Method and measurement system for real-time motion correction during scanning of a spatially moving sample

Abstract

The invention relates to a method for real-time motion correction during scanning of a spatially moving sample. An optical microscope system is provided comprising a scanner and an optical arm having a first end and a second end, arranged to guide an optical beam and to be spatially movable, the first end being optically connected to the optical scanner and the second end comprising an objective. The optical arm comprises a mechanical element providing rotation about a local optical axis. With the scanner, a scanned beam is generated and introduced into the optical arm through the first end of the optical arm, the second end of the optical arm is mechanically connected to the spatially moving sample, and by moving the optical arm the motion of the sample is tracked, the image rotation angle resulting from the position of the mechanical element is determined, and the position of the focal spot of the scanned beam exiting through the objective is modified so as to compensate the image rotation angle. The invention further relates to an optical microscope system for carrying out such a method.

Description

[0001] Method and measurement system for real-time motion correction during scanning of a spatially moving sample

[0002] The invention relates to a method for real-time motion correction during scanning of a spatially moving sample, and an optical microscope system for carrying out such a method.

[0003] In neurobiological research, brain electrophysiological (EEG, electrode) measurements and low-resolution microscopic measurements can readily be carried out on freely moving experimental animals (e.g. mice, rats). By contrast, high-resolution in vivo brain imaging, such as two-photon microscopy through a so- called craniotomy window, is performed with the head fixed. This fixed state, however, makes it difficult to conduct experiments in which the interaction of the animal with its environment, and the resulting brain activity, are to be studied.

[0004] Various approaches have been attempted to overcome the above- mentioned problem. One approach aims to create a virtual reality (VR) environment, enabling controlled animal experiments to be performed with a fixed head. One way of realising the virtual environment is to display the virtual visual content on the walls of a room surrounding the animal, using, for example, a projector or a screen. Hdlscher and co-workers (Rats are able to navigate in virtual reality. J Exp Biol (2005); 208(3): 561 -569) were the first to devise a construction that still forms the basis for most virtual environment designs for rodents. In this arrangement, the animal is surrounded by a torus-shaped dome with a diameter of 140 cm and a height of 80 cm, onto the inner surface of which the virtual visual content is projected by means of a projector. The animal stands inside the torus on a sphere with a diameter of 50 cm, the movement of which — detected via a sensor sensing the displacement of the driven surface — causes the projected virtual environment around the animal to change accordingly. Compared to this, European patent EP3996498B1 provides significantly better immersion, and thus faster learning, by attaching a virtual headset with two wings to the head-fixation mechanism of the experimental animal. The wings of the virtual headset are mechanically adjustable individually to the eyes of the experimental animal, and virtual visual content corresponding to the visual fields perceived by the animal’s eyes is projected onto the displays located in the wings. Despite the high degree of immersion, virtual reality still limits the possible observations, since the experimental animal is unable to physically interact with the virtual environment.

[0005] Another approach aims to enable the free movement of the experimental animal. Klioutchnikov, A., Wallace, D.J., Sawinski, J. et al., in “A three-photon headmounted microscope for imaging all layers of visual cortex in freely moving mice” (Nat Methods 20, 610-616 propose a solution in which a miniaturised laser scanning microscope is mounted on the head of the experimental animal (mouse), into which the laser light is delivered via an optical fibre from a remote laser light source. The drawback of this solution is, on the one hand, that this miniaturised microscope also disturbs the experimental animal, and, on the other hand, that the image quality is not sufficiently high for carrying out more sophisticated measurements.

[0006] The publication “Brain-wide neural recordings in mice navigating physical spaces enabled by a cranial exoskeleton” (James Hope, Travis Beckerle, Pin-Hao Cheng, Zoey Viavattine, Michael Feldkamp, Skylar Fausner, Kapil Saxena, Eunsong Ko, Ihor Hryb, Russell Carter, Timothy Ebner, Suhasa Kodandaramaiah; bioRxiv 2023.06.04.543578; doi: https: / / doj org / 10.1101 / 2023 06.04.543578) discloses a solution in which a microscope is mounted on an external robot arm. The robot arm, by means of a force and torque sensor, tracks the movement of the mouse’s head and keeps the microscope — which is of greater mass and correspondingly provides substantially higher resolution — in the correct position and orientation. The drawback of this solution is that the microscope remains mounted on the robot arm, which limits the mass of the microscope, so that only smaller microscopes with limited functionality can still be used.

[0007] The object of the invention is to provide a device and method which are free from the disadvantages of the solutions according to the prior art. In particular, the object of the invention is to provide a device and method which are not only suitable for scanning a moving sample, in particular a living biological sample, but also capable of performing real-time motion correction resulting from the tracking of the motion.

[0008] The problem set out above has been solved by a method serving for realtime motion correction during scanning of a spatially moving sample. The essence of the method is that an optical microscope system is provided which comprises an optical scanner and a spatially movable optical arm for guiding and focusing an optical beam, the optical arm having a first end and a second end, wherein the first end is optically connected to the optical scanner, a focusing unit comprising at least one objective is arranged at the second end, and the optical arm comprises optical elements having local optical axes defining a spatially bent optical axis, and a mechanical element providing rotation about at least one local optical axis, an optical scanning beam is generated with the optical scanner and introduced into the optical arm through the first end of the optical arm, the second end of the optical arm is mechanically connected to the spatially moving sample, and the motion of the sample is tracked by moving the optical arm, an image rotation angle relative to the sample resulting from the position of the mechanical element providing rotation about at least one local optical axis is determined, and real-time motion correction is performed by modifying the position of the focal spot of the scanning beam exiting through the objective so as to compensate for the image rotation angle.

[0009] In the course of the method, the position of the optical scanner is preferably essentially fixed.

