Microscope and methods for SPIM microscopy

Abstract

A method for SPIM microscopy with a microscope, comprising: - an illumination device comprising an illumination light source (L1, L2) and an illumination beam path for illuminating a sample (P) with a light sheet (LB); - a detection device for detecting light emitted by the sample with an objective (O); - wherein the light sheet (LB) is essentially planar at the focus (FE) of the objective (O) or at a defined plane near the focus (FE) of the imaging objective (O), and the objective (O) has an optical axis that intersects the plane of the light sheet (LB) at a non-zero angle; - wherein the sample (P) is moved through the light sheet (LB) in the direction of the optical axis of the objective (O) to detect different sample planes; - and adjustment means for adjusting the illumination beam path at least along the optical axis of the detection objective (O), characterized in thatthat - in a first step, calibration is carried out using a sample (P) to be examined by recording and storing the actual position of the light sheet (LB) in different sample planes as a function of the position of the sample (P) - and the stored position of the light sheet (LB) is used in a second step when viewing and / or detecting the sample (P) based on the values ​​stored in the first step to correct the position of the light sheet (LB) to the focal plane (FE) of the detection objective (O).

Description

[0001] The invention relates to a microscope comprising an imaging objective for imaging a sample onto a detector and means for illuminating the sample with a light sheet in the focal plane of the imaging objective or in a defined plane near this focal plane. The illumination means comprise a light source, preferably one emitting coherent light.

[0002] A microscope in which the illumination and detection beam paths are essentially perpendicular to each other, and in which the sample is illuminated by a light sheet in the focal plane of the imaging objective, i.e., perpendicular to its optical axis, is designed for the examination of samples using the selective-plane illumination microscopy (SPIM) method. Unlike confocal laser scanning microscopy (LSM), in which a three-dimensional sample is scanned point by point in individual planes of varying depths, and the resulting image information is subsequently combined to create a three-dimensional image of the sample, SPIM technology is based on wide-field microscopy and enables the imaging of the sample based on optical sections through individual planes of the sample.

[0003] The advantages of SPIM technology include the greater speed at which image information is acquired, the reduced risk of bleaching of biological samples, and an extended penetration depth of the focus into the sample.

[0004] In principle, SPIM technology excites fluorophores contained in or introduced into the sample with laser light, which is shaped into a so-called light sheet. This light sheet illuminates a selected plane within the depth of the sample, and an image of this sample plane is obtained as an optical section using imaging optics. Essentially equivalent to such excitation with a static light sheet is the rapid back-and-forth movement of a thin, rotationally symmetric laser beam in the focal plane of the imaging objective. Effectively, i.e., on average over the observation period, this also results in the shape of a SPIM light sheet.

[0005] SPIM technology is described, for example, in Stelzer et al., Optics Letters 31, 1477 (2006), in Stelzer et al., Science 305, 1007 (2004), in DE 102 57 423 A1 and in WO 2004 / 0530558 A1.

[0006] InFig. Figure 1 shows the basic structure of a SPIM microscope. The light from a light source 1 is via a lighting optic 2 shaped into a light sheet and placed on a sample 3 The sample and light sheet are located in the focal plane of an imaging objective. 4 The optical axis of the imaging lens 4 is perpendicular to the direction from which the sample was taken 3 is illuminated. The lighting optics 2 It typically comprises several optical elements that combine the coherent light from the light source. 1 collimate and form a light sheet from it. In the state of the art, the lighting optics comprise 2 usually also a cylindrical lens, whose flat side faces the sample and whose curved side faces the light source.

[0007] A schematic representation shows a sample holder PH by means of which the sample, for example controlled via a control unit A, is moved motor-driven in the direction of the optical axis of the lens. 4 is being moved.

[0008] The described “light sheet microscopy” combines optical sections with wide-field detection via a spatially resolved camera (CCD camera) by illuminating the entire lateral focal plane (xy-plane) of the detection objective with a thin sheet of light ( Fig. The light sheet is illuminated at right angles to the detection axis (z-axis).

[0009] The sample is placed in the overlapping area of ​​illumination and detection. Fluorescence signals, excited by the illumination light sheet, are projected onto the camera across the entire field of view of the detection objective. Due to the perpendicular illumination with a thin light sheet, only a small portion of the axial extent of the detection optics is illuminated, thus creating an optical section. To observe a different area within the sample, the sample is moved through the light sheet using a sample positioning unit, independent of the optics. By acquiring optical sections at various sample positions along the detection axis, it is possible to record three-dimensional image stacks. These image stacks can then be reconstructed into a 3D image.

