Patent Description:
Oblique plane microscopy, described for example in document <CIT>, is a technique for volumetric imaging of a specimen by means of a light sheet. The light sheet is directed into the specimen by an objective and illuminates a plane that is tilted with respect to the focal plane of said objective. Accordingly, most parts of the illuminated plane are outside the focal plane of the objective and are thus subject to defocus aberrations.

In order to achieve fast and aberration free imaging of the illuminated plane, a method called remote focusing is used which is described in <NPL>). This method makes use of a telescope system. In order to correct the above-mentioned defocus aberrations, the magnification of the telescope system is set to a ratio of two refractive indices, one of which being associated with an object side of the telescope system and the other being associated with an image side of the telescope system. If this condition is met, the image of the illuminated plane is free from defocus aberration.

Another factor contributing to the quality of an image formed by the oblique plane microscope are spherical aberrations, which occur as a result of inhomogeneities in an object to be imaged or refractive index mismatch. These spherical aberrations can be corrected by adjustable optical correction means, e.g. a correction lens. However, these two factors are not independent of each other. Adjusting the optical correction means might move the focal plane of the optical imaging system, for example. A proper adjustment of the optical correction means can be performed, if the imaging depth into the specimen and the refractive index of the specimen are known. Additionally, adjusting the magnification of the optical zoom system requires knowledge of the two refractive indices, on the object side and on the image side, respectively.

Document <CIT> discloses an oblique plane microscope comprising an optical imaging system. The imaging system may comprise a zoom lens for adapting the magnification of a scan and tube lens to objective lenses having different properties. In particular, in the setup disclosed in D1, the zoom lens is used to adapt the light path to the back aperture of a specific objective.

It is an object of the present invention to provide a method and an oblique plane microscope that allow for fast and easy correction of spherical aberrations and defocus aberrations.

The aforementioned object is achieved by the subject-matter according to the independent claims. Advantageous embodiments are defined in the dependent claims and the following description.

An oblique plane microscope comprises an optical imaging system configured to form an image of an object. The optical imaging system comprises a telescope system with an optical zoom system, which is adjustable for adapting the magnification of the telescope system to a ratio between two refractive indices, one of which being associated with an object side of the telescope system and the other being associated with an image side of the telescope system. The oblique plane microscope further comprises a control unit being configured to evaluate an image quality of the image formed by the optical imaging system and to adjust the optical zoom system based on said evaluation.

Both conventional spherical aberrations and spherical aberrations due to a breakdown of the remote focusing condition (from here on denoted defocus aberrations, not to be confused with defocus aberrations in a centered optical system without remote focusing) have a characteristic influence on the quality of the image formed by the optical imaging system. Defocus aberrations manifest themselves as a position depended coma in the image formed by the optical imaging system. The coma will be more pronounced in areas of the image corresponding to regions of the object that are more out of focus, i. would require more remote focusing. Therefore, defocus aberrations have a greater impact on the image quality in image areas that are more distant from image areas corresponding to the focal plane of the optical imaging system in the image. On the other hand, spherical aberrations, e.g. due to refractive index mismatch, are mainly position independent, given that the imaging depth into the specimen is large compared to the amount of remote focusing required for imaging the field of view. Thus, spherical aberrations have a relatively homogeneous impact on the image quality. It is therefore possible to distinguish and determine the types of aberration present in the image by evaluating image quality.

The control unit of the oblique plane microscope is configured to evaluate the image quality and to adjust the optical zoom system based on said evaluation. Most notably, no measurement of the two refractive indices is needed in order to adapt the magnification of the telescope system to a ratio between both refractive indices. Such a measurement would have to be performed either in advance, which is time consuming and unreliable since conditions in situ, i.e. inside the microscope sample space where the object is located, are different from conditions ex situ, i.e. outside the microscope. On the other hand, measuring both refractive indices in-situ requires additional microscope components. Thus, the oblique plane microscope described herein is able to correct spherical aberrations and defocus aberrations fast and easily.

In a preferred embodiment, the telescope system comprises optical correction means which is adjustable for correcting a spherical aberration of the optical imaging system. The control unit is configured to adjust the optical correction means based on the evaluation of the image quality of the image. This allows to correct conventional spherical aberrations, e.g. due to refractive index mismatch. Thereby the overall quality of the image formed by the optical system is further increased.

