Light sheet microscope and method for operating a light sheet microscope

Abstract

The invention relates to a light sheet microscope and a method for operating a light sheet microscope. In particular, it relates to a microscope comprising: an illumination objective with a first optical axis formed for generating a light sheet, and a detection objective with a second optical axis formed for detecting light emanating from a sample plane. The illumination objective and the detection objective are oriented relative to each other and to the sample plane in such a way that the first optical axis and the second optical axis intersect in the sample plane and form a substantially right angle with each other. The optical axes form an angle different from zero with a reference axis pointing orthogonally to the sample plane. In addition, there is an overview illumination device formed for far-field illumination of the sample plane, which has an illumination optical device with a third optical axis, so that the detection objective provides and forms light for the detection light sheet as well as light of the illumination optical device. The invention further relates to a method for operating a light sheet microscope.

Description

[0001] This application is a divisional application of the original application with a filing date of March 21, 2017, a national application number of 201710169469.1, and an invention name of “Light Sheet Microscope and Method for Operating a Light Sheet Microscope”. Technical Field

[0002] The invention relates to a light sheet microscope and a method for operating a light sheet microscope. Background Art

[0003] Light sheet microscopy is mainly used for imaging medium-sized samples, such as organisms, whose size ranges from a few hundred micrometers to a few millimeters. Usually, these samples are embedded in agar and arranged in a glass capillary. To examine the sample, the glass capillary is placed in a sample chamber filled with water and agar and sample are pressed out of the glass capillary in a block. The sample is illuminated by the light sheet and the fluorescence emitted by the sample is imaged onto a camera with a detection objective perpendicular to the light sheet and thus also perpendicular to the light sheet optics.

[0004] According to the prior art, a microscope structure for a light sheet microscope (SPIM structure, Single Plane Illumination Microscopy) comprises an illumination optical device 2 and a detection optical device 3 (hereinafter also referred to as SPIM objective), which are arranged at an angle of 45° to a sample plane 4 and point perpendicularly to each other from above onto the sample plane 4 (see FIG. 1a). The sample 5 arranged in the sample plane 4 is, for example, located at the bottom of a sample holder 7 formed as a culture dish. The sample holder 7 is filled with a liquid 8, for example water, and the two SPIM objective lenses 2, 3 are immersed in the liquid 8 during the use of the light sheet microscope. The sample plane 4 extends in a plane XY spanned by the X-axis X and the Y-axis Y of a Cartesian coordinate system. The first optical axis A1 and the second optical axis A2 run in a plane XY spanned by the X-axis X and the Y-axis Y of a Cartesian coordinate system.

[0005] This approach offers the advantage of high resolution in the axial direction, since a thin light sheet 6 can be generated by the illumination objective 2. Due to the higher resolution, smaller samples 5 can be examined. Furthermore, interfering background fluorescence is significantly reduced and the signal / background ratio is thus improved.

[0006] According to the prior art, an overview image is generated parallel to the sample plane 4 and the sample holder 7 by recording a far-field image vertically through the transparent bottom of the sample holder 7 with a far-field objective 20 located below the sample 5. Transmitted light illumination of the sample 5 and recording of the transmitted light are not possible here, since the two SPIM objectives 2, 3 are arranged side by side too close to each other.

[0007] In order to achieve a simpler sample preparation in a standard sample holder, such as a multiwave plate, the two SPIM objectives 2, 3 can be directed from below through the transparent bottom of the sample holder 7 in an opposite arrangement into the sample plane 4 (see FIG. 1b ), although the 45° configuration is maintained. In this arrangement, the aberrations caused by the sample holder 7, which is tilted relative to the optical axes A1 and A2 and is in the form of a cover glass, must be corrected by special optical elements (DE 10 2013 107 297 A1, DE 102013 112 596 A1). The sample 5 arranged in the sample plane 4 is illuminated through the bottom of the sample holder 7 and the excited fluorescence of the sample is detected. The sample holder 7, such as a multiwave plate, a culture dish and / or an object carrier, can be used and contamination of the sample, in particular during high throughput scanning, can be avoided.

[0008] The problem with an upright and inverted configuration is that the region of interest must be imaged as an overview image through the sample by means of the far-field objective. For an upright arrangement, this means that the far-field objective must usually be viewed through a few millimeters of liquid, for example an aqueous solution such as a buffer. If, for example, a multi-wave plate is used, a pronounced meniscus is formed on the liquid surface in the individual recesses of the multi-wave plate, which produces a prism effect, so that this imaging is primarily possible only with very low NA. Another solution proposes to move, rotate and / or tilt the illumination and / or detection objective so that the far-field objective can be positioned.

[0009] Contrast methods are often used to visualize contrast-poor objectives (e.g. phase objectives) in microscopes and are often supplemented with fluorescence imaging. Hereby, samples such as cells or tissue sections are imaged in their entirety in transmitted light and their state analyzed.

[0010] Recently, further possibilities for contrast enhancement of objectives with poor contrast (phase objectives) have been developed which in another way provide the same or similar information content as the "classical methods". Examples are half pupil contrast, oblique illumination and TIE (Transport of Intensity Equation).