[0010] This requires capturing multiple three-dimensional image stacks from different angles. One image stack, for example, comprises 200 images. At least four different illumination angles are needed for a three-dimensional image.

[0011] For good image quality and clean sectioning, perfect overlap of the illumination and detection planes is particularly critical. Due to changing samples and refractive indices, this is a recurring task in daily work.

[0012] The well-known relative movement of the light sheet to the sample and objective lens causes the illuminated z-plane within the sample, and thus the sample plane used for evaluation, to vary during adjustment. If such an adjustment procedure is applied to a real sample, this z-movement leads to significant variations in the information within the individual adjustment images, making evaluation of the adjustment image impossible. This problem also affects adjustment procedures with a reference sample, albeit to a lesser extent. However, these samples are more homogeneous, so it is rarely noticeable.

[0013] Manual, user-controlled adjustment is time-consuming and requires considerable experience. For easier evaluation, the user typically focuses on highly structured image areas, the information content of which, however, depends significantly on the z-plane of the sample. If this z-plane varies during the adjustment process, it often leads to incorrect evaluation. The prior art is further described in patent applications DE 10 2007 017 598 A1 and DE 10 2007 045 897 A1. Known adjustment methods require, firstly, that a fluorescent reference object (e.g., beads / fiducials or a homogeneous fluorescent object) is positioned instead of or within the sample. These reference objects are illuminated with the planar light sheet, and the optimal adjustment point is determined based on the contrast or the PSF (e.g., in the case of beads).Secondly, some methods involve illuminating the fluorescent reference object with a reference structure. This is achieved, for example, by a grating in a plane conjugate to the object plane or by modulating the scanned light sheet.

[0014] Adjustment methods using a reference sample require that the reference sample be completely identical to the actual user sample in all optical properties (refractive index, surface curvature, penetration depth, etc.). This is not feasible for the existing range of suitable samples. Therefore, these adjustment methods accept deviations from the optimal alignment. Optical effects caused by inhomogeneous sample structures, such as different cell structures, cannot be taken into account at all. Similar effects occur, for example, when the penetration depth into the sample medium changes significantly during the acquisition of an image stack.

[0015] General light sheet alignment according to the state of the art is described, for example, by Greger et al. (Greger et al., Rev. Sci. Instr. 78, 023705, 2007) (Section IIB). This method uses a gimbal mount and a telescope to move the light sheet along the z-direction of the detection optics by means of an angular movement. This alignment of the illumination optics relative to the detection optics also corresponds to the earlier state of the art according to Voie et al. (Journal of Microscopy 170, 229, 1992; section “illumination system”) or Santi et al. (BioTechniques 46, 287, 2009, Suppl. Mat.). Krzic et al. (Nat Methods 9, 730, 2012, Suppl.) describe the three-dimensional light sheet alignment for a scanning light sheet using a "parked" laser beam illuminating a fluorescent solution. This method utilizes, among other things, the waist of the laser beam. The utilization of scattered light is also described.This section also describes how the light sheet alignment is achieved by moving the detection optics relative to the light sheet in a fluorescent solution or reference sample. Keller et al. (Science 322, 1065, 2008, Supp) describe a setup with movable detection optics, but this is not described here in the context of light sheet alignment. Presentation of the inventive solutions and exemplary embodiments

[0016] The invention is characterized, among other things, by the features of the independent and dependent patent claims which are included in the present disclosure.

[0017] A beneficial prerequisite for good, automated adjustment is the choice of the right evaluation criterion and its applicability to the actual sample instead of a reference structure.

[0018] No method is known in the prior art in which the sample to be examined is used directly in the light sheet adjustment, whereby it is moved together with the light sheet relative to the objective or the detection plane.

[0019] The invention consists in particular of a movement of the light sheet and the sample relative to the predetermined focal plane of the detection unit, preferably coupled via a control unit.

[0020] Conversely, the detection lens can also be moved independently, or all elements can be coordinated and moved relative to each other via a common control unit to achieve the inventive effect. If the sample is moved after or simultaneously with an adjustment of the light sheet, the observed sample plane and thus the fundamental image content remain identical. Differences in the image are caused solely by the adjustment position. To ensure correctly coupled movement, the two individual movements can advantageously be calibrated beforehand to achieve sufficient accuracy.