[0010] Preferably, the second end of the optical arm is rigidly connected to the sample, i.e. the sample can move or rotate only together with the element located at the end of the optical arm.

[0011] Preferably, the optical scanner is a laser scanner with acousto-optic deflectors, comprising a first pair of acousto-optic deflectors for deflecting the focal point of the scanning beam exiting the objective in an X-Z plane defined by an optical Z axis of the objective and an X axis perpendicular thereto, and a second pair of acousto-optic deflectors for deflecting the focal point of the scanning beam in a Y-Z plane defined by a Y axis perpendicular to the X and Z axes and the Z axis.

[0012] Preferably, within the moving sample a reference object is designated and three mutually perpendicular straight lines passing through the reference object are designated, and during scanning of the moving sample the straight lines passing through the reference object are scanned in such a way that the laser beam is focused to one end of the straight line, and linear or non-linear chirp signals are provided as acoustic frequency signals to the deflectors deflecting in the X-Z plane and to the deflectors deflecting in the Y-Z plane, thereby continuously moving the focal point along the straight line, determining the translation of the reference object along the mutually perpendicular x, y and z axes fixed to the sample, wherein the z axis fixed to the sample preferably coincides with the optical Z axis of the objective, and during scanning of the moving sample the position of the focal spot is also modified to compensate the translation of the reference object.

[0013] Preferably, scanning of the reference object and modification of the position of the focal spot are repeated during scanning of the moving sample with such frequency as to provide essentially real-time motion correction.

[0014] Preferably, the modification of the position of the focal spot compensating the linear displacement of the reference object is corrected at least partly by controlling the acousto-optic deflectors of the laser scanner.

[0015] Preferably, the image rotation angle is compensated with image rotation optics arranged rotatably about the optical axis.

[0016] Preferably, the image rotation optics comprises a K-mirror, the K-mirror comprising a first and a second mirror arranged at an obtuse angle relative to each other and facing in the direction of the obtuse angle, and a third mirror facing the first and second mirrors and arranged parallel to the line of intersection of the planes of the first and second mirrors, and further comprising an afocal relay arranged on the optical axis of the image rotation optics, the afocal relay comprising a lens element before the K-mirror and a lens element after the K-mirror, arranged such that a rear focal plane of the lens element before the K-mirror and a front focal plane of the lens element after the K-mirror substantially coincide with the plane of the third mirror.

[0017] Preferably, the image rotation angle is compensated by controlling the acousto-optic deflectors of the laser scanner.

[0018] Preferably, the mechanical element providing rotation about at least one local optical axis is a plurality of mirrors arranged in the optical arm to bend the optical beam at right angles and mounted rotatably with bearings coaxial with the incoming local optical axis, and the image rotation angle caused by the rotatable mirrors is determined as the algebraic sum of the mechanical rotation angles of the individual mirrors.

[0019] Preferably, the rotatable mirrors are motorised or moved by an external motorised unit, for example a robot arm, and the mechanical position of the optical arm is at least partly determined from the motor positions.

[0020] Preferably, an objective is arranged at the second end of the optical arm, and the mechanical element providing rotation about at least one local optical axis is a head bearing arranged at the second end of the optical arm, rotatable about the axis of the objective while leaving the axis of the objective free, the sample being fixed to the head bearing, and during movement of the sample the rotation of the head bearing about the axis of the objective, as a local optical axis, is determined and the resulting image rotation is compensated.

[0021] Preferably, the rotation of the head bearing is determined by a rotation sensor arranged at the head bearing.

[0022] Preferably, during movement of the sample the actual mechanical position of the optical arm is determined, and by optical ray tracing calculation at least two beams passing through the microscope system are determined, preferably a beam starting along the bent optical beam path and a beam deflected slightly along a first perpendicular axis relative to the bent optical beam path, and it is determined with what rotation and displacement these beams reach the sample, and real-time motion correction is carried out by modifying the position of the focal spot of the beam exiting through the second end of the optical arm so as to compensate for the image rotation and displacement.

[0023] Preferably, the optical microscope system is calibrated in such a way that the second end of the optical arm is mechanically connected to a static sample, the optical arm is moved through its N-dimensional range of motion defined by its possible mechanical positions with a given resolution, an image of the static sample is acquired in each mechanical position, and image rotation and displacement are measured on the acquired image relative to a reference image, and during measurement of the moving sample the image rotation and displacement corresponding to the actual mechanical position of the optical arm are determined from the calibration measurements, and real-time motion correction is performed by modifying the position of the focal spot of the beam exiting through the second end of the optical arm so as to compensate for the image rotation and displacement.

[0024] Preferably, the image rotation angle resulting from the position of the mechanical element providing rotation about at least one local optical axis is determined by designating a plurality of reference objects within the moving sample, measuring their positions, and determining such an image rotation with which the relative positions of the reference objects remain unchanged, and the position of the focal spot of the beam exiting through the second end of the optical arm is modified so as to compensate for the thus determined image rotation angle.

[0025] Preferably, the optical arm comprises, for guiding the optical beam, an output section, a shoulder unit, an upper arm, an elbow unit, a forearm, and a wrist joint unit with an optical output arranged in sequence, the shoulder unit mechanically connecting the output section and the upper arm rotatably relative to each other about at least a first and a second rotation axis perpendicular to each other, the elbow unit mechanically connecting the upper arm and the forearm rotatably relative to each other about at least a third rotation axis, and the wrist joint unit mechanically connecting the forearm and the optical output of the wrist joint unit rotatably relative to each other about at least a further rotation axis.

[0026] Preferably, the output section, the upper arm, the forearm and the optical output of the wrist joint unit are optically connected by mirrors arranged in the shoulder unit, the elbow unit and the wrist joint unit.

[0027] Preferably, an afocal lens system is arranged in the optical arm, wherein a rear focal plane of each lens coincides with a front focal plane of the subsequent lens.

