阅读说明：本技术 用于借助光片显微镜对样本成像的方法 (Method for imaging a sample by means of a light-sheet microscope ) 是由 西奥·拉瑞欧 克里斯蒂安·舒曼 于 2019-01-29 设计创作，主要内容包括：介绍一种用于借助光片显微镜(10)对样本成像的方法。其中,利用两个光片(58、60)从两个不同的照明方向对所述样本照明,这些光片具有不同的偏振状态,并且在所述样本的目标区域(E)中彼此共面地重叠。借助所述光片显微镜(10)的成像光学机构(14)产生被照明的所述目标区域(E)的图像。利用两个所述光片(58、60)在被照明的所述目标区域(E)中产生干涉图案(I),由此对所述目标区域(E)的图像施加与所述干涉图案(I)相应的图像调制。分析所述图像调制。根据所分析的所述图像调制,相对于所述成像光学机构(14)的清晰区域(F)来调节被照明的所述目标区域(E)。(A method for imaging a sample by means of a light sheet microscope (10) is described. Wherein the sample is illuminated from two different illumination directions by means of two light sheets (58, 60) which have different polarization states and which overlap one another coplanarly in a target area (E) of the sample. An image of the illuminated target region (E) is generated by means of an imaging optical system (14) of the light sheet microscope (10). An interference pattern (I) is generated in the illuminated target area (E) by means of the two light sheets (58, 60), whereby an image modulation corresponding to the interference pattern (I) is applied to the image of the target area (E). The image modulation is analyzed. Adjusting the illuminated target region (E) relative to a clear region (F) of the imaging optics (14) as a function of the analyzed image modulation.)

1. A method for imaging a sample by means of a light sheet microscope (10), wherein,

illuminating the sample from two different illumination directions with two light sheets (58, 60) having different polarization states and overlapping each other coplanar in a target area (E) of the sample; and the number of the first and second electrodes,

generating an image of the illuminated target region (E) by means of an imaging optics (14) of the light sheet microscope (10),

it is characterized in that the preparation method is characterized in that,

generating an interference pattern (I) in the illuminated target area (E) with the two light sheets (58, 60), whereby an image modulation corresponding to the interference pattern (I) is applied to the image of the target area (E),

the image modulation is analyzed and, in addition,

adjusting the illuminated target region (E) relative to a clear region (F) of the imaging optics (14) as a function of the analyzed image modulation.

2. Method according to claim 1, characterized in that the image modulation is analyzed in such a way that the amplitude of the image modulation is determined and the illuminated target region (E) is adjusted relative to a clear region (F) of the imaging optics (14) in such a way that the amplitude is maximized.

3. Method according to any one of the preceding claims, characterized in that features of the interference pattern (I) are specified and the image modulation is analyzed with the aid of the specified features.

4. The method as claimed in any of the preceding claims, characterized in that the interference pattern (I) is produced in the form of a fringe pattern whose interference fringes extend parallel to a bisector of an angle subtended by the illumination directions of the two light sheets (58, 60) with respect to one another, wherein the fringe pattern is characterized by a modulation period in accordance with the following relationship: where f is the modulation period, λ is the wavelength of the light sheet, and α is the angle.

5. The method according to any of the preceding claims, wherein the image modulation is analyzed by fourier-analyzing the image.

6. The method as claimed in one of the preceding claims, characterized in that the illuminated target region (E) is adjusted relative to a clear region (F) of the imaging optics (14) in such a way that the two light sheets (58, 60) overlapping one another coplanar move jointly along the optical axis (O') of the imaging optics (14).

7. The method as claimed in one of the preceding claims, characterized in that the two light sheets (58, 60) are transferred into an interference-capable polarization state before the illuminated target region (E) is adjusted relative to a clear region (F) of the imaging optics (14).

8. The method according to claim 7, characterized in that the two light sheets (58) are linearly polarized non-orthogonally to one another before the illuminated target area (E) is adjusted relative to a clear area (F) of the imaging optics (14).

9. The method as claimed in claim 7 or 8, characterized in that after adjusting the illuminated target area (E) relative to the clear area (F) of the imaging optics (14), the two light sheets (58, 60) are brought into a polarization state which cannot interfere.

10. Method according to any of the preceding claims, characterized by a pre-adjustment in which the illuminated target region (E) is adjusted relative to a clear region (F) of the imaging optics (14) in dependence on the brightness of the image.