In another preferred embodiment, the telescope system comprises an objective arranged at an object side of the telescope system. The control unit is configured to divide the image formed by the oblique plane microscope into two or more areas. A first area comprises an image of a first region and a second area comprises an image of a second region. The first region and the second region of the object are positioned at different distances from the objective along its optical axis.

Preferably, the control unit is configured to divide the image formed by the oblique plane microscope into three or more areas. The first area comprises an image of the first region of the object, said first region being located on a side of a focal plane of the objective facing away from objective. The second area comprises an image of the second region of the object, said second region being located on a side of the focal plane facing the objective. A third area comprises an image of a third region of the object, said third region being intersected by the focal plane.

The defocus aberrations affect different parts of the image that correspond to the first region and the second region of the object differently, since these object regions are located at different depth within the object, i.e. at different distances from the focal plane of the objective. This fact is exploited by dividing the image into two or more areas. For example, the magnification of the optical zoom system might be adjusted based on one of the areas corresponding to a region more distant from the focal plane of the objective. The correction means on the other hand might be adjusted based on an evaluation of the image quality of an area which is closer to the focal plane and thus subject to little or no interfering defocus aberrations. Therefore, dividing the image into two or more areas and evaluating the image quality of at least one of the areas allows for an adjustment of the optical correction means and the optical zoom system based on said evaluation. This means a better correction of the spherical aberrations and the defocus aberrations which increases the overall quality of an image created by the oblique plane microscope according to this preferred embodiment.

In another preferred embodiment, the control unit is configured to evaluate an image quality of the third area of the image and to adjust the optical correction means on said evaluation. The third area is intersected by the focal plane. Thus, the third area is subject to little or no defocus aberrations. This means that nearly all aberrations affecting the image quality of the image of the third area are caused by spherical aberrations. By, adjusting the optical correction means such that e.g. the image quality of the third area is maximized, a fast correction of the spherical aberrations present in the image formed by the optical imaging system is achieved.

In another preferred embodiment, the control unit is configured to evaluate an image quality of the first area and/or the second area, and to adjust the optical zoom system means based on said evaluation. The first and second areas each correspond to regions of the object which are not intersected by the focal place, respectively. The first and second areas are therefore subject to defocus aberrations. Adjusting the magnification of the optical zoom system such, that e.g. the image quality of the first area and/or the second area is maximized, will result in a fast correction of the defocus aberrations present in the image formed by the optical imaging system.

In another preferred embodiment, the control unit is configured to evaluate the image quality by determining a Strehl ratio (image intensity), a contrast value, an image sharpness measure and/or a width of an autocorrelation function of the image.

It is advantageous to configure the control unit for determining a direction dependent image quality of the first area and/or the second area. The first area and the second area each include parts of the image that correspond to regions of the object on different sides of the focal place. The coma induced by defocus aberrations is oriented in the first area in a manner different from the coma in the second area. Since the coma depends on direction, the control unit can reliably identify the coma based on an evaluation of the direction dependent image quality.

In another preferred embodiment, the optical zoom system is configured to render the telescope system telecentric over the entire magnification range with respect to both the object side and the image side. This means that the position of a pupil of the optical zoom system is fixed even if the magnification of the optical zoom system is adjusted. Thus, the focal plane of the optical imaging system is always imaged onto the same image plane. This allows for volumetric imaging without the need for additional components for detecting different image planes or for correcting the position of said pupil plane.

In another preferred embodiment, the magnification range of the telescope system corresponds to a range in which the ratio of the two refractive indices is between <NUM> and <NUM>. This allows for a wide variety of combinations of objectives, cover slips, and samples to be used in combination with the oblique plane microscope according to this preferred embodiment, and also to compensate for manufacturing tolerances of the optical components of the telescope system, which manifest in a tolerance of the magnification.

In another preferred embodiment, the telescope system is formed by a Keplerian telescope comprising the optical zoom system.

In another preferred embodiment, the control unit is configured to evaluate an image quality of the image formed by the optical imaging system and to adjust the optical correction means and the optical zoom system based on said evaluation in an iterative process. Adjusting the optical correction means and the optical zoom system is an optimization problem. One or more parameters are maximized (or minimized) with respect to a setting of the correction means and with respect to the magnification of the optical zoom system. The parameters correspond to the image quality of the image formed by the optical imaging system and/or the image quality of one or more areas of said image. The setting of the correction means may be a position of the correction lens along the optical axis of the optical imaging system, for example.

This optimization problem can be solved fast with iterative methods know from the prior art.