[0021] Furthermore, for the procedures described below, it is possible and advantageous to determine the sample structures to be used for adjustment beforehand using another contrast method and to align the sample relative to detection thereafter.

[0022] In Fig. Figure 2 shows the basic structure of a SPIM microscope for carrying out the inventive process.

[0023] A sample P, which may be located in a sample chamber PK, wherein the sample or sample chamber is arranged to be adjustable in the direction of a Z axis and is also arranged to be rotatable around the optical axis of the detection objective, is detected by a detection beam path which consists of a vertically adjustable detection objective O whose detection axis or optical axis runs in the Z direction, to which a preferably replaceable light filter F, a tube lens TL and an area receiver CCD are connected in the detection direction.

[0024] Essentially perpendicular to the detection axis Z, here in the X direction, is an illumination beam path, consisting here of two lasers L1, L2 coupled via beam splitter BS, which via an AOTF for wavelength selection and intensity adjustment, a deflecting mirror S and a beam expander BE as well as an anamorphic optic such as here a cylindrical lens ZL for beam shaping, which generates a planar light distribution that penetrates the sample.

[0025] A schematic representation shows an adjustment unit BLjust, which can, for example, adjust or tilt the elements S, BE, and ZL in several directions. Preferably, the illumination is adjusted along the Z-axis, represented by the vertical arrow, and rotated about the Z-axis, and tilted, for example, about the Y-axis. The pivot point of the tilt can also be located in the sample, for example, in the optical axis CA of the detection objective, through a coupled Z-axis adjustment and rotation about the mirror axis.

[0026] A common control and control unit CU, usually formed by a computer and a display unit, is connected to all adjustment devices such as the AOTF, the sample chamber adjustment PK and the illumination adjustment BLJust in order to be able to carry out the method according to the invention.

[0027] In Fig. 3– Fig. 6 is an excerpt from the Fig. 2 shown: O: Detection lens P: As a sample, a tissue with labeled cell nuclei for exemplary samples. K: Camera image of the sample with cell nuclei LB: Area of ​​light-sheet illumination (e.g., Gauss beam) OA: optical axis of illumination (dotted) FE focal plane of the detection optics

[0028] Fig. , b shows in Fig. the initial state of a defocused light sheet. and in Fig. B. the adjusted state by moving the light sheet. This becomes clear in Fig. and Fig. Different sample areas P1, P2 are imaged by the objective lens O.

[0029] The invention now ensures that the condition in Fig. This ensures that the same results are obtained for each sample level traversed, in order to achieve a perfect sample section.

[0030] The following advantageous methods are proposed for this purpose: Procedure 1:

[0031] When the user focuses the sample by moving the sample or sample holder in the direction of the detection axis of the lens (Z-axis), the actual differences in the refractive index of the sample or refractive index jumps can cause the light sheet to no longer lie exactly in the focal plane of the detection lens.

[0032] Therefore, in a calibration step prior to the actual sample measurement, a calibration table is recorded which performs a Z-adjustment for the actual sample used, in which the actual position of the light sheet is recorded as a function of the vertical position of the sample (and thus as a function of the refractive index of the sample).

[0033] For this purpose, the respective height position of the light sheet, together with its position on its adjustment means, is recorded and stored for different sharply focused, i.e., focused sample areas of a real sample.

[0034] The recording of the light leaf position is carried out, for example, as follows: The sample undergoes a Z-adjustment and the respective Z positions form a storage value with Z1 – Zi (i = 1 – n).

[0035] For the Z1 – Zi, the light sheet is adjusted at least vertically, preferably also tilted before or after (by the control unit Cu in Fig. 2 for example).

[0036] The adjustment and / or tilting preferably takes place around the respective value Zi.

[0037] Based on the sharpness determination or contrast analysis by the CCD in Fig. 2 (also conceivable visually by the viewer) and the evaluation unit CU will determine the value of the highest sharpness / optimal contrast with the corresponding values ​​set by the adjustment unit BLJust in Fig. 2. Cu, assigned to the respective value Zi, is stored (in the calibration table) or the light sheet position set with it is also used directly for a measurement.