[0028] Preferably, each mirror is arranged at an angle of 45° to the rotation axis passing through it, wherein in the shoulder unit there is a first shoulder mirror arranged at an angle of 45° to the output section and rotatable relative to the output section, and a second shoulder mirror arranged at an angle of 45° to the upper arm and rotatable relative to the first shoulder mirror, and the output section and the upper arm are optically connected by a shoulder afocal relay comprising a lens element arranged before the shoulder unit in the output section and a lens element arranged after the shoulder unit in the upper arm, arranged such that a rear focal plane of the lens element before the shoulder unit coincides with a front focal plane of the lens element after the shoulder unit in a common plane in the vicinity of the first and second shoulder mirrors.

[0029] Preferably, in the elbow unit there is a first elbow mirror arranged at an angle of 45° to the upper arm and preferably connected to the upper arm without rotation, and a second elbow mirror arranged at an angle of 45° to the forearm and rotatable relative to the first elbow mirror, and the upper arm and the forearm are optically connected by an elbow afocal relay comprising a lens element arranged before the elbow unit in the upper arm and a lens element arranged after the elbow unit in the forearm, arranged such that a rear focal plane of the lens element before the elbow unit coincides with a front focal plane of the lens element after the elbow unit in a common plane in the vicinity of the first and second elbow mirrors.

[0030] Preferably, in the wrist joint unit there is a first wrist mirror arranged at an angle of 45° to the forearm and rotatable relative thereto, and a second wrist mirror arranged rotatable relative to the first wrist mirror, the wrist joint unit comprising a wrist section connecting the first and second wrist mirrors, the wrist section being arranged at an angle of 45° to both wrist mirrors, and a wrist lens is arranged in the vicinity of the wrist section, preferably in the wrist section itself.

[0031] Preferably, a rear focal plane of the lens element after the shoulder unit coincides with a front focal plane of the lens element before the elbow unit, and a rear focal plane of the lens element after the elbow unit coincides with a front focal plane of the wrist lens.

[0032] Preferably, the system comprises an image rotation section arranged optically before the output section, the image rotation section comprising image rotation optics arranged rotatably about the optical axis of the image rotation section, for example a K-mirror, the K-mirror comprising a first and a second mirror arranged at an obtuse angle relative to each other and facing in the direction of the obtuse angle, and a third mirror facing the first and second mirrors and arranged parallel to the line of intersection of the planes of the first and second mirrors, and further comprising an afocal relay arranged on the optical axis of the image rotation section, the afocal relay comprising a lens element before the K-mirror and a lens element after the K-mirror, arranged such that a rear focal plane of the lens element before the K-mirror and a front focal plane of the lens element after the K-mirror substantially coincide with the plane of the third mirror, and a rear focal plane of the lens element after the K-mirror coincides with a front focal plane of the lens element before the shoulder unit.

[0033] Preferably, the system comprises a focusing unit connected to the optical output of the wrist joint unit, the focusing unit comprising an objective, at least a first detector and a beam splitter unit, wherein a main dichroic mirror is arranged to optically connect the objective with the second wrist mirror and with the first detector, and preferably the focusing unit is rotatable about the optical axis of the optical output of the wrist joint unit, and the dichroic mirror is arranged at an angle of 45° to the optical axis of the optical output of the wrist joint unit.

[0034] The objectives set out above are further achieved by an optical microscope system for scanning a spatially moving sample, comprising an optical scanner. The essence of the microscope system is that it comprises a spatially movable optical arm for guiding and focusing an optical beam, the optical arm having a first end and a second end, wherein the first end is optically connected to the optical scanner, a focusing unit comprising at least one objective is arranged at the second end, and the optical arm comprises optical elements having local optical axes defining a spatially bent optical axis, and a mechanical element providing rotation about at least one local optical axis, and the optical microscope system is configured to perform the following steps: generating an optical scanning beam with the optical scanner and introducing it into the optical arm through the first end, tracking the spatially moving sample mechanically connected to the second end of the optical arm by moving the optical arm, determining the image rotation angle resulting from the position of the mechanical element providing rotation about at least one local optical axis between the first and second ends of the optical arm, and performing real-time motion correction by modifying the position of the focal spot of the beam exiting through the second end of the optical arm so as to compensate for the image rotation angle.

[0035] Preferably, the optical scanner is a laser scanner with acousto-optic deflectors, comprising a first pair of acousto-optic deflectors for deflecting the focal point of the scanning beam exiting the objective in an X-Z plane defined by the optical Z axis of the objective and an X axis perpendicular thereto, and a second pair of acousto-optic deflectors for deflecting the focal point of the scanning beam in a Y-Z plane defined by a Y axis perpendicular to the X and Z axes and the Z axis.

[0036] Preferably, the optical microscope system is further configured to perform the following steps: designating within the moving sample a reference object and three mutually perpendicular straight lines passing through the reference object, scanning during the scanning of the moving sample the straight lines passing through the reference object by focusing the laser beam to one end of the straight line, and providing linear or non-linear chirp signals as acoustic frequency signals in the x-axis deflectors and the y-axis deflectors, thereby continuously moving the focal point along the straight line, determining the linear displacement of the reference object along the local optical axis corresponding to the optical axis of the laser scanner and along the local axes corresponding to the x and y axes of the laser scanner, which are perpendicular thereto, and modifying the position of the focal spot during scanning of the moving sample so as also to compensate the linear displacement of the reference object.

[0037] Preferably, the modification of the position of the focal spot compensating the linear displacement of the reference object is configured to be corrected at least partly by controlling the acousto-optic deflectors of the laser scanner.

[0038] Preferably, the system comprises image rotation optics arranged rotatably about the optical axis, and is configured to compensate the image rotation angle with the image rotation optics.