The correction means may be configured such, that adjusting the correction means does not affect the position of the focal plane of the optical imaging system. Such a correction means is known e.g. from the document <CIT>. In this advantageous embodiment, adjusting the correction means does not introduce additional (conventional) defocus aberrations, i. does not shift the object plane along the optical axis. This means, the volume of the object imaged is invariant under adjustment of the correction means, making the analysis of the image quality more robust and thus facilitating an image-based adjustment of the correction means.

According to another aspect, a method for correcting an aberration in an oblique plane microscope is provided. The method comprising evaluating an image quality of the image formed by an optical imaging system of the oblique plane microscope and adjusting optical correction means and an optical zoom system of the oblique plane microscope based on said evaluation.

The method has the same advantages as the oblique plane microscope and can be supplemented using the features described herein with reference to the microscope.

<FIG> shows a schematic diagram of an oblique plane microscope <NUM> according to an embodiment. The oblique plane microscope <NUM> comprises an optical imaging system <NUM> configured to form an image of an object <NUM> and a control unit <NUM>.

The optical imaging system <NUM> comprises an illumination system <NUM> and a telescope system <NUM>. The illumination system <NUM> is configured to form a light sheet in an intermediate image space <NUM>. The telescope system <NUM> is configured to form an image of the light sheet within the object <NUM> and to form an image <NUM> (see <FIG>) of an object plane <NUM> (see <FIG>) within the object <NUM> in the intermediate image space <NUM>. The oblique plane microscope <NUM> further comprises an optical detection system <NUM> configured to detect the image <NUM> formed by the telescope system <NUM>. In an alternative embodiment, the light sheet may be guided into the object <NUM> by a dichroic beam splitter arranged in telescope system <NUM>.

The illumination system <NUM> comprises a light sheet forming unit <NUM> configured to form the light sheet. The light sheet forming unit <NUM> comprises a light source, in particular laser light source, and light sheet forming elements, for example a cylindrical lens or a scanning element. The illumination system <NUM> further comprises an illumination objective <NUM> configured to direct the light sheet into the intermediate image space <NUM>.

The telescope system <NUM> forms an optical transport system in the sense that it is configured to transport the light sheet from the intermediate image space <NUM> into the object <NUM> and to create the image <NUM> of the object plane <NUM> illuminated by the light sheet in the intermediate image space <NUM>. In other words, the telescope system <NUM> transports illumination light and detection light from the intermediate image space <NUM> to the object <NUM> and back, respectively.

In the present embodiment, the telescope system <NUM> is telecentric and formed by a Keplerian telescope system. The telescope system <NUM> comprises an image side objective <NUM>, a tube lens <NUM>, a first ocular <NUM>, scanning element <NUM>, a second ocular <NUM>, an optical zoom system <NUM>, and an object side objective <NUM>, in this order from the intermediate image space <NUM>.

The scanning element <NUM> is configured to move the light sheet through the object <NUM> along a direction perpendicular to the optical axis O of the objective <NUM>. The optical zoom system <NUM> is configured adjustable for adapting the magnification of the telescope system <NUM> to a ratio between two refractive indices. One refractive index is associated with the object side of the telescope system <NUM> and the other refractive index is associated with the image side of the telescope system <NUM>. More specifically, the refractive index associated with the object side of the telescope system <NUM> is the refractive index of the object <NUM>, and the refractive index associated with the image side of the telescope system <NUM> is the refractive index of an optical medium, e.g. air, being present within the intermediate image space <NUM>. In the present embodiment, the magnification range of the optical zoom system <NUM> corresponds to a range in which the ratio of the refractive indices is between <NUM> and <NUM>. The objective <NUM> comprises correction means <NUM>, for example a movable correction lens, configured to correct a spherical aberration of the optical imaging system <NUM>. In another embodiment, the corrections means <NUM> may be arranged in the image side objective <NUM> instead.

The optical detection system <NUM> comprises a detection objective <NUM>, a tube lens <NUM>, and a detector element <NUM>. The detection objective <NUM> and the tube lens <NUM> are configured to image the intermediate image space <NUM> onto the detector element <NUM>. This means that the image <NUM> of the object plane <NUM> formed by the telescope system <NUM> within the intermediate image space <NUM> is object onto the detector element <NUM>. Thus, the image <NUM> is detected by the detector element <NUM>. In an alternative embodiment, the detection objective <NUM> may be configured as an objective with a finite conjugate length. In this alternative embodiment, the optical detection system <NUM> does not comprise the tube lens <NUM>. In another embodiment, the detection objective <NUM> may comprise concentric front lenses configured to correct spherical aberrations. In another embodiment, the role of detection objective <NUM> may be fulfilled by the image side objective <NUM> and a mirror and beam splitting arrangement, as known from prior art.

