Patent Description:
Light sheet microscopes of the art are known, wherein parameters of the microscopes like magnification, numerical aperture and the like are commonly fixed. Modification of such a parameter of a light sheet microscope is cumbersome and time consuming.

In <CIT>, <CIT> and <CIT>, a SCAPE microscope, a surgical microscope and a microscope assembly are disclosed, respectively. A dual lens camera with movable mirror is shown in <CIT> and an infrared spectrophotometer is described in <CIT>.

<CIT> discloses an optical arrangement for imaging a sample. The optical arrangement comprises one first objective lens and one second objective lens, one illumination source for producing an illumination beam, a detector for imaging radiation from the sample, and at least one mirror for reflecting the radiation from one of the first objective lens or the second objective lens into the detector. The one mirror is double-sided and dependent on the illumination beam at the other one of the first objective lens and the second objective lens.

It is one aspect of the present invention to provide a light sheet microscope that facilitates modification of the microscope parameters and that increases the microscope's versatility.

It is the object of the present invention to provide an improved light sheet microscope with increased versatility.

This object is solved by the subject matter of claim <NUM>.

Preferred embodiment are defined by the dependent claims.

The present invention solves the above problem for the light sheet microscope mentioned in the beginning, in that the mirror assembly, in a first operational state is in a first operational position and in a second operational state is moved into a second operational position, wherein in the first operational state of the microscope, the optical system is configured to transmit illumination light along a first illumination light path through the at least one first optical element into pa first sample volume and configured to transmit observation light along a first observation light path from the first sample volume to a detector device, and in that, in the second operational state of the microscope, the optical system is configured to transmit the illumination light along a second illumination light path via the mirror assembly through the at least one second optical element into a second sample volume and configured to transmit observation light along a second observation light path from the second sample volume to the detector device.

The inventive light sheet microscope has thus the advantage that it allows for selecting at least from the first optical element and the second optical element an optical element through which a sample volume is illuminated and through which observation light from said sample volume is received for transmitting it towards the detector device.

The inventive light sheet microscope may be further improved by additional technical features described in the following. Those additional technical features may be arbitrarily combined with each other or may be omitted if the technical effect obtained with the omitted technical feature is not relevant to the present invention.

The inventive light sheet microscope may be embodied as an oblique plane microscope or as a SCAPE microscope.

The light sheet microscope may comprise three or more optical elements, wherein the invention allows for selecting one of said optical elements. The light sheet microscope thus may allow for selecting one optical element out of an arbitrary number of optical elements. Selecting one of said optical elements is to be understood as providing an operational state of the microscope in which an illumination light path and an observation light path pass through said optical element.

The optical element or optical elements may be objectives. The optical elements may differ from each other in at least one of the direction of the corresponding optical axis, a magnification, a numerical aperture and the material and corresponding optical properties of the optical elements. This list of properties is not completed and further properties of the optical elements may differ for at least two optical elements used in the light sheet microscope.

A light sheet microscope being in a first or second operational state may be understood as one or several features of the light sheet microscope being in a first or second position.

The inventive light sheet microscope thus allows to switch between a first and a second illumination light path of at least two different illumination light paths. An arbitrary number of different light paths is conceivable, wherein the number may be limited by the geometry of the microscope and the available space.

The at least two different illumination light paths may be, at least in portions, oriented collinearly to each other, i.e. be identical. Preferably, the light paths may be identical from an illumination source to the second operational position of the mirror assembly.

Each of the observation light paths may be defined by the corresponding illumination light path. A first illumination light path, along which illumination light is transmitted into the first sample volume thus defines the first observation light path or may at least define limitations of the position of the first observation light path. Preferably, in the sample volume, the observation light path may be oriented essentially perpendicular to the illumination light path. More preferably, an angle between observation light path and illumination light path is between <NUM>° and <NUM>°. Even more preferably, this angle is between <NUM>° and <NUM>°, for example around <NUM>°.