[0039] Preferably, the image rotation optics comprise a K-mirror, the K-mirror comprising a first and a second mirror arranged at an obtuse angle relative to each other and facing in the direction of the obtuse angle, and a third mirror facing the first and second mirrors and arranged parallel to the line of intersection of the planes of the first and second mirrors, and further comprising an afocal relay arranged on the optical axis of the image rotation optics, the afocal relay comprising a lens element before the K-mirror and a lens element after the K-mirror, arranged such that a rear focal plane of the lens element before the K-mirror and a front focal plane of the lens element after the K-mirror substantially coincide with the plane of the third mirror.

[0040] Preferably, the system is configured to compensate the image rotation angle by controlling the acousto-optic deflectors of the laser scanner.

[0041] Preferably, the optical scanner has an aperture diaphragm imaged through the optical arm onto the entrance pupil of the objective.

[0042] Preferably, the mechanical element providing rotation about at least one local optical axis is a head bearing arranged at the second end of the optical arm, rotatable about the axis of the objective while leaving the axis of the objective free.

[0043] Preferably, a rotation sensor for measuring the rotation of the head bearing is arranged at the head bearing.

[0044] Preferably, the mechanical element providing rotation about at least one local optical axis is a plurality of mirrors arranged in the optical arm to bend the optical beam at right angles and mounted rotatably with bearings coaxial with the incoming local optical axis.

[0045] Preferably, the rotatable mirrors are motorised or movable by an external motorised device, for example a robot arm.

[0046] Preferably, the optical arm comprises, for guiding the optical beam, an output section, a shoulder unit, an upper arm, an elbow unit, a forearm and a wrist joint unit with an optical output arranged in succession, the shoulder unit mechanically connecting the output section and the upper arm rotatably relative to each other about at least a first and a second mutually perpendicular rotation axis, the elbow unit mechanically connecting the upper arm and the forearm rotatably relative to each other about at least a third rotation axis, and the wrist joint unit mechanically connecting the forearm and the optical output of the wrist joint unit rotatably relative to each other about at least a further rotation axis, the output section, the upper arm, the forearm and the optical output of the wrist joint unit being optically connected by mirrors arranged in the shoulder unit, the elbow unit and the wrist joint unit, and an afocal lens system being arranged in the optical arm such that a rear focal plane of one lens coincides with a front focal plane of a subsequent lens.

[0047] Preferably, each mirror is arranged at an angle of 45° to the rotation axis passing through it, wherein in the shoulder unit there is a first shoulder mirror arranged at an angle of 45° to the output section and rotatable relative to the output section, and a second shoulder mirror arranged at an angle of 45° to the upper arm and rotatable relative to the first shoulder mirror, and the output section and the upper arm are optically connected by a shoulder afocal relay comprising a lens element arranged in the output section before the shoulder unit and a lens element arranged in the upper arm after the shoulder unit, arranged such that a rear focal plane of the lens element before the shoulder unit coincides with a front focal plane of the lens element after the shoulder unit in a common plane in the vicinity of the first and second shoulder mirrors.

[0048] Preferably, in the elbow unit there is a first elbow mirror arranged at an angle of 45° to the upper arm and preferably connected to the upper arm without rotation, and a second elbow mirror arranged at an angle of 45° to the forearm and rotatable relative to the first elbow mirror, and the upper arm and the forearm are optically connected by an elbow afocal relay comprising a lens element arranged in the upper arm before the elbow unit and a lens element arranged in the forearm after the elbow unit, arranged such that a rear focal plane of the lens element before the elbow unit coincides with a front focal plane of the lens element after the elbow unit in a common plane in the vicinity of the first and second elbow mirrors.

[0049] Preferably, in the wrist joint unit there is a first wrist mirror arranged at an angle of 45° to the forearm and rotatable relative thereto, and a second wrist mirror arranged rotatable relative to the first wrist mirror, the wrist joint unit comprising a wrist section connecting the first and second wrist mirrors, the wrist section being arranged at an angle of 45° to both wrist mirrors, and a wrist lens is arranged in the vicinity of the wrist section, preferably in the wrist section itself.

[0050] Preferably, a rear focal plane of the lens element after the shoulder unit coincides with a front focal plane of the lens element before the elbow unit, and a rear focal plane of the lens element after the elbow unit coincides with a front focal plane of the wrist lens.

[0051] Preferably, the focusing unit is connected to the optical output of the wrist joint unit, the focusing unit comprising an objective, at least a first detector and a beam splitter unit, wherein a main dichroic mirror is arranged to optically connect the objective with the second wrist mirror and with the first detector, and preferably the focusing unit is rotatable about the optical axis of the optical output of the wrist joint unit, and the dichroic mirror is arranged at an angle of 45° to the optical axis of the optical output of the wrist joint unit.

[0052] Preferably, the focusing unit comprises a second detector, and in the beam splitter unit a secondary dichroic mirror is arranged between the main dichroic mirror and the second detector.

[0053] Preferably, the system comprises a robot arm with at least three degrees of freedom, and the optical arm is mounted on the robot arm so as to be movable by the robot arm.

[0054] Preferably, the system comprises an examination table arranged beneath the robot arm.

[0055] Preferably, the mechanical element providing rotation about at least one local optical axis is a head bearing arranged at the second end of the optical arm, rotatable about the axis of the objective while leaving the axis of the objective free, and the robot arm is provided with a force and torque sensor for measuring force and torque acting at the head bearing, and the system is configured to move the robot arm on the basis of the signals of the force and torque sensor so as to continuously predict the motion of an experimental animal connected to the head bearing on the basis of the measured forces and torques, and to move the focusing unit to the position and desired orientation intended to be assumed by the animal.

[0056] Preferably, in the shoulder unit, the elbow unit and the wrist joint unit, motorised bearings with rotary drives connect the output section and the upper arm, the upper arm and the forearm, and the forearm and the optical output of the wrist joint unit rotatably relative to each other.