The control unit <NUM> is connected to the correction means <NUM>, the optical zoom system <NUM>, the scanning element <NUM>, the detector element <NUM>, and the light sheet forming unit <NUM>. The control unit <NUM> is configured to control the aforementioned elements of the oblique plane microscope <NUM>. Further, the control unit <NUM> is configured to divide the image <NUM> of the object plane <NUM> formed by the optical imaging system <NUM> into three areas <NUM>, <NUM>, <NUM> (see <FIG>) each of these areas <NUM>, <NUM>, <NUM> corresponding to a different region <NUM>, <NUM>, <NUM> (see <FIG>) of the object <NUM>. These three areas <NUM>, <NUM>, <NUM> and the three regions <NUM>, <NUM>, <NUM> of the object <NUM> are described in more detail below with reference to <FIG> and <FIG>. The control unit <NUM> is further configured to evaluate an image quality of the image <NUM> and/or an image quality of the three areas <NUM>, <NUM>, <NUM> and to adjust the optical correction means <NUM> and the optical zoom system <NUM> based on said evaluation. The adjustment based on the evaluation of the image quality will be described in more detail below with reference to <FIG>.

<FIG> shows a schematic diagram of an object side end <NUM> of the optical imaging system <NUM> of the oblique plane microscope <NUM> according to <FIG>. The optical axis O of the objective <NUM> of the telescope system <NUM> is illustrated in <FIG> as a dash-dotted line. The focal plane <NUM> of the objective <NUM> is shown in <FIG> is a dashed line. The position of the object plane <NUM> is shown in <FIG> as a solid line.

A first region <NUM> of the aforementioned three regions is located on a side of the focal plane <NUM> facing away from the objective <NUM>. A second region <NUM> is located on a side of the focal plane <NUM> facing the objective <NUM>. A third region <NUM> is being intersected by the focal plane <NUM> of the objective <NUM>. Accordingly, the three regions <NUM>, <NUM>, <NUM> are located at different distances from the objective <NUM> along the optical axis O thereof. In other words, the three regions <NUM>, <NUM>, <NUM> are located at different depths within the object <NUM>.

<FIG> shows a schematic diagram of the image <NUM> of the object plane <NUM> formed by the optical imaging system <NUM>. The image <NUM> is divided into the three areas. A first area <NUM> corresponds to the first region <NUM>, a second area <NUM> corresponds to the second region <NUM>, and a third area <NUM> corresponds to the third region <NUM> of the object <NUM>. As can be seen in <FIG>, the three areas <NUM>, <NUM>, <NUM> do not cover the complete image <NUM>. In the present embodiment, the three areas <NUM>, <NUM>, <NUM> are rectangular. However, the three area may have any other suitable shape.

The first and second regions <NUM>, <NUM> of the object <NUM> are not intersected by the focal plane <NUM> of the objective <NUM>. Thus, the areas <NUM>, <NUM> of the image <NUM> corresponding to the first and second regions <NUM>, <NUM> are subject to defocus aberrations. The defocus aberrations manifest themselves as coma in the image <NUM>. The amount of coma depends on the position with respect to a line <NUM> in the image <NUM> corresponding to the focal plane <NUM> of the objective <NUM>. The amount of coma further depends on the mismatch between the magnification of the telescope system <NUM> and the ratio between the two refractive indices. Thus, the more distant the first and second regions <NUM>, <NUM> are located from the focal plane <NUM> of the objective <NUM>, the stronger the coma will be in the first and second areas <NUM>, <NUM>. The coma due to defocus aberrations lowers the image quality within the first and second regions <NUM>, <NUM>.

In contrast, the third region <NUM> is being intersected by the focal plane <NUM> and thus mostly in focus. Consequently, the third area <NUM> of the image associated with the third region <NUM> is not subject to defocus aberrations. However, the third region <NUM> is subject the spherical aberrations, e.g. due to refractive index mismatch, affecting the image quality in all areas of the image <NUM> equally.

In order to correct both defocus aberrations and spherical aberrations, the control unit <NUM> is configured to adjust the optical correction means <NUM> and the optical zoom system <NUM> based on the evaluation of the image quality. This process in described in the following with reference to <FIG>.