11. A light sheet microscope (10), comprising:

an illumination unit (12) designed for illuminating a sample from two different illumination directions with two light sheets (58, 60) having different polarization states and overlapping each other coplanarly in a target area (E) of the sample;

imaging optics (14) designed to produce an image of the illuminated target region (E); and

a control unit (44) for controlling the operation of the motor,

it is characterized in that the preparation method is characterized in that,

the control unit (44) is designed to control the illumination unit (12) such that an interference pattern (I) is generated in the illuminated target area (E) by means of the two light sheets (58, 60), whereby an image modulation corresponding to the interference pattern (E) is applied to the image of the target area (E),

the control unit (44) is designed to analyze the image modulation, and the control unit (44) is designed to control the illumination unit (12) and/or the imaging optics (14) in order to adjust the illuminated target region (E) relative to a clear region (F) of the imaging optics (14) in accordance with the analyzed image modulation.

12. The light sheet microscope (11) as claimed in claim 10, characterized in that the illumination unit (12) comprises:

a light source (16) designed for generating an illumination beam (52);

a first polarization component (20) which is designed to split the illumination light beam (52) into two differently polarized partial beams (54, 56); and

an illumination optics (14) which is designed to produce two light sheets (58, 60) from the two partial beams (54, 56) for illuminating the target region (E).

13. The light-sheet microscope (10) as claimed in claim 11, characterized in that the illumination unit (12) comprises a deflection unit (26) which can be controlled by the control unit (44) and by means of which two light sheets (58, 60) overlapping one another in a coplanar manner can be moved jointly along the optical axis (O') of the imaging optics (14).

14. A light sheet microscope (10) as claimed in claim 11 or 12, characterized in that the illumination unit (12) comprises a second polarization component (22) which can be controlled by the control unit (44) and is designed to selectively transition the two light sheets (58, 60) into an interference-capable polarization state and into an interference-incapable polarization state.

15. The light sheet microscope (10) as recited in claim 14, wherein the second polarizing component (22) is a retardation plate.

Technical Field

The invention relates to a method for imaging a sample by means of a light sheet microscope, wherein the sample is illuminated from two different illumination directions by means of two light sheets, and an image of the illuminated target region is generated by means of the imaging optics of the light sheet microscope, the light sheets having different polarization states and overlapping one another in a coplanar manner in the target region of the sample. The invention also relates to a correspondingly operating light sheet microscope.

Background

In so-called light sheet or light plate microscopy, a target area of a sample is illuminated with a thin light sheet by means of illumination optics and the thus illuminated target area is imaged by means of imaging optics, the optical axis of which is perpendicular to the optical axis of the illumination optics. Three-dimensional imaging can be achieved by moving the target region illuminated with the light sheet, ideally coinciding with the clear region of the imaging optics, continuously along the optical axis of the imaging optics through the sample. The advantage of this method, which is used in wide-field fluorescence microscopy, is in particular also that the light load on the sample is particularly low.

However, the problem is that the illumination light propagates perpendicularly to the optical axis of the imaging optics. The illumination light is scattered or absorbed by scattering centers or absorbers inside the specimen, which can be detected in the resulting image in the form of streak artifacts along the propagation direction of the illumination light.

In order to reduce such artifacts, it is proposed in patent DE 102016108384B 3 to illuminate the target area of the specimen not with only one light sheet, but with two light sheets, which are directed from the same side, but from different illumination directions, toward the target area and overlap there coplanar with one another, i.e. in a common illumination plane. This light sheet illumination is realized in that the illumination beam passes through a Wollaston prism, which splits the illumination beam into two differently linearly polarized partial beams, which are deflected away from the optical axis of the illumination beam path and thus enter the illumination plane, i.e. into the target region of the sample, from different illumination directions. If the illumination light obscuration now occurs in one of the two illumination directions due to the scattering centers or the absorber, sufficient light sheet illumination of the target region is always ensured by the other illumination direction which is not influenced by the scattering centers or the absorber.

In order to ensure as sharp an image as possible in a light sheet microscope, a precise spatial overlap is required between the illumination plane, i.e. the target region defined by the light sheet thickness, and the focal plane, i.e. the clear region of the imaging optics defined by the clear depth. In the prior art, the fine adjustment, referred to below as overlap adjustment, required for this purpose is mostly carried out by visual evaluation, for example using a reference specimen with small fluorescent particles, by means of which a spatial overlap is produced between the illuminated target region and the clear region of the imaging optics. The microscopically imaged sample image itself may also be visually evaluated for overlay adjustment. However, this procedure only allows relatively coarse overlap adjustment. In particular, the introduction of the reference specimen does not reflect the real imaging situation in which aberrations occur due to refractive index misadjustments, for example, due to the sample. In contrast, a relatively complicated adjustment method provides for a mirror to be introduced into the illumination beam path, which, once adjusted, deflects the light sheet into the reference position of the detector arranged in the image plane of the imaging beam path.

