sample, having a detection objective, the focal plane of which lies in the sample volume parallel to or in the object plane, and an area detector having a detector plane. In this case, the focal plane is understood to mean that region which is extended along the optical axis of the detection objective and which is imaged sharply with inclusion of the depth of focus of the detection objective. The light coming from the sample is preferably fluorescence light excited in the sample by the illumination with the first light sheet, the wavelengths or wavelength ranges of said fluorescence light being different than a first wavelength of excitation light used for generating the first light sheet. The invention furthermore relates to a method for light sheet microscopy. BACKGROUND OF THE INVENTION In light sheet microscopy, a thin layer, typically a few micrometers of a sample marked with fluorescence markers, is illuminated by means of a light sheet generated by an illumination device. As a result, the sample is excited in the layer and fluorescence light is emitted from the layer. The fluorescence light can be imaged by a detection device, whereby a fluorescence image representation of the layer is captured on a detector. If a plurality of layers of a sample are recorded sequentially, even relatively large sample volumes can be imaged. Light sheet microscopy is used in particular when examining biological samples. It is usually the case here that the light sheet which intersects the sample volume in a light sheet plane is oriented parallel to the object plane or forms an illumination angle with the object plane which is different than zero. Using this technique, which is also referred to as SPIM (Selective Plane Illumination Microscopy), spatial recordings even of relatively thick samples can be produced within a relatively short time. A visual, spatially extended representation of the sample is possible on the basis of optical sections combined with a relative movement in a direction perpendicular to a light sheet plane. The SPIM technique is preferably used in fluorescence microscopy; in that context it is also referred to as LSFM (Light Sheet Fluorescence Microscopy). In that case, the sample is illuminated uniformly in a lateral direction. A selective imaging, i.e. an imaging associated with a small depth of focus, is then possible in the direction perpendicular to the light sheet plane. The LSFM technique has a number of advantages over other established methods such as confocal laser scanning microscopy or two-photon microscopy. Since the detection can be undertaken in the wide field, it is possible to capture relatively large sample regions. Even though the resolution is somewhat lower than in confocal laser scanning microscopy, it is possible to analyze thicker samples using the LSFM technique because the penetration depth is greater. Moreover, this method has the lowest light exposure of the samples, which inter alia reduces the risk of bleaching of the sample since the sample is only illuminated by a thin light sheet at a non-zero angle with respect to the detection direction. The SPIM technique is now widely described in the literature, for example in DE 102 57 423 A1 and in WO 2004/053558 A1 based thereon, or in the review article “Selective Plane Illumination Microscopy Techniques in Developmental Biology” by J. Huisken et al., published in 2009 in the journal Development, vol. 336, page 63. One of the main applications of light sheet microscopy is in the imaging of medium-sized organisms having a size of a few 100 μm up to a few millimeters. Said organisms are generally embedded in an agarose gel, which is in turn situated in a glass capillary. This glass capillary is introduced into a water-filled sample cha