[0057] Preferably, the output section, the upper arm and the forearm are configured as rigid tubes.

[0058] Preferably, the optical microscope system is a laser scanning microscope system comprising a laser scanning microscope, preferably a multiphoton microscope, further comprising a laser scanner, and a microscope objective arranged at the second end of the optical arm.

[0059] Further details of the invention will be described, by way of embodiments, with reference to the drawings. In the drawings:

[0060] Figure 1 a is a side view of a laser scanning microscope system according to the invention,

[0061] Figure 1 b is a perspective view of the laser scanning microscope system, Figure 2 is a schematic view of a laser scanning microscope system comprising acousto-optic deflectors,

[0062] Figure 3 is a perspective view of the optical arm and the robot arm of the laser scanning microscope system of Figure 1 ,

[0063] Figure 4 is an enlarged perspective view of the focusing unit of the laser scanning microscope system of Figure 1 ,

[0064] Figure 5 is a schematic view showing the optical beam path of the scanner and the optical arm of a laser scanning microscope system according to the invention,

[0065] Figure 6A is an enlarged view of the part of the optical arm of Figure 5 containing the image rotation section,

[0066] Figure 6B is an enlarged view of the part of the optical arm of Figure 5 containing the shoulder unit,

[0067] Figure 6C is an enlarged view of the part of the optical arm of Figure 5 containing the elbow unit,

[0068] Figure 6D is an enlarged view of the part of the optical arm of Figure 5 containing the wrist joint unit,

[0069] Figure 7 is a schematic view showing the optical beam path of the scanner and the optical arm of another laser scanning microscope system according to the invention,

[0070] Figure 8 is an image illustrating reference objects recorded in a field of view of the sample,

[0071] Figure 9 is a diagram illustrating the reference measurement according to the invention,

[0072] Figure 10A is a schematic view of sparse neuron-level labelling on the left, and on the right an example of a maximum intensity z-projection of an imaging field of view,

[0073] Figure 10B shows fluorescent signals detected with JEDI-2P from a region of interest delineated on dendrites,

[0074] Figure 10C shows the power spectrum of real-time motion-corrected measurements and the power spectrum of residual motion after real-time motion correction,

[0075] Figure 10D shows the cumulative density distribution of soma displacements.

[0076] Definitions:

[0077] In the context of the present description, a lens (in the case of afocal relays, a lens element) is understood to mean any optical lens operating on a refractive, reflective and / or diffractive principle, for example a converging lens, a diverging lens, a compound lens, an objective lens, and the like.

[0078] In the context of the present description, for lenses (in the case of afocal relays, lens elements), a distinction is made between a front focal plane and a rear focal plane. The front focal plane, along the optical beam path in the direction towards the object under examination by the microscope, is the focal plane preceding the lens, whereas the rear focal plane is the focal plane following the lens.

[0079] In the context of the present description, the pupil of a lens or an imaging system is understood to mean the image of the aperture stop, as is well known to a person skilled in optical design.

[0080] In the context of the present description, a freely moving experimental animal is understood to mean a quasi-freely moving experimental animal whose movement is not significantly influenced by a focusing unit connected to the head of the experimental animal.

[0081] In the context of the present text, scanning (sweeping) is understood to mean a targeted change in the direction and / or divergence of an optical beam, resulting in a displacement of the focal point of the beam as required. Scanning is typically used for the successive (or partially successive) laser excitation of different points or volumes of a sample. A device that produces the deflection or change in divergence is referred to as a scanner.

[0082] Figures 1a, 1 b and 2 show, by way of example, an embodiment of a microscope system 10 according to the invention, designed for the examination of a quasi freely moving experimental animal.

[0083] The microscope system 10 comprises an optical scanner 100 and an optical arm 200 arranged to guide and focus an optical beam. A first end 200a of the optical arm 200 is optically connected to the optical scanner, while at a second end 200b a focusing unit 280 is arranged, comprising at least one objective 282. The optical arm 200 comprises optical elements having local optical axes which define a spatially bent optical axis, such as one or more lenses, relays, mirrors, polarisers, A / 2 plates, prisms, and the like. The spatially bent optical axis is formed from the local axes of the individual optical elements; bending of the optical axis is typically effected by mirrors.

[0084] The optical arm 200 further comprises at least one mechanical element providing rotation about a local optical axis. This may be, for example, a mirror arranged in the optical arm 200 to deflect the optical beam guided through the optical arm 200 at a right angle (or another angle), and arranged to be rotatable by means of a bearing coaxial with the incoming local optical axis, optionally a motorised mirror.

[0085] The at least one mechanical element providing rotation about a local optical axis may also be, for example, a bearing arranged at the second end 200b of the optical arm 200 about the axis of an objective 282 (which is, from the perspective of the optical arm 200, a local optical axis), rotatable while leaving the axis of the objective 282 free, to which a sample can be fixed (for example the head of an experimental animal, and therefore hereinafter referred to as a head bearing in order to distinguish it from other bearings). In this way, the head bearing results in an image rotation relative to the examined sample between the first end 200a and the second end 200b of the optical arm 200.

[0086] Naturally, a plurality of different mechanical elements may be simultaneously present in the optical arm 200, each of which individually also results in an image rotation between the first end 200a and the second end 200b of the optical arm 200. For example, the optical arm 200 preferably comprises several rotatable mirrors as well as the head bearing arranged at the second end 200b.

[0087] The microscope system 10 according to the invention will hereinafter be described by way of a laser scanning microscope system 10; however, the invention can similarly be implemented with other types of optical microscope.

[0088] The laser scanning microscope system 10 comprises a laser scanning microscope 11 having a laser scanner 100 and one or more laser sources 101 optically connected thereto. The laser scanning microscope 11 may, for example, be a confocal laser scanning microscope or a multiphoton laser scanning microscope, such as a two-photon laser scanning microscope.