[0011] A microscope is known from WO 2012 / 110488 A2, which has an optical path for illuminating a sample and at least one detection optical path. The microscope comprises a focusing unit in the illumination optical path for focusing the illumination radiation. The focusing unit defines a substantially two-dimensional illumination area, which extends in the direction of the illumination optical path and transversely to the illumination optical path. In addition, a delimiting element is arranged in the illumination optical path, which is formed as a part for selectively illuminating the illumination area. WO 2012 / 110488 A2 also discloses a microscopy method.

[0012] All of these contrast methods require, in the case of largely transparent samples 5 , transmitted illumination, which for the reasons described above cannot be easily integrated into a microscope 1 with a 45° SPIM arrangement. Summary of the invention

[0013] The object of the present invention is to propose the possibility of a light sheet microscope by which an overview image parallel to the sample plane can be recorded, wherein the overview image has a high information content, in particular a high contrast.

[0014] This object is achieved by a microscope according to independent claim 1 and by a method according to independent claim 11. Advantageous embodiments are the subject matter of the dependent claims.

[0015] The microscope comprises an illumination objective with a first optical axis formed for generating a light sheet, wherein the light sheet is generated or can be generated at least partially in a sample plane, and a detection objective with a second optical axis formed for detecting light emanating from the sample plane, wherein the illumination objective and the detection objective are arranged relative to each other and to the sample plane in such a way that the first optical axis and the second optical axis intersect in the sample plane and form a right angle with each other, and such that the first optical axis and the second optical axis form an angle different from zero with a reference axis pointing orthogonally to the sample plane. Furthermore, there is an overview illumination device formed for far-field illumination of the sample plane, which has an illumination optic with a third optical axis.

[0016] According to the invention, the detection objective provides and is formed for detecting the light of the light sheet and the light of the overview lighting device.

[0017] The sample plane is used as the reference plane in the following text.

[0018] The microscope can be formed as an upright microscope.The microscope can be formed as an inverted microscope in a further configuration.

[0019] Since the third optical axis of the illumination optics is oriented at an angle different from 0° to the second optical axis of the detection objective, oblique overview illumination or far-field illumination (hereinafter referred to as "illumination") is necessarily achieved without having to introduce a shutter in the light path of the illumination optics as is usually the case in conventional tripods and devices.

[0020] Oblique illumination enables the recording of overview images or far-field images with greater contrast (compared to illumination parallel to the optical axis of the detection objective). The illumination optics of the microscope according to the invention need not necessarily be immersed in the liquid of the sample holder (so-called immersion).

[0021] Instead, the sample can be illuminated through the air-water interface with an (air) condenser. This is possible because the aberrations in the illumination that occur here are not critical for the imaging.

[0022] In an advantageous embodiment, the third optical axis coincides with the reference plane. The illumination thus takes place substantially perpendicularly to the reference plane.

[0023] Depending on the geometry of the microscope, for example, the condenser is arranged perpendicular to the sample holder, so that the region of the sample that is within the field of view of the detection objective is illuminated or can be illuminated.

[0024] The first optical axis and the second optical axis are substantially at right angles to each other. The deviation from the right angle is advantageously at most such that the deviation of the first and second optical axes caused by an angular offset in the field of view in the direction of the optical axis of the detection objective no longer exceeds the depth of field of the detection objective. Neither the first optical axis nor the second optical axis is in the sample plane.

[0025] The angle formed by the first optical axis or the second optical axis and the reference plane is substantially 90°.

[0026] The microscope according to the invention advantageously does not require an additional infinity objective for recording the overview image, whereby it requires less space, is of simpler construction and can be produced more cost-effectively than microscopes according to the prior art.

[0027] The detection of the light of the overview lighting device is provided in the transmitted light.

[0028] In a possible configuration of the microscope, the illumination optical system is formed as a condenser, an objective or a prism.

[0029] In order to achieve a high contrast in the overview image, diffuse oblique illumination can be provided in possible designs of the microscope.

[0030] In another configuration, the numerical apertures (NA) of the detection objective and the illumination optics are matched to each other so that they overlap and there is an overlapping area of ​​the numerical apertures. The overlap of the numerical apertures can be determined and / or virtually illustrated, for example, in a two-dimensional or three-dimensional graphical manner, by illustrating and / or simulating the numerical apertures of the microscope in their actual position and orientation. The plane or space used together represents the overlapping area of ​​the numerical apertures.

[0031] In order to influence the contrast of the overview image according to the principles of polarization contrast, differential interference contrast (DIC) or Hoffmann contrast, in another embodiment of the microscope, a polarizer is arranged in the light path of the illumination optical system and in the light path of the detection objective respectively. In another embodiment, at least one phase plate, Hoffmann optics or DIC prism is also arranged.

[0032] Hoffmann optics are optical elements whose arrangement and cooperation can obtain a sample image with Hoffmann contrast. In the case of Hoffmann contrast microscopy (HMC), the microscope typically has a condenser with a slit shutter and a deflector. The deflector is arranged between the condenser and the illumination light source of the illumination optics.

[0033] Another embodiment of the microscope has a diffuser between the detection objective and the optical system. The diffuser is formed, for example, by one or more static or dynamic scattering disks.

[0034] The advantage of diffuse illumination is that no expensive optics are required, such as Köhler illumination with imaging optics, condensers and shutters. Diffuse illumination is performed at many illumination angles and illumination directions. Thus, the effective numerical aperture of the illumination, which influences the image resolution in coherent or partially coherent imaging, can be increased, which is achieved by bringing the diffuser close to the sample plane and the sample.