Common to all the aforementioned methods is that they do not enable automatic overlap adjustment. It is considered here that the adjustment based on the automatic analysis of the sharpness of the sample image involves the following difficulties: the spatial spectrum of the sample to be imaged, resulting from the fourier transform of the sample image, is usually unknown. This means, in particular, that it is not known in advance whether the spatial spectrum of the sample has a high spatial frequency at all, which is due to a small sample structure which is in turn suitable for regulation.

Disclosure of Invention

The object of the present invention is to provide a method for imaging a sample by means of a light sheet microscope and a light sheet microscope itself, which allow precise and automatic adjustment of the spatial overlap between the illuminated target region of the sample and the clear region of the imaging optics.

The invention achieves this object by the subject matter of the independent claims. Advantageous developments are given in the dependent claims.

The invention provides a method for imaging a sample by means of a light sheet microscope, wherein the sample is illuminated from two different illumination directions by means of two light sheets, and an image of the illuminated target region is generated by means of the imaging optics of the light sheet microscope, the light sheets having different polarization states and overlapping one another in a coplanar manner in the target region of the sample. An interference pattern is generated in the illuminated target area using the two light sheets, thereby applying an image modulation corresponding to the interference pattern to the image of the target area. The image modulation is analyzed and the illuminated target area is adjusted relative to the clear area of the imaging optics according to the analyzed image modulation.

The invention provides for the illuminated target region to be automatically adjusted relative to the clear region of the imaging optics in that the image of the target region captured by the imaging optics is evaluated for image modulation. Image modulation is applied to the image by illuminating the imaged target area with two interfering light sheets. In this way, an interference pattern is generated which illuminates the target region and which is reflected in the image of the illuminated target region in the form of the aforementioned image modulation which is evaluated, for example, by a control unit provided for this purpose in the light-sheet microscope and is used for automatic overlay adjustment. The adjustment ends, for example, when the image modulation applied to the image of the illuminated target area is maximal. The modulation present in the image thus depends to a large extent on how accurately the target region illuminated with the light sheet and the clear region of the imaging optics spatially overlap one another. In brief, the image modulation is maximized when the illumination plane defined jointly by the two light sheets is superimposed on the focal plane of the imaging optics.

For the overlap adjustment, it is ensured that the two light sheets interfere with one another in the illuminated target region. These light sheets are thus produced: so that they have a sufficient degree of correlation with each other in time and space for interference. In addition, it is to be ensured in particular that the two light sheets have a polarization state which is fully achievable during the adjustment such that the light sheets interfere in the target region. If, for example, two light sheets are considered to be linearly polarized differently from the outset, these light sheets interfere with one another in the target region if their polarization directions are not orthogonal to one another. As long as this is ensured, there is no need for a polarizing device which is specially provided for ensuring the interference capability of the two light sheets in the target region by influencing their polarization states. Such a polarizing means, which is provided specifically for generating the interference power of the individual light sheets, can be used in an advantageous manner to generate particularly pronounced interference patterns in the target region by correspondingly influencing the polarization state. For example, a polarizing device may be used to linearly polarize two light sheets in parallel, thereby maximizing the interference of the two light sheets.

Such a polarizing device may be realized, for example, by a birefringent crystal positioned in an appropriate position in the illumination beam path, or by other types of polarizers such as retardation plates. The influence on the polarization state of the light sheet can likewise be achieved by polarization-dependent properties of the illumination optics, for example by using polarization-dependent phases of interference layers or by using stress-birefringent devices. Furthermore, electro-optical devices or devices operating on the basis of liquid crystals can be used.

Preferably, the image modulation is analyzed in such a way that the amplitude of the image modulation is determined. The illuminated target region is then adjusted relative to the clear region of the imaging optics such that the amplitude of the image modulation is maximized. The amplitude of the image modulation thus forms an optimization parameter which can be detected in a simple manner and on the basis of which automatic overlap adjustment can be carried out in a light-sheet microscope.

In an advantageous embodiment, a feature of the interference pattern is defined and the image modulation is evaluated using the defined feature. The aforementioned features are characteristics of the interference pattern, which can be derived, for example, from the selected shaping of the light sheet illumination and are therefore known in advance. This known property of the interference pattern can then be taken into account in a simple manner for the analysis of the image modulation.