[0089] The laser scanner 100 may be any known type of scanner, for example a scanner comprising acousto-optic deflectors or a scanner comprising movable (for example motorised) mirrors. The laser scanner 100 is associated with one or more laser sources 101 , which are shown schematically in Figures 1 and 2. The laser scanner 100 deflects and / or converges / diverges the beam from the laser source 101 in accordance with its control, thereby producing a scanned beam 105. In Figures 5 and 6A-6D, for illustrative purposes, three scanned beams 105 deflected to different extents are shown: a solid line for a scanned beam 105a deflected by 0° (see Figure 6A), a dashed line for a scanned beam 105b deflected to one edge of the field of view (see Figure 6A), and a dash-dotted line for a scanned beam 105c deflected to the opposite edge of the field of view (see Figure 6A), these corresponding to three different scanning settings of the laser scanner 100. The laser scanner 100 has an aperture stop 100A, which limits the size of the collimated beam (or the numerical aperture of the focusing beam) in a manner well known to a person skilled in optical design (see Figure 5). In scanners, the aperture stop is typically the optical element performing the deflection itself (mirror, acousto-optic deflector, etc.). The laser scanner 100 may also, in a known manner, comprise a built-in output lens 102 which focuses the scanned beam 105 onto a rear focal plane 102" (or, in the case of three-dimensional scanning, into the vicinity of this focal plane). Typically, the scanner field stop, which limits the scannable field of view, is arranged in this output image plane. The scanned beam 105a shown as a solid line is undeflected and is therefore focused onto the optical axis in the focal plane 102", whereas the deflected dashed and dash-dotted scanned beams 105b and 105c are focused to one side or the other at the edges of the field of view.

[0090] In the embodiment shown in Figure 2, the microscope system 10 comprises a multiphoton microscope 11 having a laser source 102. However, microscope systems 10 are also known in which multiple laser sources 102 are provided, by means of which laser beams of different wavelengths can be generated. The microscope 11 has a focusing unit 280 defining an optical Z axis, which in the present case comprises a microscope objective 282. Between the laser source 102 and the microscope objective 282, the common bent optical beam path 16 defined by individual optical elements extends, which defines an optical beam path incident on the optical axis. The bent optical beam path 16 is indicated in Figure 1 by a dashed line and extends from an output aperture of the laser source 102 through a rear aperture 282a of the microscope objective 282 into the sample 60. The bent optical beam path 16 is defined by local optical axes of various optical elements, including beam axes defined after optical deflections caused by refraction, reflection, diffraction and the like. Such optical elements in the present embodiment include mirrors 20, 212, a main dichroic mirror 292, a secondary dichroic mirror 293 (beam splitter), a mirror 294 or a dichroic mirror 294, a beam manipulator 22, lenses 24, and a telecentric relay 26. The beam manipulator 22 may comprise, for example, a Faraday isolator, a dispersion-compensation module, a beam stabiliser and a beam expander, which are individually disclosed, for example, in patent document WO2016 / 079547A1 , and are well known from the prior art. The microscope system 10 also comprises one or more detectors 284, 285, such as a photomultiplier tube (PMT) and / or a camera. The main dichroic mirror 292 serves to direct fluorescent light emitted by the sample 60 and arriving through the microscope objective 282 into the detectors 284, 285. Of course, other known optical elements may also be used, and the optical elements presented here may also be arranged in other configurations, as will be apparent to the skilled person.

[0091] In the preferred embodiment shown in Figure 2, the laser scanner 100 comprises acousto-optic deflectors 40. The laser scanner 100 has a first pair of acousto-optic deflectors comprising first and second deflectors X1 and X2, which deflect, in an X-Z plane defined by the optical Z axis of the microscope objective 282 and an X axis perpendicular thereto, a focal point 19 of the scanning beam 18 passing through deflectors X1 and X2 and through the optical arm 200 and exiting the microscope objective 282. Furthermore, the laser scanner 100 has a second pair of acousto-optic deflectors comprising third and fourth deflectors Y1 and Y2, which deflect, in a Y-Z plane defined by a Y axis perpendicular to the X and Z axes and the Z axis, the focal point 19 of the scanning beam 18 passing through deflectors Y1 and Y2 and through the optical arm 200 and exiting the microscope objective 282. It should be noted that by the scanning beam 18 is meant the useful beam, i.e. the beam belonging to that diffraction order which actually passes through the microscope system 10 and which the microscope objective 282 focuses into the focal point 19, thereby allowing actual use for scanning.

[0092] The X and Y axes are preferably defined in the focal plane of the microscope objective 282, so that the origin of the coordinate system defined by the X, Y and Z axes also lies in the focal plane. Each of the deflectors X1 , X2, Y1 , Y2 comprises an acousto-optic (AO) crystal 41 and a piezoelectric transducer 42 arranged at one end of it. In the arrangement shown in Figure 1 , the transducer 42 is located at the same end of the AO crystal 41 of both deflectors X1 and X2, and likewise at the same end of the AO crystal 41 of both deflectors Y1 and Y2, so that the generated acoustic wave propagates in essentially the same direction in the deflectors X1 and X2, and likewise in the deflectors Y1 and Y2.

[0093] It is also well known to the skilled person that the deflectors X1 , X2, Y1 , Y2 can be arranged in a different sequence or with a different orientation. Such alternative arrangements are disclosed, for example, in publication WO / 2010 / 076579 A1 and in publication WO / 2013 / 102771 A1 , the contents of which are incorporated herein by reference.

[0094] The coordinate system spanned by the X, Y and Z axes is a relative coordinate system which moves together with the microscope objective 282 such that the Z axis always coincides with the optical Z axis of the microscope objective 282. At the output aperture 102 of the laser scanner 100, the deflection directions of the deflectors 40 define axes X', Y' and Z', the coordinate system spanned by which is absolute (provided that the laser scanner 100 itself is not moved).