[0035] The effective illumination NA can thus also be increased in a simple manner by means of a nominally specified value for the illumination optics, for example for an overview of the illumination optics.

[0036] Furthermore, the spatial angle and direction of the illumination can be adjusted, for example by illuminating only a ring or a ring section of the diffuser. This can be achieved simply by a corresponding mask arranged directly on the diffuser or by imaging the mask on the diffuser, wherein the latter case can be achieved, for example, by illuminating the diffuser with a conventional transmitted light condenser with a mask for contrast microscopy. Advantageously, diffuse illumination can be combined with the above-described oblique illumination.

[0037] In one embodiment, the diffuser can be arranged directly in front of an illumination source, which is formed, for example, as a light emitting diode (LED) or OLED (organic LED), an LED array or an OLED array and / or a halogen lamp. In addition, the diffuser can be illuminated or illuminated by an existing (Koehler) microscope illumination device.

[0038] In a further possible embodiment, the diffuse illumination is achieved by using a self-illuminating diffuse illumination light source as a diffuser, for example an LED and / or an OLED with a focusing screen arranged next to it.

[0039] In a further possible embodiment, the microscope has a dimming element, for example a mask, in the beam path of the overview illumination, by which the overlapping region of the numerical apertures is dimmed, so that a dark far-field illumination is or can be generated in the sample plane.

[0040] In a further embodiment of the microscope, the mask is arranged as a dimming element in the pupil of the overview illumination or the illumination optics.

[0041] Dark far-field illumination can be performed by an illumination objective which can be formed as a SPIM illumination objective. Transmitted light illumination can also be performed parallel to the reference axis, for example from above, wherein the overlapping area of ​​the numerical apertures is blocked or can be blocked, for example by a mask, for example by a suitable shutter.

[0042] In a further embodiment, the microscope has a movable mask in the illumination pupil of the overview illumination device, by which the illumination pupil is or can be covered exactly halfway.

[0043] The so-called half-pupil contrast method (Mehta, S. et al. 2009, Optics Letters 34: 1924-1926) realizes the visual illustration of the phase objective in a simple way. For this purpose, a mask is introduced into the illumination pupil, wherein the mask covers half of the illumination pupil. Then, a transmitted light image is taken. In the next step, the mask is introduced into the illumination pupil so that the opposite half of the illumination pupil is covered. The image is taken again. If the mask is exactly in the pupil in the axial direction in Kohler illumination, a uniform illuminated image of the sample with half intensity is generated respectively. However, the phase gradient in the sample causes a lateral shift of the imaging of the illumination pupil on the objective pupil, which is proportional to the phase gradient. The two obtained partial images are then subtracted from each other and standardized, so that the phase gradient can be illustrated.

[0044] The pupil division must be carried out precisely, ie the mask must cover exactly half of the pupil and be located exactly at the position of the pupil in the direction of the optical axis, since otherwise strong artifacts would result in the calculated image.

[0045] In the light sheet arrangement, the above-described method of half pupil contrast is not possible in the manner known from the prior art. In the case of half-sided coverage (half-division) of the pupil, a strongly asymmetrical intensity distribution always results between the partial images in the illumination beam path and in the detection beam path.

[0046] However, this asymmetrical intensity distribution can be avoided if half of the illumination pupil is covered exactly in the plane spanned by the optical axis of the detection objective and the optical axis of the illumination objective.

[0047] In the implementation of a microscope with oblique illumination, the coverage of the illumination pupil can be performed in the illumination light path as well as in the detection light path.

[0048] If collinear illumination is implemented, the illumination pupil can also be covered in the illumination beam path and in the detection optical axis. In this case, the divided orientation principle does not play a role.

[0049] The mask can be configured to be movable, for example, and arranged in the pupil of the detection objective. Through the movable mask, the pupil is precisely covered by half or can be covered by half.

[0050] The object of the invention is further solved by a method for operating a light sheet microscope. The method comprises the steps of illuminating a sample in a sample plane with light from an overview illumination device along a reference axis pointing essentially orthogonally to the sample plane, and detecting the light from the overview illumination device as transmitted light by a detection objective having a detection axis referred to as a second optical axis, wherein the second optical axis of the detection objective forms an angle different from zero with the reference axis. Furthermore, the method comprises the step of generating an overview image of the sample or at least a region of the sample in said step depending on the light from the overview illumination device recorded by the detection objective. Furthermore, advantageously as an alternative to the aforementioned step, the light of the light sheet generated in the reference plane (sample plane) is recorded by the detection objective for recording the light from the overview illumination device. Here, it is sufficient for the light sheet to intersect the sample plane to be regarded as generating a light sheet in the sample plane.

[0051] In a further embodiment of the method, the overview image is generated by TIE (transport of intensity equation). Advantageously, the Z-stack required for TIE is always available and no additional method steps have to be performed.

[0052] The image is captured during the mutual movement of the sample and the focus of the detection objective in the sample plane, for example in the direction of the X-axis. Here, an image of the sample is captured at each capture position and the image is stored as required. The captured images are combined into an image stack, which is usually called a Z stack because it is generated in conventional microscopes by movement of the objective or the sample along the Z axis. In the present case, the image plane of the detection objective, in which the field of view extends and in which the focus is located, is inclined relative to the sample plane and extends along the first optical axis. In this regard, reference is also made to FIGS. 7a and 7b as well as Figures 8a to 8c and description of the belonging.