For example, the interference pattern is produced in the form of a fringe pattern whose interference fringes run parallel to an angle bisector of an angle enclosed by the illumination directions of the two light sheets, wherein the fringe pattern is characterized by a modulation period according to the following relationship: and f is lambda sin (alpha/2). Where f is the modulation period, λ is the wavelength of the light sheet, and α is the aforementioned angle that those illumination directions enclose with each other.

In this example, the modulation period f is a characteristic of the interference pattern known from the outset, which is produced by the defined wavelength λ of the light sheets and by the likewise defined angle α between the two illumination directions of the two light sheets. In addition to the modulation period, the shape of the interference pattern illuminated by the light sheets, i.e. the illumination direction of both light sheets, is also known. Due to the knowledge of the modulation period and the direction of the interference pattern, the amplitude of the image modulation can now be determined simply and reliably, for example by fourier analysis of the image. In the frequency domain generated by fourier analysis, the narrowly located modulation periods of the interference fringes are convolved with the spatial spectrum of the sample, which represents the previously unknown structure of the sample. However, since the captured image of the target region is represented by data which are positive real numbers, the sum of the constant components of these data which occupy the fourier spectrum is always also positive real numbers, so that a priori knowledge of the modulation period f of the interference pattern and its direction allows the amplitude of the image modulation to be reliably determined even in the case of the high-frequency spatial spectrum of the sample.

In the preceding example, the interference fringes of the interference pattern can thus be detected simply and reliably in the form of a modulation of the captured image by the imaging optics of the light-sheet microscope for a not too large angle α between the illumination directions of the two light sheets. The amplitude of the image modulation applied to the image by the interference fringes can thus be used as an optimization parameter or quality criterion for the coplanarity between the illumination plane and the focal plane of the imaging optics. Since the amplitude of the image modulation is the only parameter to be optimized, the parameter space for adjusting the coplanarity is one-dimensional. A simple linear search algorithm can be used to maximize the amplitude of the image modulation.

Preferably, for the overlap adjustment, the two coplanar overlapping light sheets are jointly moved along the optical axis of the imaging optics. This can be done, for example, by a deflection element arranged in the illumination beam path, which is controlled as a function of the evaluated image modulation. It is also possible, however, to move the imaging optics along its optical axis instead of the light sheet illumination, in order to spatially coincide the clear region with the illuminated target region. The light sheet can be moved simultaneously, and the clear area of the imaging optical mechanism can be moved.

In a preferred embodiment, the two light sheets are brought into an interference-capable polarization state before the illuminated target region is adjusted relative to the clear region of the imaging optics. For example, the aforementioned polarization states are selected such that the two light sheets are polarized linearly, non-orthogonally, in particular parallel to one another, in the target region. This ensures that a very pronounced interference pattern is formed in the target region, by means of which interference pattern a correspondingly pronounced image modulation is produced in the captured image.

In a further advantageous embodiment, the two light sheets are brought into a polarization state which cannot interfere after the illuminated target region has been adjusted relative to the clear region of the imaging optics. This avoids modulation of the microscope image which would interfere with the actual imaging. This step can be automated by means of fourier analysis of the image.

Provision is preferably made for a preliminary adjustment in which the illuminated target region is adjusted relative to the clear region of the imaging optics as a function of the image brightness. This preconditioning can be carried out in particular also in the case of the spatial spectrum of the high frequencies of the sample, depending on the energy content of the detected total spectrum resulting from the convolution of the spatial spectrum and the illumination spectrum.

The light sheet microscope according to the present invention includes: an illumination unit designed for illuminating the sample from two different illumination directions with two light sheets having different polarization states and overlapping each other coplanarly in a target area of the sample; imaging optics configured to produce an image of the illuminated target area; and a control unit. The control unit is designed to control the illumination unit such that an interference pattern is generated in the illuminated target area by means of the two light sheets, whereby an image modulation corresponding to the interference pattern is applied to the image of the target area. The control unit is also designed to analyze the image modulation and to control the illumination unit and/or the imaging optics in order to adjust the illuminated target region relative to the clear region of the imaging optics in accordance with the analyzed image modulation.

In a preferred design, the lighting unit comprises: a light source designed to generate an illumination beam; a first polarization component, which is designed to split the illumination light beam into two differently polarized partial beams; and an illumination optics, which is designed to produce two light sheets from the two partial beams for illuminating the target region. The first polarization component is designed, for example, such that it deflects the two partial beams away from the optical axis of the illumination unit, preferably at opposite equal angles. If the angle at which the two partial beams enclose one another is denoted by β (that is to say the equal angle opposite with respect to the optical axis is denoted by ± β/2) and the magnification caused by the imaging optics is denoted by γ, two light sheets are generated in this embodiment within the sample, which light sheets propagate inside the sample at an angle of ± β/2 γ with respect to the optical axis of the illumination unit. The angle β >0 between the two propagation directions reduces the formation of streak artefacts due to scattering and absorption.