[0038] This determination of the position of the light sheet relative to the Zi values ​​is subsequently used to make a corresponding correction when the user views or detects a specific Z-plane in the sample, i.e., to adjust the actual light sheet position for exactly this focused sample plane so that the actual light sheet is always in the actually viewed or detected focal plane despite the aforementioned fluctuations in the refractive index. Procedure 2:

[0039] During the sample adjustment to a new Z-plane, the light sheet is moved in relation to the sample simultaneously or immediately afterwards (for example, to determine the optimal contrast), at least in the Z direction, preferably also tilted around a rotation axis, for example in the middle of the focal plane of the detection objective.

[0040] For example, a curved surface or interface in the sample can, due to its refractive index profile, cause the light sheet to be "bent away" from a horizontal position to a tilted position.

[0041] This is advantageously compensated for by the procedure.

[0042] This can be done visually or automatically (analogous to autofocus methods that work with contrast adjustment),

[0043] The linking of the sample movements in the Z-direction and the light sheet adjustment, as well as the evaluation of the sample contrast, preferably takes place in the control and computing unit XX.

[0044] Advantageously, methods 1 and 2 can also be applied in a combined form.

[0045] In Fig. – Fig. This figure illustrates the effects that can occur due to the optical properties of the sample, for example, during the aforementioned image stack acquisition. Three positions of the sample P relative to the detection objective O are shown. Any additional equipment, such as a sample chamber with immersion medium, has been omitted from the illustration. The sample could, for example, be a section of cell tissue in which the cell nuclei are visualized using a fluorescent marker.

[0046] In the position after Fig. and b) the area of ​​the light sheet illumination completely overlaps with the focal plane of the detection objective, so that the cell nuclei ZK located in the focal plane of the detection optics O are registered with approximately the same contrast in the camera image K.

[0047] In the position after Fig. and b) due to the altered optical path length caused by the different sample position, two effects occur: Firstly, the focal plane FE of the detection shifts in the z-direction relative to the nominal optical axis of the illumination LB. The cell nuclei located in the focal plane can no longer be visualized with full contrast due to the reduced overlap with the light sheet illumination. Furthermore, a focus shift along the x-direction can also occur in the illumination, resulting in corresponding image inhomogeneity due to the inhomogeneous intensity distribution in the illumination beam.

[0048] In the position after Fig. 6a) and b) in addition to an extended focus shift in the detection, a tilting of the light sheet also occurs, since the illumination beam is deflected at the sample surface by a refractive index jump.

[0049] The effects described are shown as examples; of course, the situation is normally even more complex, as the sample is sometimes characterized by a complicated, inhomogeneous permittivity, which can also manifest itself, for example, in the occurrence of several optical interfaces within the sample volume.

[0050] No methods are known in the prior art that directly compensate for such artifacts like the methods described above. Further advantageous effects and embodiments of the invention:[A]

[0051] The coupled movement of the light sheet and sample relative to each other greatly simplifies the manual adjustment procedure. In this method, the user generally assesses the image quality visually. Since they can now concentrate on the image information in a stationary sample plane, the adjustment procedure becomes more intuitive and goal-oriented. The problems described above no longer occur. [B]

[0052] A second aspect is that the coupled movement enables automatic light sheet adjustment in real samples. The aid of a reference sample, whether a homogeneously fluorescent sample or specific reference objects such as beads, is no longer necessary. Since the coupled adjustment movement always uses the same sample plane for evaluation, the generally available image information is irrelevant. The coupled movement is advantageous for all automatic adjustment methods, whether using a planar or structured light sheet, and when evaluating contrast, modulation depth, or PSF. [C]

[0053] Light sheet microscopy methods are based on the principle that a stack of images (z-stack) is acquired through a relative movement of the sample and the detection focal plane. Due to sample inhomogeneities or the penetration depth into the sample medium, the optimal adjustment position for the light sheet depends on the z-plane. An advantageous aspect of the invention is that, through the coupled movement and thus the applicability of the adjustment methods to all samples – especially real samples – object-specific inhomogeneities of different sample planes, which require different optimal adjustment positions depending on the plane, can be calibrated out. This method is implemented, for example, as follows: 1. For a subset of z-planes (= sample planes), the optimal adjustment position is determined within the real sample using the preferred evaluation algorithm. 2. The optimal adjustment position for the z-planes between these support planes is interpolated using a suitable function (polynomial, spline, ...). 3. While the z-stack is being acquired, the specific optimal adjustment position of the light sheet is set for each z-plane. This also corresponds to a coupled movement, for example, of the sample and the light sheet. In particular, this can also include an angular adjustment (see > Fig. 6). 4. By optimally adjusting each individual image plane, a z-stack with optimal image quality is achieved. 5. Any distortions in the overall 3D image of the sample caused by the light sheet tracking can be additionally corrected computationally. [D]