<FIG> is a flowchart of a process for correcting the defocus aberrations and the spherical aberrations using the oblique plane microscope <NUM> according to <FIG>.

The process starts in step S10. In step S12 the image <NUM> of the object plane <NUM> formed by the telescope system <NUM> in the immediate image space is detected by the optical detection system <NUM>. Then, in step S14 the control unit <NUM> divides the detected image <NUM> into the three areas <NUM>, <NUM>, <NUM> shown in <FIG>.

In step S16, the control unit <NUM> evaluates the image quality of the image <NUM> and the three areas <NUM>, <NUM>, <NUM> by evaluating a Strehl ratio, a contrast value, an image sharpness measure and/or a width of an autocorrelation function, or any other suitable image quality metric known from prior art, of the image <NUM> and/or the three areas <NUM>, <NUM>, <NUM>. In particular, the control unit <NUM> evaluates a direction dependent image quality of the first and seconds areas <NUM>, <NUM>.

In step S18, the control unit <NUM> adjusts the optical correction means <NUM> and the optical zoom system <NUM> based on the evaluation of the image quality in step S16. The adjustments may be performed at the same time or sequentially. The optical zoom system <NUM> is adjusted such that the magnification of the optical zoom system <NUM> is adapted to the ratio of the two refractive indices associated with the object side and the image side, respectively. This adjustment is performed in order to correct the defocus aberrations. In the present embodiment, the control unit <NUM> adjusts the optical zoom system <NUM> by maximizing the image quality of the first and second areas <NUM>, <NUM>, since the image quality depends mainly on the amount of coma due to the defocus aberration. This can be done in a single step or in an iterative process in which the steps S16 and S18 are repeated until the optical zoom system <NUM> has been fully adapted. The control unit <NUM> adjusts the optical correction means <NUM> in the present embodiment by maximizing the image quality of the third area <NUM>. The third area <NUM> is less affected by coma due to defocus aberration, so the image quality mainly depends on the spherical aberrations. This can be done in a single step or in an iterative process in which the steps S16 and S18 are repeated until the optical correction means <NUM> has been fully adapted. The process is then stopped in step S20.

<FIG> shows a schematic diagram of an oblique plane microscope <NUM> according to another embodiment. The oblique plane microscope <NUM> according to <FIG> is distinguished from the oblique plane microscope <NUM> according to <FIG> in terms of how the light sheet is coupled into the telescope system <NUM>. Identical or equivalent elements are designated in <FIG> and <FIG> by the same reference signs.

A telescope system <NUM> of the oblique plane microscope <NUM> according to the present embodiment comprises a dichroic beam splitter <NUM> which is arranged between the scanning element <NUM> and the first ocular <NUM>. The dichroic beam splitter <NUM> is configured to reflect the light sheet formed by the illumination system <NUM> onto the scanning element <NUM>. Further, the dichroic beam splitter <NUM> is configured to transmit the detection light originating in the object <NUM> towards the optical detection system <NUM>. <FIG> shows a schematic diagram of an oblique plane microscope <NUM> according to a further embodiment. The oblique plane microscope <NUM> according to <FIG> is distinguished from the oblique plane microscope <NUM> according to <FIG> in that an image side objective <NUM> of a telescope system <NUM> comprises adjustable focus means <NUM>. Identical or equivalent elements are designated in <FIG> and <FIG> by the same reference signs.

The adjustable focus means <NUM> are controlled by a control unit <NUM> and configured to be adjustable for adjusting the position of the focal plane <NUM> along the optical axis O of the objective <NUM>. Since the focal plane <NUM> can be moved through the object <NUM> by the adjustable focus means <NUM>, the telescope system <NUM> according to <FIG> does not comprise the scanning elements.

Claim 1:
An oblique plane microscope (<NUM>, <NUM>, <NUM>), comprising:
an optical imaging system (<NUM>) configured to form an image (<NUM>) of an object (<NUM>), said optical imaging system (<NUM>) comprising a telescope system (<NUM>) with an optical zoom system (<NUM>), which is adjustable for adapting the magnification of the telescope system (<NUM>) to a ratio between two refractive indices,
one of which being associated with an object side of the telescope system (<NUM>) and the other being associated with an image side of the telescope system (<NUM>); and
a control unit (<NUM>, <NUM>),
wherein the control unit (<NUM>, <NUM>) is configured to evaluate an image quality of the image (<NUM>) formed by the optical imaging system (<NUM>) and to adjust the optical zoom system (<NUM>) based on said evaluation.