The first polarization component is preferably designed such that the two partial beams are linearly polarized, wherein their polarization directions are orthogonal to one another. These orthogonal polarization directions have the following advantages in true imaging: image modulation due to interference between the two light sheets is avoided. Furthermore, by illumination with these two polarization directions, the light-selective effect in the excitation of fluorophores is reduced.

In a preferred embodiment, the first polarization component is a Wollaston prism. Such a prism is formed, for example, by two perpendicular prism prisms glued to one another on their base surfaces. The optical axes of the two prisms are orthogonal to each other.

Preferably, the illumination unit comprises a deflection member which can be controlled by the control unit and by means of which the two coplanar overlapping light sheets can be moved jointly along the optical axis of the imaging optics. The deflection member is, for example, a mirror which is driven by a motor controlled by a control unit so as to move two coplanar light sheets along the optical axis of the imaging optical mechanism.

In a particularly preferred embodiment, the illumination unit comprises a second polarization component which can be controlled by the control unit and which is designed to selectively switch the two light sheets into an interference-capable polarization state and into an interference-incapable polarization state. In particular in combination with the aforementioned first polarization means, this embodiment enables both a precise overlap adjustment and a high-resolution imaging which is not disturbed by interference effects, in particular by image modulation.

Preferably, the light sheet microscope according to the invention has two separate specimen-facing objective lenses, one of which is assigned to the illumination unit and the other to the imaging optics, and whose optical axes are perpendicular to one another. The objective assigned to the illumination unit is preferably such that the two light sheets are directed from the same side toward the target area of the sample.

The light sheet microscope can also be designed as a so-called tilted-plane microscope having a single objective facing the sample for both illumination and detection. The imaging optics of the light sheet microscope are in this embodiment designed as transmission optics which image the light sheet into the target region of the sample and at the same time produce an image of the illuminated target region. The transmission optical unit preferably has a scanning device which is designed for an axial and/or transverse scanning process for stereoscopic imaging in such a way that it moves the light sheet correspondingly through the sample.

Drawings

The invention is described in detail below with the aid of the accompanying drawings. Wherein:

FIG. 1 shows an embodiment of a light sheet microscope according to the invention in a schematic sectional view;

FIG. 2 is a further schematic sectional view of the light sheet microscope according to FIG. 1;

FIG. 3 is a flow chart illustrating, by one embodiment, a method for overlap adjustment according to the present invention;

FIG. 4 is a graph showing the magnitude of image modulation in relation to the offset between an illuminated target area of a light sheet microscope and a clear area of an imaging optical mechanism;

FIG. 5 illustrates an interference pattern generated in a target region;

FIG. 6 shows the spatial spectrum of the interference pattern according to FIG. 5 by Fourier transformation;

FIG. 7 shows an image of a target area having an image modulation corresponding to an interference pattern for overlay adjustment;

FIG. 8 shows the spatial spectrum of the image according to FIG. 7 obtained by Fourier transformation; and

fig. 9 shows the target area image after the image modulation is removed.

Detailed Description

Fig. 1 and 2 show cross-sectional views of a light sheet microscope 10.

The light sheet microscope 10 includes an illumination unit 12 and an imaging optical mechanism 14. In the present exemplary embodiment, illumination unit 12 and imaging optics 14 are oriented relative to one another such that their optical axes O or O' are perpendicular to one another in the region of the specimen not explicitly shown in fig. 1 and 2. In fig. 1 and 2, reference is made to a vertical xyz coordinate system, whose z axis coincides with the optical axis O of the illumination unit 12. Thus, the light sheet microscope 10 is shown in x-z section in fig. 1 and in y-z section in fig. 2. The views according to fig. 1 and 2 are simplified and purely schematic. And thus only those components necessary for an understanding of the present invention are shown.

The illumination unit 12 has a light source 16 and illumination optics, generally indicated at 18 in fig. 1 and 2. The illumination optics 18 comprise a first polarization component in the form of a Wollaston prism 20, a motorized second polarization component in the form of a compensator 22, an anamorphic focusing system in the form of a cylindrical lens 24, a motorized adjusting mirror 26, an eyepiece lens 28, a deflecting mirror 30, a tube lens 32 and an illumination objective 34 with an objective pupil 36. The aforementioned compensator is constituted, for example, by a birefringent crystal, in particular a retardation plate.