[0095] For the measurement, a third coordinate system also plays a role, which is defined downstream of the microscope objective 282 by the sample 60 mechanically connected to the second end 200b of the optical arm 200. If at the second end of the optical arm 200 there is an element allowing rotation of the sample 60 about the optical axis, then the sample 60 is rigidly connected to this element, since no further rotation has to be permitted. Naturally, an embodiment is also conceivable in which the element allowing rotation is not part of the optical arm 200, but belongs, for example, to a sample holder, in which case its rotation can be measured, for example, with a rotation sensor or in another way. The third coordinate system defined by the sample 60 is taken such that its z axis coincides with the optical Z axis of the microscope objective 282, while its x and y axes are mutually perpendicular and also perpendicular to the z axis. The coordinate system spanned by the x, y and z axes is also a relative coordinate system, but it is fixed to the sample 60.

[0096] When the optical arm 200 is moved, the coordinate system defined by the X, Y and Z axes also moves in space and may rotate about the Z axis of the microscope objective (and thus about the z axis of the sample 60), as is schematically shown in Figure 2. This results in the coordinate system defined by the deflection axes of the laser scanner 100, and the coordinate system defined by the axes x, y and z fixed to the sample 60, rotating relative to each other about the common Z, z axis, which without correction would cause an image rotation within the sample 60. One task is the real-time compensation of this image rotation. Preferably, vibrations of biological or mechanical origin are also compensated in real time, which in the absence of such compensation would result in a small translational displacement of the two coordinate systems relative to each other, that is, an image displacement within the sample 60. Compensation of image rotation and image displacement likewise falls within the scope of motion correction, since both result from the movement of the microscope system 10 and the sample 60.

[0097] The microscope system 10 further comprises an optical arm 200 according to the invention connected to the optical output of the microscope 11 , at a second end 200b of the optical arm 200 opposite the microscope 11 a focusing unit 280 being arranged. The objective 282 of the microscope system 10 is located here. For this reason, the microscope 11 forming part of the microscope system 10 preferably does not comprise its own objective (i.e. it is an objective-less microscope 11 ), but is complemented by the objective 282 arranged at the end of the optical arm 200.

[0098] In the optical arm 200, by means of optical elements, an optical beam path is formed which guides the laser beam arriving from the laser scanner 100 into the focusing unit 280.

[0099] The optical arm 200 is shown in detail with reference to Figures 3-7.

[0100] In the present embodiment, starting from the direction of the laser scanner 100, the optical arm 200 according to the invention comprises the following main units: an image rotation section 210, an output section 220, a shoulder unit 230, an upper arm 240, an elbow unit 250, a forearm 260, a wrist joint unit 270 and a focusing unit 280. The optical output of each main unit is optically connected to the optical input of the next unit, thereby jointly forming the optical beam path in the optical arm 200.

[0101] In other embodiments, the optical arm 200 does not comprise an image rotation section 210, image rotation being implemented by software instead. An embodiment is also conceivable in which the optical arm 200 does not comprise the focusing unit 280, but the focusing unit 280 is connected as a separate unit to an optical output 270A of the wrist joint unit 270, the optical output 270A being schematically indicated in Figure 4.

[0102] In a preferred embodiment, the microscope system 10 comprises a robot arm 300 with at least three degrees of freedom, and the focusing unit 280 is mounted on a movable end of the robot arm 300, so that the focusing unit 280 is not held and moved by the optical arm 200 but by the robot arm 300, which in the present embodiment is suspended from a stand 400.

[0103] The optical arm 200 terminates in the objective 282 forming part of the focusing unit 280, and in the optical arm 200 an optical beam path is formed which images an aperture diaphragm 100A of the laser scanner 100 onto an entrance pupil 282P of the objective 282.

[0104] The optical arm 200 is an opto-mechanical system by means of which the scanned beam 105 from the laser scanner 100 can be directed into the objective 282, the position of which can be varied dynamically, in such a manner that the aperture stop 100A of the laser scanner 100 is imaged onto the entrance pupil 282P of the objective 282. In this way, the arrangement functions as if the objective 282 were directly connected to the laser scanner 100 (optionally via a tube lens in the case of an infinity-corrected objective). The objective 282 focuses the scanned beams 105, entering through the pupil 282P as collimated beams, into the focal plane 282F. Thus, within the optical arm 200, in front of the objective 282, there are optical elements arranged for guiding the scanned beam 105, so that the properties of the scanned beam 105 travelling through the optical arm 200, such as deflection and divergence, are preserved (or, if required, rescaled by applying non-unity magnification relays), such that the scanned beam 105 enters the objective 282 with these properties. This is achieved by the optical elements arranged within the optical arm 200 appropriately tracking the mechanical movement of the optical arm 200. It is noted that the scanned beams 105 illustrated in the figures also represent the beam path defined by the optical elements, since by “beam path” is meant the path traversed by the scanned beams 105. In the case of a non-scanning microscope, the scanned beam 105 corresponds to the optical beam emerging from the aperture stop of the microscope 11 , and its optical path is defined by the optical elements of the optical arm 200.

[0105] As shown in Figure 3, the shoulder unit 230 mechanically connects the output section 220 and the upper arm 240 to each other in a rotatable manner about at least a first rotation axis A and a second rotation axis B perpendicular thereto. The elbow unit 250 mechanically connects the upper arm 240 and the forearm 260 to each other in a rotatable manner about at least a third rotation axis C. The wrist joint unit 270 mechanically connects the forearm 260 and the optical output 270A of the wrist joint unit 270, and the focusing unit 280 connected thereto, to each other in a rotatable manner about at least one, preferably two, rotation axes D and E indicated in the figure.