Further, the first illumination light path and the first observation light path are assigned to the first sample volume. Accordingly, the second illumination light path and the second observation light path are assigned to the second sample volume. Accordingly, a third or fourth sample volume with the corresponding light paths may be provided. The number of possible sample volumes is only limited by geometrical limitations of the light sheet microscope.

In the first operational position of the mirror assembly, the first illumination light path may bypass the mirror assembly.

In one embodiment of the inventive light sheet microscope, the mirror assembly may be configured to be at least one of linearly and rotatably moveable from the first operational position to the second operational position.

Thus, in the first operational state, the first illumination light path and the first observation light path may bypass the mirror assembly, whereas in the second operational state the mirror assembly may be located in the second illumination light path and in the second observation light path.

As mentioned above, the two or more illumination light paths and the two or more observation light paths may overlap in section, wherein the second operational position of the mirror assembly may define a branching point for the illumination light paths, respectively a combination point for the observation light paths. Preferably, according to the operational state of the light sheet microscope, only one illumination light path and only one observation light path is selected by means of the operational position of the mirror assembly.

The mirror assembly may comprise at least one mirror. It is also conceivable that the mirror assembly comprises two or more mirrors for redirecting the illumination light path and the corresponding observation light path to a corresponding optical element. Apart from the first illumination light path and the first observation light path (both bypassing the mirror assembly), the mirror assembly may define further at least a second illumination and observation light path by a second operational position. The mirror assembly of the light sheet microscope may further define a third illumination and observation light path by a third operational position. Accordingly, a fourth, fifth, up to n-th operational position of the mirror assembly may be possible. In each operational position a different mirror (or different number of mirrors) may be provided in the illumination and observation light path.

For each operational position, the mirror assembly may be in a pre-adjustment-state. If the inventive light sheet microscope has more than two operational positions, e.g. three operational positions, a first mirror of the mirror assembly may be provided in the illumination and observation light path in the second operational position of the mirror assembly, whereas, a second mirror of the mirror assembly may be provided in the illumination and observation light path in the third operational position of the mirror assembly. Thus, in the third operational position of the mirror assembly, the third illumination light path and the third observation light path may bypass the first mirror of the mirror assembly. Consequently, different mirrors, each of which may provide a preadjustment, may be used for directing illumination and observation light along the second, third, up to n-th illumination and observation light path.

Preferably a back focal plane/a pupil of the first optical element and the second optical element (in other embodiments of the microscope also of the further optical elements like the third, fourth, up to n-th optical element) are located at the same position in the light sheet microscope. In general, the back focal plane of an optical element is the Fourier plane of the focal plane of the optical element lying in the sample or in a further plane conjugate to this plane. Thus, e.g. for the first optical element, the corresponding back focal plane is a focal plane located opposite the first sample volume. This definition of the back focal plane may be applied to a second optical element and a second sample volume; as well as to possible further, e.g. third etc optical elements and sample volumes. It is further particularly advantageous if the back focal plane/pupil of all optical elements, i.e. the first, second, third, etc n-th optical elements do coincide with a further focal plane of an objective that generates an oblique virtual image, which is subsequently imaged onto the detector device by an optical assembly oriented in an oblique manner with respect to the other optical components.

The focusing angles of the optical elements (first, second, third, etc.) may be the same, i.e. M_1 / n_1 = M_2 / n_2 may apply where M is the magnification given by the ratio of the tube lens and the objective M = f_Tube / f_Objective. Thus, as an example: f_Tube=<NUM>, f_Objective=<NUM> -> M=<NUM> the variable n is the refractive index that the lens is optimized for, i.e. n_1=<NUM> for a water immersion lens. In cases in which the equation above does not match, an intermediate telescope may be required. According to this disclosure, the suggested way to match the equation is to have matching focal lengths of the tube lenses f_tube in each of the beam paths.

The optical elements are preferably fixed in their position and configured i.e., adjusted such that their focal planes/pupils overlap as described above. Thus, by a guided linear movement of the optical assembly, changing the operational states of the light sheet microscope is easily performed without the need of readjustment. Prior art solutions that e.g. apply a rotatable revolver may not provide such an accuracy and require cumbersome and time-consuming readjustment.