[0053] The method can be carried out in another configuration by recording a Z-stack of the plane, i.e., individual images of the plane, wherein when the plane is tilted relative to the third optical axis and the sample plane, the plane is transformed into a Z-stack in which the plane is not tilted relative to the third optical axis and the sample plane, by virtually surrounding the recorded Z-stack, whose individual planes are shifted (skewed) with respect to each other, with a grid having X-axes, Y-axes and Z-axes running orthogonally to each other, wherein the X-axis and the Y-axis are oriented parallel to the sample plane and the Z-axis is oriented perpendicular to the sample plane.

[0054] The distance of the planes of the normalized Z-stack in the direction of the Z-axis is chosen such that it corresponds to the one of the lateral resolution of the recorded Z-stack, so that:

[0055] Δx'=Δx

[0056] Δy' = Δy, and

[0057] Δz'=Δx or Δy.

[0058] Then calculate the new grid points P of the unshifted normalized Z-stack (de-skewed) x,y,z , and through the adjacent grid points P of the recorded Z stack x’,y’,z’ The three weighted interpolation calculations of each new grid point P x,y,z The strength on.

[0059] The specifications X, Y and Z relate to the directions of the axes of a Cartesian coordinate system.

[0060] The symbols x', y' and z' represent the coordinates of the grid points. The symbols x, y and z represent the coordinates of the grid point P in the unshifted (deskewed, de-skewed) grid. x’,y’,z’ The coordinates of .

[0061] The symbols Δx, Δy, Δz and Δx', Δy', Δz' denote the difference between coordinates in the direction of one of the axes X, Y and Z. The distance between adjacent XY planes of the Z stack in the direction of the Z axis is also given by the symbols Δz' and Δz and is called the step size.

[0062] In a variant embodiment of the method, a Z-stack of a plane is recorded, wherein the plane is transformed into the XY plane of a standardized Z-stack, the recording being carried out as follows:

[0063] The recorded Z-stack, whose individual planes are shifted (skewed) relative to one another, is virtually surrounded by a grid with mutually orthogonal X-, Y- and Z-axes, wherein the X- and Y-axes are oriented parallel to the sample plane and the Z-axis is oriented perpendicular to the sample plane,

[0064] The distance of the XY plane of the normalized Z-stack in the direction of the Z-axis is chosen such that the distance corresponds to the one of the lateral resolution of the recorded Z-stack (skew), such that:

[0065] Δx'=Δx

[0066] Δy'=Δy

[0067] Δz'=Δy*sin(α1),

[0068] The angle α1 is formed by the first optical axis and the third optical axis.

[0069] Calculate the new grid points P of the unshifted normalized Z-stack (deskewed) x,y,z , and through the adjacent grid points P of the recorded Z stack x’,y’,z’ The three weighted interpolation calculations of each new grid point P x,y,z The strength on.

[0070] In a further embodiment of the method, the acquisition speed of the planar recording is adjusted as follows:

[0071] Adjust the step length Δz' between two planes to be recorded, or

[0072] recording a Z-stack with a first step size Δz', selecting a region of interest (ROI) and recording the selected ROI with a second step size Δz', wherein the second step size Δz' is smaller than the first step size Δz', and thereby the resolution of the ROI recorded with the second step size Δz' is higher than the resolution of the ROI recorded with the first step size, or

[0073] Only one XY plane parallel to the reference plane is calculated and displayed separately.

[0074] It is also possible to select a single row of regions of interest in the direction of the X-axis or the Y-axis and to record a single row of regions of interest in the direction of the Z-axis for each XY plane.

[0075] Each XY plane moves, for example, by an amount of Δ=Δz / tan(α1) relative to the previous XY plane. BRIEF DESCRIPTION OF THE DRAWINGS

[0076] The present invention is explained in detail below based on embodiments and drawings. The drawings are:

[0077] 1 a shows a schematic illustration of a microscope according to the prior art with a 45° arrangement of illumination and detection objectives above the sample plane and a far-field objective below the sample;

[0078] 1 b shows a schematic illustration of a microscope according to the prior art in reverse with a 45° arrangement of the illumination objective and the detection objective below the sample plane and a far-field objective above the sample;

[0079] Figure 2 a shows a schematic illustration of a first embodiment of a microscope according to the present invention;

[0080] Figure 2 b shows a schematic illustration of the numerical aperture of a first embodiment of a microscope according to the invention;

[0081] FIG. 3 a shows a schematic illustration of a second embodiment of a microscope according to the invention with a diffuser according to the invention;

[0082] FIG3 b shows a schematic illustration of the numerical aperture of a second embodiment of a microscope according to the present invention;

[0083] FIG. 4 a shows a schematic illustration of a third embodiment of a microscope with a mask according to the present invention;

[0084] FIG4 b shows a schematic illustration of the numerical aperture of a third embodiment of a microscope according to the present invention;

[0085] FIG. 5 a shows a schematic illustration of a fourth embodiment of a microscope according to the present invention with a half-side mask;

[0086] FIG. 5 b shows a schematic illustration of a fourth embodiment of a microscope according to the invention in a side view, with a schematically illustrated pupil overlay;

[0087] Figure 6 shows a schematic illustration of a (fifth) embodiment of a microscope according to the invention with a symbolized recording area;

[0088] FIG. 7 a shows a schematic illustration of a Z-stack observed along the optical axis of the detection objective of a microscope according to the present invention;

[0089] FIG7 b shows a schematic illustration of a Z-stack in a side view of the sample;

[0090] Figure 8a Schematic illustration of a moving recorded Z-stack (skew) and a virtual grid is shown;

[0091] Figure 8b shows a schematic illustration of a first transformation 1; and

[0092] Figure 8c A schematic illustration of a second transformation 2 is shown.