The imaging optics 14 comprise an imaging objective 38 facing the sample to be imaged, a tube lens 40 and a position-resolving detector in the form of a camera 42.

The light sheet microscope 10 also contains a control unit 44 which controls the overall microscope operation. In the present exemplary embodiment, the control unit 44 serves in particular to control the compensator 22, the motorized adjustment mirror 26 and the camera 42, and to carry out the image analysis described in detail below. Accordingly, the control unit 44 is connected via control lines 46, 48, 50 to the compensator 22, the adjusting mirror 26 or the camera 42.

The light source 16 emits a collimated illumination beam 52 onto the Wollaston prism 20, which is formed, for example, by two perpendicular prisms, such as a Calcit prism, glued to one another on their base surfaces. The Wollaston prism 20 splits the incident illumination light beam 52 into two partial beams 54, 56 with different polarization states, as shown in fig. 2. The Wollaston prism 20 splits the illumination light beam 52 into two partial beams 54, 56 in a plane which is parallel to the y axis here, i.e. in the drawing plane in the sectional view according to fig. 2 and perpendicular to the drawing plane in the sectional view according to fig. 1.

Subsequently, the two partial beams 54, 56 pass through the compensator 22, with which the polarization states of the partial beams 54, 56 can be influenced as required. For this purpose, a servomotor, not shown in fig. 1 and 2, is provided in the light-sheet microscope 10, which servomotor acts on the compensator 22 under the control of the control unit 44, so that it influences the polarization state of the two partial beams 54, 56 in a desired manner or leaves it unchanged.

The partial beams 54, 56 then pass through the cylindrical lens 24. The cylindrical lens has the following characteristics: it causes the partial beams 54, 56 to be focused only in a direction parallel to the x-axis, respectively, whereas it has no optical effect on the partial beams 54, 56 in a direction parallel to the y-axis. The cylindrical lens 24 thus produces in the region of its focal plane a light-sheet-like illumination light distribution from the partial beams 54, 56, respectively, which is focused in the direction of the x axis and extends in the direction of the x axis in a planar manner. It is relevant to note that the associated drawings in fig. 1 and 2 are simplified for ease of understanding. Thus, for example, in fig. 2, the focal points of the two partial beams 54, 56 emerging from the cylindrical lens 24, which correspond to planes conjugate to the cylindrical lens 24, are arranged on the surface of the motorized adjusting mirror 26. In practice, however, one of these focal points is located in front of the surface and the other focal point is located behind the surface in the direction of light propagation. The same applies to the focal point representation on the surface of the deflecting mirror 30. In addition, the deflection of the light on the steering mirror 26 and the deflection mirror 30 is shown in the same manner in fig. 1 and 2, although this deflection of the light is produced in the illustrated manner either only in the x-z plane or in the y-z plane.

After reflection on the adjusting mirror 26, the two partial beams 54, 56 pass through the eyepiece lens 28 and are reflected on the deflecting mirror 30. The partial beams 54, 56 then enter the entrance pupil 36 of the illumination objective 34 after passing through the tube lens 32, which directs the partial beams 54, 56 toward the sample, so that the partial beams 54, 56 illuminate the target region E of the sample from two different illumination directions.

The illumination optics 18 of the light sheet microscope 10 generate two light sheets 58, 60 which propagate in different illumination directions and which overlap one another in a coplanar manner in the target region E of the specimen to be illuminated. In this particular exemplary embodiment, it is assumed that the compensator 22 has not initially influenced the polarization states of the partial beams 54, 56, the two light sheets 58, 60 being linearly orthogonally polarized relative to one another in the target region E. Thus, for example, the light sheet assigned to the partial beam 54 is p-polarized, while the light sheet assigned to the partial beam 56 is s-polarized. The eyepiece lens 28, the tube lens 32 and the illumination objective 34 form an intermediate imaging optics inside the illumination optics 18, the cylindrical lens 24 producing light sheets by focusing the partial beams 54, 56 at the position of the adjusting mirror 26, which intermediate imaging optics image these light sheets into the target area E of the specimen.

In the exemplary embodiment according to fig. 2, the two illumination directions, from which the light sheets 58, 60 are directed toward the target area E of the specimen, enclose an angle α with one another. This angle α is related to the angle β which the two partial beams 54, 56 enclose with one another after being separated by the Wollaston prism 22. Specifically, the angle α is obtained from the relationship α β/γ, where γ refers to the magnification of the intermediate imaging optical mechanism composed of the eyepiece lens 28, the tube lens 32, and the illumination objective lens 34.