In another embodiment of the light sheet microscope, the mirror assembly may be configured to be moved along an insertion direction into the first illumination light path, wherein the insertion direction is one of.

The mirror assembly may be preferably moved linearly into the first illumination light path.

In another embodiment of the light sheet microscope, the optical system may comprise a first tube lens and a second tube lens, wherein the first tube lens may be provided in the first illumination light path, and wherein the second tube lens may be provided in the second illumination light path. Accordingly, a third or fourth tube lens may be provided in the third or fourth illumination light path, respectively. Preferably, in each operational state of the light sheet microscope one tube lens, through which the illumination light path (preferably also the observation light path) is directed, is applied.

The tube lens may be adapted in terms of numerical apertures, focal distance or magnification to the corresponding first, second, third or n-th optical element. The mirror assembly in the first, second, third or n-th operational position as well as the first, second, third or n-th tube lens may be pre-adjusted. Such a preadjustment may preferably result in a partially collinear arrangement of all illumination light paths from a light source to a branching point (at this point at least a first mirror of the mirror assembly is located in the second, third or n-th operational position) and in a partially collinear arrangement of all observation light paths from a combination point to the detector device.

Most preferably, the optical element provided in one of the plurality of illumination light paths and/or the corresponding tube lens are attached at a fixed position with respect to the microscope and only the mirror assembly is moved.

However, at least one of the second tube lens and the at least one second optical element may be configured to be moved together with the mirror assembly from the first operational position into the second operational position, wherein in the second operational position the second tube lens and/or the at least one second optical element is inserted into the second illumination light path. The mirror assembly may comprise a further tube lens (e.g. a third tube lens). Said one or more further tube lens(es) may be configured to be moved together with the mirror assembly.

It is also conceivable that the mirror assembly comprises different, independently movable mirrors. Together with each of said independently movable mirrors an accordingly independently movable tube lens may be provided.

Exemplarily, a first tube lens may be provided in the first illumination light path (preferably also in the first observation light path), i.e. when the mirror assembly is bypassed. In the second operational position of the mirror assembly, a first mirror may be moved (optionally together with a second tube lens) into the first illumination light path, thereby defining the second illumination light path. A third operational position of the mirror assembly is conceivable, in which a second mirror may be moved (optionally together with a third tube lens) into the first illumination light path, thereby defining a third illumination light path. Movement of the first mirror (and the second tube lens) may be independent on a movement of the second mirror (and the third tube lens). Said movement may be in particular along different directions. Said directions may be oriented perpendicular to each other.

In a further embodiment of the light sheet microscope, in the first illumination light path, the first tube lens of the optical system may be located between the at least one first optical element and the second operational position of the mirror assembly. This allows to bypass the first tube lens if the mirror assembly is moved into the second operational position.

Thus, if an inventive light sheet microscope is considered that comprises the first operational state and the second operational state (without excluding further operational states) different possibilities for switching between the states are possible. First, only the mirror assembly is moved from the first operational position to the second operational position for transmitting the illumination light along the second illumination light path wire the mirror assembly through the at least one second optical element into the second sample volume. Here, the second optical element (which may be an objective) and the second tube lens are stationary with respect to the light sheet microscope and are not moved when changing the operational state. Second, the second tube lens may be moved together with the mirror assembly, wherein the second optical element remains stationary. Third, the mirror assembly, the tube lens and the second optical element are altogether moved into the second operational position.

Thus, for each operational state the light sheet microscope may provide a combination of tube lens and optical element.

Each of these combinations is configured for observation of a sample volume. The sample volumes are different in at least the position and optionally also in orientation for each operational state.