[0093] In the following embodiments and schematic diagrams, the same reference numerals refer to the same elements. DETAILED DESCRIPTION

[0094] From the prior art, an upright microscope 1, schematically illustrated in FIG. 1 a, is known, which has an illumination objective 2, a detection objective 3 and a far-field objective 20. A light sheet 6 is or can be generated by the illumination objective 2 along a first optical axis A1, which can be used to examine a sample 5 arranged in a sample plane 4. The detection objective 3 has a second optical axis A2, along which light emitted from the sample plane 4 can be recorded. The first optical axis A1 and the second optical axis A2 are oriented orthogonally to each other and each form an angle of 45° with the sample plane 4, which serves as a reference plane B.

[0095] The far-field objective 20 has a third optical axis A3, which is oriented orthogonally to the sample plane 4 and serves as a reference axis B. The first optical axis A1 to the third optical axis A3 intersect in the region of the extension of the light sheet 6 within the sample 5. Furthermore, the first optical axis A1 forms a first angle α1 with the reference axis B and the second optical axis forms a second angle α2 with the reference axis B of 90°-α1, for example 45° respectively.

[0096] The sample 5 is arranged in a sample holder 7 which is filled with a liquid 8 .

[0097] 1 b schematically shows a microscope 1 with an inverse arrangement of illumination objective 2 and detection objective 3 , wherein illumination objective 2 and detection objective 3 are arranged below sample plane 20 and far-field objective 20 is arranged above sample plane 20 . Angles α1 and α2 are again each 45°.

[0098] The exemplary embodiments described below are shown by way of example with the aid of an inverted microscope 1 and can also be designed as an upright microscope 1 in other embodiments.

[0099] exist Figure 2 In the reverse microscope 1 schematically illustrated in a, the illumination 2 objective and the detection objective 3 are arranged at angles α1 and α2 of 45°.

[0100] In other embodiments of the microscope 1 , the angles α1 and α2 have angle values ​​that deviate from 45°, wherein the angles substantially add up to 90°.

[0101] As an alternative to the far-field objective 20 (see FIGS. 1a and 1b ), the illumination optics 9 of the overview illumination device are present in the form of a condenser which is formed to send light into the sample plane 4 along a third optical axis A3 coinciding with the reference axis B. The condenser is formed as an air condenser in a further embodiment. The illumination optics 9 is formed as an optical lens in a further embodiment.

[0102] In a further embodiment, the illumination optical system 9 is formed by an illumination objective 20. The illumination objective can be formed for observation and / or imaging of the sample 5 in addition to illumination.

[0103] The region of the sample 5 that is within the field of view of the detection objective 3 is illuminated by the illumination optical system 9. Since the third optical axis A3 of the illumination optical system 9 is oriented at an angle not equal to 0° or 180° to the second optical axis A2, oblique illumination is necessarily present, unlike the introduction of a mask into the beam path of the illumination optical system 9 in a corresponding manner as in conventional tripods.

[0104] The oblique illumination enables the recording of the far-field image as an overview image with enhanced contrast by the detection objective 3. The illumination optical device 9 does not necessarily have to be immersed in the liquid 8, for example water, of the sample holder 7 (water immersion). Instead, the (air) condenser can illuminate the sample 5 through the air-liquid interface. This is possible because the aberrations that occur in the illumination here are not critical for the imaging.

[0105] In a further embodiment of the microscope 1 , polarizers, Hoffmann optics and / or DIC prisms are selectively arranged in suitable combinations in the beam path of the illumination optics 9 and in the beam path of the detection objective 3 , respectively, as schematically indicated by the blocks designated by reference numeral 10 .

[0106] exist Figure 2 b shows the opening cone of the illumination optical device 9, namely the numerical aperture NA1, and the numerical aperture NA2 of the detection objective lens 3, as well as the region where the numerical apertures NA1 and NA2 overlap (overlap region). The first numerical aperture NA1 and the second numerical aperture NA2 do not extend parallel to each other.

[0107] The numerical aperture NA1 of the illumination optics 9 is schematically illustrated as an opening angle on the objective side and as an opening angle on the image side along the third optical axis A3 .

[0108] This graphical representation of numerical aperture is also used in the following embodiments.

[0109] A diffuser 12 in the form of a diffuser disk is arranged in the beam path of the illumination optics 9 between the illumination optics 9 and the sample 5 (FIG. 3a). The light of the illumination optics 9 scattered by the diffuser 12 is illustrated by a semi-ellipse enclosed by a dashed line (FIG. 3b).

[0110] In relation to the numerical apertures NA1, NA2 and their mutual overlap, only the portion of the illumination light from the overlapping area can be detected by the detection objective 3. The degree of overlap can be adjusted by matching the numerical aperture NA1 of the illumination objective 20 or the illumination optical device 9. In order to achieve the highest possible resolution, it is necessary to work with high numerical apertures NA1 and NA2 on the illumination side and on the detection side. In the case of samples with low structural density, such as samples with discrete small objects, a strong overlap of NA1 and NA2 is disadvantageous because small changes must be detected in front of a bright background.