With reference to the flowchart according to fig. 3, it will be described in the following by way of example how the overlap adjustment with the light sheet 10 can be carried out by spatially coinciding the target region E of the sample illuminated with the two coplanar light sheets 58, 60 with the clear region F of the imaging optics 14, which is located in the focal region of the probe beam 62 along the optical axis O' of the imaging optics 14 according to fig. 1.

In a step S1 of the flowchart according to fig. 3, the light sheet illumination is first automatically pre-adjusted to the clear region F of the imaging optics 14. This pre-adjustment may be made, for example, based on the brightness of the image captured by the camera 42. For this purpose, the adjusting mirror 26 is brought under the control of the control unit 44 into a position in which the light sheet illumination is responsible for the maximum image brightness.

After the preliminary adjustment, in step S2, the polarization states of the two partial beams 54, 56 and thus of the two optical sheets 58, 60 are adjusted by the compensator 22 under the control of the control unit 44, so that the two optical sheets 58, 60 produce the greatest interference in the target area E. In the present exemplary embodiment, the compensator 22 adjusts the polarization states of the two partial beams 54, 56 to this end in such a way that they are linearly polarized parallel to one another. In this way, an interference pattern is generated in the target region E by interference of the two light sheets 58, 60, as is shown purely exemplarily in fig. 5. Here, for the sake of clarity, fig. 5 shows the interference pattern I on the assumption that the sample is completely uniform.

The interference pattern I according to fig. 5 has a plurality of interference fringes which, with reference to fig. 2, run in a direction which runs parallel to the angle bisector of the angle α. The angle α is enclosed by two illumination directions in which the two light sheets 58, 60 propagate. In the exemplary embodiment shown, the aforementioned angle bisector of the angle α thus coincides with the optical axis O of the illumination optics 18. Thus, as shown in fig. 5, the interference fringes extend in the direction of the optical axis O of the illumination optical mechanism 18.

The actual adjustment is performed in subsequent steps S3 to S5. First in step S3 an image of the target area E of the sample illuminated with the two light sheets 58, 60 is recorded by the camera 42. Since the two optical sheets produce the interference pattern I shown in fig. 5, an image modulation corresponding to the interference pattern is applied to the image picked up by the camera 42. This is illustrated in fig. 7, which shows an image of the target region taken by the camera 42, in which image modulation corresponding to the interference pattern I can be clearly seen in the form of horizontal fringes.

In step S4, the control unit 44 analyzes the image modulation contained in the captured image. For this purpose, the control unit 44 makes use of a priori knowledge of the characteristics of the interference pattern I as a result of the defined light sheet configuration. In the present exemplary embodiment, this feature is specified by the modulation period, i.e. the spacing between adjacent interference fringes, and by the orientation of the interference pattern according to fig. 5. The modulation period is obtained according to the following relation: where f is the modulation period, λ is the wavelength of the optical sheets, and α is the angle between the propagation directions of the two optical sheets 58, 60. The direction of the interference pattern is also directly produced by the prescribed light sheet molding. As described above, in the present embodiment, the interference fringes extend parallel to the optical axis O of the illumination optical mechanism 18.

The features of the interference pattern according to fig. 5 are reflected on the spatial frequency spectrum shown in fig. 6, which it produces by fourier analysis. In the graph according to fig. 6, the horizontal spatial frequency is marked along the horizontal axis and the vertical spatial frequency is marked along the vertical axis. The unit is given by the characteristics of discrete fourier-transform, and the signal intensity is expressed as a gray scale. In the case of the spatial frequencies resulting from the modulation period of the interference pattern shown in fig. 5, the spatial spectrum according to fig. 6 has two signals. Precisely, the spatial frequencies in fig. 6 each form the inverse of the modulation period of the interference pattern I in accordance with the fourier transform. For clarity, the signal strength representing the constant component of the spatial spectrum at spatial frequency zero is omitted from fig. 6, since this constant component far exceeds the signal strength shown in fig. 6. With reference to the horizontal axis, a constant component with spatial frequency zero-the signal lies between the two signals shown in fig. 6. It is contemplated herein that the spatial frequencies in fig. 6 are given in arbitrary units.

In step S4, the image shown in fig. 7 is thus fourier-transformed, resulting in its spatial spectrum shown in fig. 8, which image has an image modulation corresponding to the interference pattern I according to fig. 5. The spatial frequency spectrum according to fig. 8 shows, along the vertical axis, two spatial frequency signals which are arranged at equal distances on both sides of a main central signal which represents a constant component of the spatial frequency spectrum when the spatial frequency is zero. The two aforementioned signals reflect the image modulation caused by the interference pattern I. In particular, the spatial frequency of these signals, i.e. the (positive) spacing that these signals have from the center along the vertical axis of the constant component-the signal has, is determined by the modulation period of the interference pattern. The larger the spatial frequency, the smaller the modulation period of the interference pattern I, that is, the smaller the spacing of adjacent interference fringes in fig. 7. The directions of the two spatial frequency signals representing the image modulation in fig. 8 correspond to the directions of the image modulation in fig. 7. It can thus be seen in fig. 7 that the horizontally running interference fringes succeed one another in the vertical direction.