It is for instance conceivable that an inventive light sheet microscope comprises three operational states, wherein in the first operational state the light sheet microscope is configured for observation from a side (i.e. perpendicularly with respect to an up down direction defined by gravity), whereas in the second operational state a sample provided in the second sample volume may be observed from above, i.e. with illumination along the direction of gravity. Additionally, in a third operational state the light sheet microscope may be configured to observe a sample from below, i.e. with illumination against the direction of gravity.

In a further embodiment of the inventive light sheet microscope, a ratio of focal lengths of the second optical element and the second tube lens may be essentially equal to the ratio of focal lengths of the at least one first optical element and the first tube lens. This provides equal path lengths of the illumination and observation light path for both (preferably more general: for all) operational states.

Further, the mirror assembly may comprise at least one adjustable mirror, which is configured to be adjusted in at least one of an angle to and a position along the second illumination light path. This may facilitate any readjustment necessary after changing the operational state of the light sheet microscope from the first operational state into the second operational state.

The optical system of the inventive light sheet microscope may comprise a pair of adjustment mirrors, each of which is configured to be adjusted in at least one of an angle to and a position along the first and the second illumination light path. With such a pair of adjustment mirrors not only the position of the light path in the optical element or the tube lens may be adjusted but also the angle under which light path passes through said element.

In another advantageous embodiment of the light sheet microscope, the first optical element may be a first objective, wherein the second optical element may be a second objective and wherein the first objective differs from the second objective in at least one of.

Further, an optical axis of the first optical element and an optical axis of the second optical element may be oriented one of.

As explained above, a direction under which the sample volume is observed may be different for different operational states of the light sheet microscope. In the parallel orientation the observation occurs in the same direction for the two (respectively all) operational states of the light sheet microscope, whereas in the antiparallel orientation the direction of observation is opposite, e.g. downwards and upwards for the first operational state and the second operational state, respectively. The third case above refers to an observation of the sample volume from the side, wherein in the other operational state the observation is performed downwards or upwards.

In another advantageous embodiment of the inventive light sheet microscope, the optical system may comprise at least two scan mirrors, which are configured to be adjustable in at least one of angle and position with respect to the first and the second illumination light path and which are configured to scan the first and the second sample volume with a light sheet generated in said sample volume.

The program code may, for example, be stored on a machine-readable carrier.

Other embodiments comprise the computer program for performing one of the methods described herein, stored on a machine-readable carrier.

A further embodiment comprises a processing means, for example, a computer or a programmable logic device configured to or adapted to perform one of the methods described herein.

In the following, the inventive light sheet microscope will be described with reference to the attached figures in the figures, same technical features and features having the same technical effect will be referred to with the same reference numeral. Technical features of different figures may be arbitrarily combined or may be omitted. The figures show exemplary examples of the invention which do not limit the scope of protection which is defined by the claims.

In the following description of the figures, each technical feature is provided with a preceding number that indicates in which figure said technical feature is given. This, however, does not exclude that for instance a feature with, as an example, reference numeral <NUM> is present in <FIG>. Neither does this reference numeral <NUM> exclude the presence of said feature in preceding figures <NUM>, <NUM> etc. One and the same technical feature, which is present in more than one figure may thus be referred to by reference numerals <NUM>, <NUM>, <NUM>, <NUM>,. x05, with 'x' being the overall number of figures provided.

<FIG> shows a light sheet microscope <NUM>, in particular an oblique plane microscope <NUM>, which comprises an optical system <NUM> with at least one first optical element <NUM>, at least one second optical element <NUM> and a mirror assembly <NUM>.

<FIG> indicates two operational states of the oblique plane microscope <NUM>, namely a first operational state <NUM>, in which the optical system <NUM> is configured to transmit illumination light <NUM> from a light source <NUM> along a first illumination path <NUM> through the first optical element <NUM> into a first sample volume <NUM>.

Within the first sample volume <NUM> a light sheet <NUM> is generated that illuminates a planar portion within the first sample volume <NUM>.

<FIG> also shows a coordinate system indicating directions y and z. in the first operational state <NUM> the first sample volume <NUM> is illuminated along the z direction, which may correspond to an illumination from the side. This may for instance be applied when examination of for instance root growth is desired.