[0111] This disadvantageous effect is avoided by arranging a mask 11 in the pupil of the overview illumination device or the pupil of the illumination optics 9 which precisely blocks the overlapping region of NA1 and NA2 , as schematically illustrated in FIG. 4 a .

[0112] In FIG. 4 b , the region of the numerical aperture NA1 that is masked by the mask 11 is labeled NA1mask, and the region that is not masked is labeled NA1unmask.

[0113] The mask can thus block the overlapping area as depicted, thereby achieving a dark field, wherein only scattered light is detected by the detection objective 3 within the sample 5. Alternatively, the non-overlapping area can be blocked by the mask 11, thereby achieving an optimal oblique illumination.

[0114] In a fourth embodiment of a microscope 1 schematically illustrated here in FIG. 5 a , a mask 11 is arranged in the beam path of the overview illumination, which results in half-side coverage of the overview illumination pupil and can produce a half-pupil contrast, as described above.

[0115] FIG. 5 b shows diagrammatically the overlap in the beam path of the overview illumination device and the illumination optics 9 and the resulting obstruction in the beam path of the detection objective 3 .

[0116] Each of the aforementioned embodiments may comprise a control unit 13 (illustrated only in FIG. 5 a ), which may be formed for controlling an illumination light source, an illumination objective 2, a detection objective 3 and / or an illumination optics 9, not illustrated in detail. Furthermore, the control unit 13 may be formed for evaluating the recorded overview image and / or the image of the light sheet 6. The control unit 13 may be connected to a display for graphically illustrating the recorded overview image and / or the image of the light sheet 6.

[0117] By implementing the microscope 1 according to the invention, fundamentally different possibilities for image recording are possible. Figure 6 The examples explain some of the possibilities.

[0118] The simplified illustration of the light sheet 6 also shows the objective plane of the detection objective 3. The sample scanning can be carried out with a first scanning movement SB1 in the direction of the sample plane 4. In this case, a sample volume enclosed by a solid circle and shown as a rectangle for simplified illustration is scanned.

[0119] In a further possibility, a relative movement between the sample 5 and the light sheet 6 or the objective plane in the direction of the second optical axis A2 is performed as a second scanning movement SB2. In this case, a sample volume, which is shown enclosed by a solid circle, is scanned.

[0120] A third possibility is to produce a relative movement in the direction of the third optical axis A3 as a third scanning movement SB3 , wherein a sample volume indicated by the dash-dot circle is scanned.

[0121] Furthermore, combinations of scanning movements SB1 , SB2 and / or SB3 are also possible.

[0122] The scanned sample volume can then be transferred into a Z-stack by the transformation described below.

[0123] A person skilled in the art may consider combinations of the embodiments.

[0124] The method according to the invention can be implemented with each of the above-described embodiments of the microscope 1 .

[0125] According to the accompanying drawings, in particular according to Figures 7a, 7b and Figure 8a , Figure 8b and Figure 8c The construction of the method is described below.

[0126] 7a schematically and for reasons of illustration, a Z-stack is shown only in perspective, as would be possible in the coordinate system of the detection objective 3 and of a detector (not shown). The mutual distances of the recorded individual images EB in the direction of the Z axis Z are marked with Δz' (see below).

[0127] FIG. 7 b shows a Z-stack in the coordinate system of the sample 5 in a side view rotated relative to FIG. 7 a .

[0128] exist Figure 8a A virtual grid is shown by way of example and schematically in , wherein the X-axis X and the Y-axis Y are oriented parallel to the sample plane 4 and the Z-axis Z is oriented perpendicular to the sample plane 4. The recorded individual images EB forming a shifted Z-stack are schematically illustrated in the figure.

[0129] Figure 8b schematically illustrates a transformation 1 described in detail below, and Figure 8c Transformation 2 is schematically illustrated.

[0130] In the method, firstly, a single image EB is obtained (FIG. 7a and FIG. 7b), which is recorded with the above-described implementation of the microscope 1. The single image EB is perpendicular to the second optical axis A2 of the detection objective 3 (FIG. 7a) and is oriented at an angle α2 relative to the sample plane 4 (FIG. 7b). A plurality of single images EB are recorded at recording positions, wherein the recording positions are each spaced apart by a pre-adjusted or selectable step size (marked with Δz') from one another. By moving the sample 5 in the X direction X (scanning) and recording the single image EB at each recording position, a Z stack (3D volume stack) can be generated, which in this case is a shifted Z stack, since the single image EB is oriented at an angle α2 relative to the sample plane 4 (FIG. 7b).

[0131] A Z-stack is a sequence of individual images EB which follow one another in the direction of the Z-axis Z. This can be easily implemented for samples 5 which have no preferred orientation.

[0132] If, for example, the sample 5 does not have an arbitrary position and / or extension in space due to external and / or practical circumstances, the sample 5 has a preferred direction. For example, a cell resting or growing on a base, such as a glass plate. The shape of the cell along its contact surface with the base is predetermined by the base and is essentially flat. The cell therefore has a preferred direction facing away from the base.