Based on the features of the interference pattern I, which are specified by the optical sheet modeling, it is known a priori at which positions, i.e. at which spatial frequencies, the spatial spectrum according to fig. 8 is analyzed in order to quantitatively detect the image modulation. In the present example, the spatial spectrum shown in fig. 8 is analyzed at exactly the following locations: at these locations two signals are found above or below the central constant component signal (e.g., only the upper signal has a positive spatial frequency). This signal represents the amplitude of the image modulation and is therefore used later as an optimization parameter for the overlap adjustment.

In step S5, the motorized adjustment mirror 110 is shifted by a predetermined threshold value under the control of the control unit 44, so that the two optical sheets 58, 60 overlapping one another are jointly moved along the optical axis O' of the imaging optical system 14. Next, the control flow returns to step S3.

The steps S3 to S5 are repeated, for example, using a linear, i.e. one-dimensional, search algorithm (taking into account suitable interruption criteria, if necessary) for a period of time until the optimization parameters given by the spatial frequency signal representing the image modulation amplitude detected in step S4 are maximized.

Fig. 4 shows purely exemplarily that the amplitude of the image modulation varies as a function of the offset which occurs between the illuminated target region E and the clear region F of the imaging optics along the optical axis O' thereof. If the offset is equal to zero, the image modulation amplitude is maximized and the overlap adjustment is ended.

Finally, in step S6, the compensator 22 is controlled by the control unit 44 such that it brings the partial beams 54, 56 and thus the light sheets 58, 60 into a polarization state in which the light sheets 58, 60 do not interfere with one another. In the present exemplary embodiment, the light sheets 58, 60 are linearly orthogonally polarized relative to one another in the polarization state. By this polarization adjustment, the interference pattern is canceled in the target area E. Accordingly, in the image of the target area E taken by the camera 42, the image modulation is eliminated, as shown in fig. 9. Here, the elimination of the image modulation can likewise be carried out as follows: in one of the steps S3 to S5, the amplitude of the image modulation is used as an optimization parameter in accordance with a one-dimensional search method, with the difference of course that the control unit 44 does not control the adjusting mirror 26 in this case, but controls the compensator 22, and the amplitude of the image modulation should not be maximal, but minimal. The image of the target region E from which the image modulation has been removed in this way can then be taken into account for the actual image generation.

The present invention is not limited to the above-described embodiments. For example, it is possible to perform the overlap adjustment in a different manner from the exemplary embodiment in which the two light sheets 58, 60 are moved along the optical axis O' of the imaging optics 14. It is thus likewise possible, for example, to move the clear region F of the imaging optics 14 for the overlap adjustment. The polarization states of the light sheets 58, 60 can also be influenced in a manner different from the exemplary embodiment described, as long as it is ensured that the two light sheets 58, 60 interfere with one another in the target region E during the presetting. It is possible in particular for the compensator 22 to act on only one of the two partial beams 54, 56. The invention is also not limited to having the two light sheets 58, 60 face the target area E from the same side as in the previous embodiments. Thus, for example, it is also possible for the two light sheets 58, 60 to be focused into the target region from different sides by means of suitable deflection means. Such a deflection component can be realized, for example, by a so-called mirror cap arranged on the sample-facing illumination objective 34.

The light microscope can also be designed as a tilted-plane microscope of the type described above, which has a single specimen-facing objective for illumination and detection.

List of reference numerals

10 light sheet microscope

12 Lighting Unit

14 imaging optical mechanism

16 light source

18 illumination optical mechanism

20 Wollaston prism

22 motorized compensator

24 cylindrical lens

26 motorized adjustable mirror

28 eyepiece lens

30 deflecting mirror

32 tube lens

34 illumination objective

36 pupil of objective lens

38 imaging objective

40 tube lens

42 vidicon

44 control unit

46 control circuit

48 control circuit

50 control circuit

52 illumination beam

54 sub-beams

56 sub-beams

58 light sheet

60 light sheet

62 Probe light Beam

Optical axis of an O-lighting unit

Optical axis of O' imaging optical mechanism

E target area

F clear region

Angle alpha

Angle beta

I interference pattern