Within the first sample volume <NUM> observation light <NUM> is generated and transmitted via a first observation light path <NUM> through the first optical element <NUM>, which generates an oblique virtual image 128a, which is then correctly (i.e. planar and undistorted) imaged by an optical detector assembly 112a onto a detector device <NUM>.

In the first operational state <NUM> the mirror assembly <NUM> is in the first operational position <NUM>. In the first operational position <NUM> the mirror assembly <NUM> is bypassed by the first illumination light path <NUM>, i.e. the mirror assembly <NUM> may for instance be located in a bypass position 114b, which is indicated in <FIG>. Different bypass positions 114b, like for instance inside or outside of the drawing plane are conceivable.

The mirror assembly <NUM> may be moved into a second operational position <NUM> along an insertion direction <NUM>. This may be performed by a translation device <NUM> like a linear translation stage <NUM>.

The insertion direction <NUM> may be oriented parallel to a reflection surface 114a. The insertion direction <NUM> may also be directed perpendicular to the first illumination light path <NUM> or essentially perpendicular to a reflection plane <NUM> at the mirror assembly <NUM> in the second operational state <NUM>.

In the second operational state <NUM> of the microscope <NUM>, the optical system <NUM> is configured to transmit the illumination light <NUM> along a second illumination light path 108a via the mirror assembly <NUM>. The illumination light <NUM> is transmitted through the at least one second optical element <NUM> into a second sample volume 109a. As with the first sample volume <NUM>, the light sheet <NUM> is generated and the observation light <NUM> is transmitted along a second observation light path 111a from the second sample volume 109a to the detector device <NUM>.

As shown in <FIG>, in a first portion <NUM> of the microscope <NUM> the first illumination light path <NUM> at least partially corresponds, overlaps and is collinear with the second illumination light path 108a. Similarly, in the first portion <NUM>, also the first observation light path <NUM> and the second observation light path 111a are collinear with each other.

<FIG> shows a second embodiment of the oblique plane microscope <NUM> that is similar to the one shown in <FIG>.

The oblique plane microscope <NUM> of <FIG> further comprises a first tube lens <NUM> and a second tube lens <NUM>. The first tube lens <NUM> is provided in the first illumination light path <NUM> as well as in the first observation light path <NUM>.

The second tube lens <NUM> is applied (i.e. light is transmitted there through) in the second operational state <NUM>, i.e. when the mirror assembly <NUM> is moved in the second operational position <NUM>.

It is noted that the refraction of the light paths is shown in a schematic manner. Further, the operational state referred to as first operational state <NUM> may, in a different embodiment of the inventive light sheet microscope <NUM> correspond to the second operational state <NUM>. Consequently, in this (not shown) different embodiment of the light sheet microscope <NUM>, the mirror assembly may be provided in the first observation light path <NUM> and in the first illumination light path <NUM> in the first operational state <NUM>, whereas the mirror assembly <NUM> may be bypassed in the second operational state <NUM>.

<FIG> shows an inventive light sheet microscope <NUM> in the form of a scape microscope <NUM>.

The SCAPE microscope <NUM> comprises the light source 307a providing the illumination light <NUM> along the first illumination light path <NUM>. Again, in the first portion <NUM>, the first illumination light path <NUM> is collinear with the second illumination light path 308a.

The illumination light <NUM> is transmitted via a further tube lens <NUM> onto a pair <NUM> of adjustment mirrors <NUM>, which may at the same time be scan mirrors <NUM> for scanning the first sample volume <NUM> or the second sample volume 309a.

The embodiment shown in <FIG> shows a second further tube lens 330a.

The mirror assembly <NUM> is shown in the first operational position <NUM> and in the second operational position <NUM>. In the first operational state <NUM> the first sample volume <NUM> is observed from below, whereas in the second operational state <NUM>, the second sample volume 309a is observed from above.

For each operational state <NUM>, <NUM> a separate optical element, namely the first optical element <NUM> and the second optical element <NUM> is provided.