[0133] In particular for samples 5 with a preferred orientation, for example grown on a cover glass as a base, it is advantageous for the user to have a nominally non-shifting Z-stack of the sample, as is usually the case, for example, in laser scanning microscopes (LSM) or when using a rotatable aperture plate (spinning disk). The individual layers of the corresponding Z-stack are oriented parallel to the sample plane 4 (XY plane). Therefore, the recorded Z-stack must be converted into a standardized non-shifting Z-stack by a suitable transformation ("de-skew"), the XY plane of which is oriented parallel to the sample plane 4. This can be achieved, for example, by the following two transformations:

[0134] Transformation 1 (xyz interpolation)

[0135] Surround the original recorded Z-stack with a grid ( Figure 8a ), where the X-axis X and the Y-axis Y are placed parallel to the sample plane 4 and the Z-axis Z is oriented perpendicular to the sample plane 4. The distance Δz' of the individual planes can be chosen to correspond to the lateral resolution of the original Z-stack to achieve isotropically identical voxel sizes:

[0136] Δx'=Δx

[0137] Δ'=Δy

[0138] Δz'=Δx or Δy

[0139] At the new grid point Px,y,z The intensity is calculated by the adjacent grid points P of the original grid x’,y’,z’ The distances subject to interpolation are marked with IP1, IP2 or IP3.

[0140] Transformation 2 (y interpolation)

[0141] Virtually surround the original recorded Z-stack with a grid ( Figure 8a ), wherein the X-axis X and the Y-axis Y are placed parallel to the sample plane 4 and the Z-axis Z is oriented perpendicular to the sample plane 4. The distance Δz' of the individual grid planes can be selected such that:

[0142] Δx'=Δx

[0143] Δy'=Δy

[0144] Δz'=Δy*sin(α1)

[0145] At the new grid point P x,y,z The intensity is calculated by the adjacent grid points P of the original grid x’,y’,z’ The weighted interpolation of Figure 8c ).

[0146] After carrying out both transformations 1 or 2 , there is a transformed and therefore normalized Z-stack with an XY plane parallel to the sample plane 4 .

[0147] The recording speed achievable by the method can be further increased by one of the following four possibilities A to D or by a combination thereof.

[0148] A. If the overview image is generated parallel to the sample plane 4, the disadvantage is that a complete Z stack must always be recorded. This results in a longer time being required to generate the overview image. The waiting time can be shortened by increasing the step length Δz between the two XY planes and thus requiring fewer images to be recorded.

[0149] B. Alternatively, a longer waiting time can also be accepted to record a large volume of the sample 5 with a lower resolution (e.g. including tilt). Then, the entire volume is virtually viewed with the help of the 3D-Viewer and the region of interest (ROI) is identified. After the overview image, this region of interest can be recorded with a higher resolution, for example with a smaller step size Δz between the XY planes.

[0150] C. A further acceleration of the method can be achieved when, instead of calculating the entire Z-stack at once, only the XY plane parallel to the sample plane 4 is calculated and immediately displayed. Each interpolation can be omitted.

[0151] D. Alternatively, it is also possible to directly read out the individual lines, for example on a camera, by selecting a single line ROI on the detector. Instead of recording a large individual image EB in the Z direction Z, many lines are now recorded in the Z direction Z. This can be done faster than recording a complete individual image EB.

[0152] In methods C and D it is possible to select which row and therefore which XY plane is displayed parallel to the sample plane 4. When displaying it must be noted that each individual XY plane must be shifted by Δ=Δz / tan(α1) relative to the previous XY plane.

[0153] List of reference numbers

[0154] 1 Microscope

[0155] 2 Illumination objective

[0156] 20 Far field objective

[0157] 3. Detection lens

[0158] 4 Sample plane

[0159] 5 Samples

[0160] 6 Light Sheet

[0161] 7 Sample holding unit

[0162] 8 Liquid

[0163] 9 Illumination optics

[0164] B Reference axis

[0165] 10 Polarizers, Hoffmann optics, DIC prisms

[0166] 11 Mask

[0167] 12 Diffuser

[0168] 13 Control unit

[0169] EB single image

[0170] NA1 Numerical aperture (of illumination optics 9)

[0171] NA2 (Numerical aperture of detection lens 3)

[0172] NA1mask (NA1's) occluded area

[0173] NA1unmask (NA1's) unmasked area

[0174] A1 First optical axis

[0175] A2 Second optical axis

[0176] A3 Third optical axis

[0177] IP1 First interpolation

[0178] IP2 Second Interpolation

[0179] IP3 third interpolation

[0180] α1 (angle between the first optical axis A1 and the third optical axis A3)

[0181] α2 (angle between the second optical axis A2 and the third optical axis A3)

Claims

1. A microscope (1), comprising: An illumination objective (2) with a first optical axis (A1) formed for generating a light sheet (6), wherein the light sheet (6) is generated or can be generated at least partially in a sample plane (4), and a detection objective (3) with a second optical axis (A2) formed for detecting light emitted from the sample plane (4), wherein: The illumination objective (2) and the detection objective (3) are oriented relative to each other and to the sample plane (4) such that the first optical axis (A1) and the second optical axis (A2) intersect in the sample plane (4) and form a right angle with each other, and The first optical axis (A1) and the second optical axis (A2) each form an angle different from zero with a reference axis (B) pointing orthogonally to the sample plane (4), An overview lighting device is formed for far-field illumination of a sample plane (4), which has an illumination optics (9) with a third optical axis (A3), and The detection objective (3) provides and forms the light for detecting the light sheet (6) and the light of the illumination optical device (9), Characterized in that the third optical axis (A3) of the illumination optical device (9) is directed along the reference axis (B) and at an angle not equal to 0° to the second optical axis (A2), thus producing an oblique overview illumination, An overview image is generated and the light of the light sheet (6) is detected by means of a detection objective (3).