[0054] Furthermore, the methods of light sheet microscopy are based on the principle that by rotating the sample relative to the detection and illumination direction and subsequently acquiring multiple z-stacks at different angles (= views), a 3D image can be reconstructed. For optimal image results, it is crucial that the light sheet is adjusted to the optimal position for each illumination direction, as the penetration depth and optical properties of the sample differ from each direction. This is only possible through simultaneous or sequential movement of the sample and the light sheet. [E]

[0055] An even better image result is achieved when sub-aspect [B], sub-aspect [C] and sub-aspect [D] are combined. [F]

[0056] In a z-stack and / or multiview image, movement of the sample can also lead to a change in focus position relative to the image field in the direction of illumination. Accordingly, tracking the light sheet position in the x-direction can be advantageous. This can, of course, be coupled with the described movement in the z-direction. QUOTES INCLUDED IN THE DESCRIPTION

[0057] This list of documents cited by the applicant was automatically generated and is included solely for the reader's convenience. The list is not part of the German patent or utility model application. The DPMA accepts no liability for any errors or omissions. Cited patent literature

[0058] DE 10257423 A1

[0005] WO 2004 / 0530558 A1

[0005] DE 102007017598 A1

[0013] DE 102007045897 A1

[0013] Cited non-patent literature

[0059] Stelzer et al., Optics Letters 31, 1477 (2006)

[0005] Stelzer et al., Science 305, 1007 (2004)

[0005] (Greger et al., Rev. Sci. Instr. 78, 023705, 2007) described (Section IIB.)

[0015] Journal of Microscopy 170, 229, 1992; section “illumination system”

[0015] Santi et al. (BioTechniques 46, 287, 2009, Suppl. Mat.)

[0015] Krzic et al (Nat Methods 9, 730, 2012, Suppl.)

[0015] Keller et al. (Science 322, 1065, 2008, Supp)

[0015]

Claims

[1] Method for SPIM microscopy using a microscope consisting of – a lighting device comprising a light source and a beam path for illuminating a sample with a light sheet, – a detection device for detecting light emitted by the sample, with a lens that is preferably adjustable in the direction of its optical axis – wherein the light sheet at the focus of the lens or a defined plane near the focus of the imaging lens is essentially planar and the lens has an optical axis that intersects the plane of the light sheet at a non-zero angle, preferably perpendicularly, – whereby the sample is moved by the light sheet in the direction of the optical axis of the objective to capture different sample planes – and adjustment means for adjusting the illumination beam path at least along the optical axis of the detection lens are provided characterized by that – In a first step, calibration is carried out using a sample to be examined by recording and saving the actual position of the flower leaf in different sample planes depending on its position in the sample. – and the stored position of the light sheet is used in a second step during the observation and / or detection of the sample, based on the values ​​stored in the first step, to correct the position of the light sheet to the focal plane of the detection objective. and / or – during the displacement of the sample, the position of the light sheet is adjusted to the focal plane of the detection objective in such a way that the light sheet performs a relative movement in at least one direction to the sample and / or the detection objective performs a relative movement to the sample. [2] Method according to at least one of the preceding claims, wherein the position of the light sheet and / or the detection objective is simultaneous with the movement of the sample or following a sample movement. [3] Method according to at least one of the preceding claims wherein the sample contrast is detected and evaluated during the relative movement. [4] Method according to at least one of the preceding claims wherein the relative movement takes place until a maximum image contrast or an image contrast optimized according to criteria is achieved. [5] Method according to at least one of the preceding claims wherein a relative movement of the light sheet around a set vertical focus position of the detection objective is carried out in order to set the maximum or optimal contrast. [6] Method according to at least one of the preceding claims wherein the coupling of the movement of the light sheet and / or the detection objective relative to the sample is carried out via a control unit which is connected to the adjustment means for the objective and / or the adjustment means for the illumination. [7] Method according to at least one of the preceding claims, wherein the illumination beam path is tilted about at least one axis, preferably about a tilting axis which lies in the optical axis of the lens. [8] Microscope and / or computer program and / or control unit, each or together for carrying out the method according to any of the preceding claims

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