The first operational position <NUM> and the second operational position <NUM> of the mirror assembly <NUM> are drawn overlapped, wherein the mirror assembly <NUM> in the first operational position <NUM> shows a first mirror <NUM> and the mirror assembly <NUM> in the second operational position <NUM> shows a second mirror <NUM>. <FIG> thus does not show one and the same mirror in different positions <NUM>, <NUM>.

By moving the mirror assembly <NUM> along the insertion direction <NUM>, which is inside the drawing plane in this embodiment, either the first mirror <NUM> or the second mirror <NUM> of the mirror assembly <NUM> may be inserted into the light paths.

The generation of the light sheet <NUM> and collecting of observation light <NUM> which is then transmitted via either a first observation light path <NUM> or via the second observation light path 311a occurs similar as described in the previous figures. In the first portion <NUM>, the first illumination light path <NUM> and the second illumination light path 308a, as well as the first observation light path <NUM> and the second observation light path 311a are collinear with each other.

The optical detector assembly 312a images the oblique virtual image 328a onto the detector device <NUM>, wherein the SCAPE microscope <NUM> further comprises a further objective <NUM> that images that oblique virtual image 328a.

<FIG> also shows a SCAPE microscope <NUM>, wherein there is not a second further tube lens 330a provided as in <FIG>, but a telescope <NUM>. Further, the SCAPE microscope <NUM> of <FIG> provides the first tube lens <NUM> and the second tube lens <NUM> in between the first optical element <NUM> and the mirror assembly <NUM> or between the second optical element <NUM> and the mirror assembly <NUM>.

Also in this embodiment, the mirror assembly <NUM> is moved along the insertion direction <NUM> into the illumination light path <NUM>, 408a. The first tube lens <NUM> and the second tube lens may be moved together with the mirror assembly <NUM> along the insertion direction <NUM>.

Again, the mirror assembly <NUM> comprises a first mirror <NUM>, which is provided in the beam paths in the first operational state <NUM>, and a second mirror <NUM>, which is provided in the beam paths in the second operational state <NUM>.

It is further conceivable, that the microscopes <NUM>, <NUM>, <NUM>, <NUM> and in particular their position of the first sample volume <NUM>-<NUM> and the second sample volume 109a-409a may be combined. Thus, the SCAPE microscope <NUM> of <FIG> may, in a different embodiment, provide three operational states and may thus combine three operational positions of the mirror assembly <NUM>, wherein three different sample volumes may be provided, e.g. the first <NUM>, the second 409a and a third sample volume 409b. The third sample volume 409b is indicated in <FIG> with a dashed line.

Claim 1:
Light sheet microscope (<NUM>), comprising an optical system (<NUM>) with at least one first optical element (<NUM>), at least one second optical element (<NUM>) and a mirror assembly (<NUM>), wherein the mirror assembly (<NUM>), in a first operational state (<NUM>) is in a first operational position (<NUM>) and in a second operational state (<NUM>) is moved into a second operational position (<NUM>), wherein,
- in the first operational state (<NUM>) of the microscope (<NUM>), the optical system (<NUM>) is configured to transmit illumination light (<NUM>) along a first illumination light path (<NUM>) through the at least one first optical element (<NUM>) into a first sample volume (<NUM>) and configured to transmit observation light (<NUM>) along a first observation light path (<NUM>) from the first sample volume (<NUM>) to a detector device (<NUM>), wherein,
- in the second operational state (<NUM>) of the microscope (<NUM>), the optical system (<NUM>) is configured to transmit the illumination light (<NUM>) along a second illumination light path (108a) via the mirror assembly (<NUM>) through the at least one second optical element (<NUM>) into a second sample volume (109a) and configured to transmit observation light (<NUM>) along a second observation light path (111a) from the second sample volume (109a) to the detector device (<NUM>), and
- wherein the first sample volume (<NUM>) and the second sample volume (109a) are nonoverlapping with one another.