2. The microscope (1) according to claim 1, characterized in that: A mask (11) is arranged in the pupil of the illumination optics (9).

3. Microscope (1) according to any one of the preceding claims, characterized in that A diffuser (12) is arranged between the detection objective (3) and the illumination optical device (9).

4. The microscope (1) according to claim 3, characterized in that The diffuser (12) is formed as an LED and / or OLED with a focusing screen arranged behind it.

5. The microscope (1) according to claim 1, characterized in that: A mask (11) is present in the beam path of the illumination optics (9), by means of which the overlapping region is darkened, so that a dark field illumination is or can be generated in the sample plane (4).

6. The microscope (1) according to claim 1, characterized in that: A movable mask (11) is located in the pupil of the illumination optical system (9), by which the pupil is precisely half-covered or can be covered.

7. A method for operating a light sheet microscope (1), the method comprising the following steps: - illuminating a sample (5) located in the sample plane (4) with light from an overview lighting device along a reference axis (B) pointing essentially orthogonally to the sample plane (4), - detecting the light of the overview illumination device as transmitted light through a detection objective (3) having a second optical axis (A2), wherein this second optical axis (A2) of the detection objective (3) forms an angle different from zero with the reference axis (B), thus generating an oblique overview illumination, - generating an overview image of the sample (5) as a function of the light of the overview illumination device recorded by the detection objective (3), and - detecting the light of the light sheet (6) generated in the sample plane (4) along the first optical axis (A1) of the illumination objective (2) by means of the detection objective (3), wherein: The first optical axis (A1) and the second optical axis (A2) intersect in the sample plane (4) and form a right angle with each other.

8. The method according to claim 7, characterized in that The symbols x, y, and z represent the grid point P in the unmoved grid. x’,y’,z’ , and wherein the symbols Δx, Δy, Δz and Δx′, Δy′, Δz′ represent the difference between the coordinates in the direction of one axis of the axes X, Y and Z.

9. The method according to claim 8, characterized in that - recording a Z stack of XY planes, wherein the XY planes with a preferred orientation are transformed into a standardized Z stack, the XY planes of which do not have a preferred orientation, which is done as follows: Virtually enclosing the recorded deflected Z-stack with a grid having mutually orthogonal X-, Y- and Z-axes (X, Y, Z), wherein the X-axis (X) and the Y-axis (Y) are oriented parallel to the sample plane (4) and the Z-axis is oriented perpendicular to the sample plane (4), The distance of the XY plane of the normalized Z-stack in the Z-axis (Z) direction is chosen such that it corresponds to one of the lateral resolutions of the recorded Z-stack, such that: Δx'=Δx Δy'=Δy, and Δz'=Δx or Δy, - Calculate the new grid points (P x,y,z ),and - through the adjacent grid points (P x’,y’,z’ ) are used to calculate the new grid points (P x,y,z ) on the strength.

10. The method according to claim 8, characterized in that - recording a Z stack of XY planes, wherein the XY planes with a preferred orientation are transformed into a standardized Z stack, the XY planes of which do not have a preferred orientation, which is done as follows: Virtually enclosing the recorded deflected Z-stack with a grid having mutually orthogonal X-, Y- and Z-axes (X, Y, Z), wherein the X-axis (X) and the Y-axis (Y) are oriented parallel to the sample plane (4) and the Z-axis (Z) is oriented perpendicular to the sample plane (4), The distance of the XY plane of the normalized Z-stack in the Z-axis (Z) direction is chosen such that it corresponds to one of the lateral resolutions of the recorded Z-stack, such that: Δx'=Δx Δy'=Δy Δz'=Δy*sin(α1), Wherein the angle α1 is formed by the first optical axis (A1) and the third optical axis (A3), - Calculate the new grid points (P x,y,z ),and - through the adjacent grid points (P x’,y’,z’ ) are used to calculate the new grid points (P x,y,z ) on the strength.

11. The method according to claim 8, characterized in that The recording speed of the XY plane can be adjusted as follows: a) adjust the step length Δz' between the two XY planes to be recorded, or b) recording a Z-stack with a first step size (Δz'), selecting a region of interest and recording the selected region of interest with a second step size (Δz'), wherein the second step size (Δz') is smaller than the first step size (Δz'), or c) Only one XY plane parallel to the sample plane (4) is calculated and displayed respectively.

12. The method according to claim 8, characterized in that A single row of regions of interest are selected in the direction of the X axis (X) or the Y axis (Y), and a single row of regions of interest are recorded for each XY plane in the direction of the Z axis (Z).

13. The method according to claim 8, characterized in that Only one XY plane parallel to the sample plane (4) is calculated and displayed respectively, wherein each XY plane is shifted by an amount Δ=Δz′ / tan(α1) relative to the previous XY plane.

14. The method according to claim 12, characterized in that Each XY plane moves by an amount of Δ=Δz′ / tan(α1) relative to the previous XY plane.

Images