[0106] The output section 220, the upper arm 240, the forearm 260 and the focusing unit 280 are able to rotate relative to one another via bearing-mounted connections, the rotation preferably being effected by the robot arm 300 through the movement of the focusing unit 280. An embodiment is also conceivable in which there is no separate robot arm 300, and the optical arm 200 itself also functions as a robot arm. In this case, at the shoulder unit 230, the elbow unit 250 and the wrist joint unit 270, motorised bearing-mounted rotary drives of known type provide for the rotation of the output section 220, the upper arm 240, the forearm 260 and the focusing unit 280 relative to one another.

[0107] A preferred arrangement is to integrate, at or near the elbow unit 250 of the optical arm 200, a lifting cable (or another lifting device) operating with a quasiconstant force and not impeding the spatial movement of the elbow, which on the one hand provides appropriate load relief for the support zones of the optical arm, and on the other hand reduces the torques generated and minimises backlash. Similarly, a mechanical component providing a quasi-constant tensile force can preferably be incorporated between the shoulder unit 230 and the wrist joint unit 270, providing the same advantages as those listed above.

[0108] The output section 220, the upper arm 240 and the forearm 260 are preferably formed as rigid tubes, whether there is a separate robot arm 300 or the optical arm 200 itself functions as a robot arm.

[0109] At the ends of the output section 220, the upper arm 240 and the forearm 260, as well as on the side of the optical output 270A opposite to the focusing unit 280, mirrors 231 , 232, 251 , 252, 271 , 272 forming part of the shoulder unit 230, the elbow unit 250 and the wrist joint unit 270, respectively, are arranged at an angle of 45° to the longitudinal axis of the adjacent tube, and at an angle of 45° to the optical axis of the optical output 270A. Optically, these mirrors 231 , 232, 251 , 252, 271 , 272 connect the output section 220, the upper arm 240, the forearm 260 and the optical output 270A to each other. The mirrors 231 , 232, 252, 271 , 272 also form an angle of 45° with the respective rotation axes A, B, C, D, E passing through them. Thus, at the adjacent ends of the output section 220 and the upper arm 240, the mirrors 231 , 232 forming part of the shoulder unit 230 form angles of 45° with the rotation axes A and B, respectively. At one end of the forearm 260, the mirror 252 forming part of the elbow unit 250 forms an angle of 45° with the rotation axis C. At the other end of the output section 260 and beside the optical output 270A, the mirrors 271 , 272 forming part of the wrist joint unit 270 form angles of 45° with the rotation axes D and E, respectively.

[0110] The focusing unit 280, in addition to the objective 282, preferably comprises at least a first detector 284 and a beam-splitter unit 290 in which a main dichroic mirror 292 is arranged that optically connects the objective 282 to the second wrist joint mirror 272 and to the first detector 284. An alternative possibility is that the detector 284 forms part of the laser scanner 100, for example in the case of a confocal laser scanning microscope 11 where it forms part of the laser scanner 100. In this case, the focusing unit 280 collects the light to be detected that returns through the objective 282 and transmits it through the optical arm 200 to the laser scanner 100.

[0111] In the case of a non-laser-scanning microscope 11 , the detector may likewise be integrated with the microscope 11 , into which the light to be detected is returned via the optical arm 200.

[0112] The focusing unit 280 is connected to the optical output 270A in a rotatable manner about a rotation axis F coinciding with the optical axis of the optical output 270A, which forms an angle of 45° with both the second wrist joint mirror 272 and the main dichroic mirror 292.

[0113] In the present embodiment, the focusing unit 280 also comprises a second detector 285, and in the beam-splitter unit 290, a secondary dichroic mirror 293 is arranged between the main dichroic mirror 292 and the second detector 285. From the secondary dichroic mirror 293, a further mirror 294 directs the light from the objective 282 to the first detector 284.

[0114] Preferably, at the end of the objective 282, there is a head bearing 286 rotatable about the optical axis of the objective 282 and leaving the optical axis of the objective 282 unobstructed, to which a head connector fixed to the head of an experimental animal, typically a small experimental animal (such as a mouse), can be attached in a rotatable manner about the optical axis. The head connector is a plate which preferably - and in a customary manner - contains an observation aperture formed for the objective 282, through which craniotomy-based brain imaging in vivo on the experimental animal can be performed, as is known to the skilled person.

[0115] The head bearing 286 is preferably provided with a rotation sensor 287, which measures the rotation of the head bearing 286 and thereby of the head connector (not shown) affixed to the head of the experimental animal. The rotation sensor 287 may be any known type of rotation sensor, for example, an optical, magnetic, inductive, or other sensor. The signal from the rotation sensor 287 may be used to control the image rotation optics, which in the present embodiment is a K-mirror 211 , which is arranged in an optional image rotator section 210 to be rotatable in a motorised manner about its optical axis, thereby ensuring that the microscope image remains stationary even when the head bearing 286 rotates. The K-mirror 211 comprises, in a known manner, a first mirror 211 a and a second mirror 211 b enclosing an obtuse angle with each other and facing towards the obtuse angle, and further comprises a third mirror 211c facing towards the two mirrors 211 a, 211 b and parallel to the line of intersection of the planes of the two mirrors 211 a, 211 b. The K-mirror 211 is preferably provided with a control unit (which may coincide with the control unit of the entire microscope system 10) which controls the rotation of the K-mirror 211 in accordance with the signal of the rotation sensor 287. In a known manner, rotation of the K-mirror 211 by an angle alpha rotates the microscope image by twice the angle alpha about the optical axis. Accordingly, the K-mirror 211 must be rotated by half of the rotation angle of the head bearing 286 in order to prevent the microscope image from rotating relative to the brain area of the experimental animal under examination.

[0116] As is known to the skilled person, image rotation can also be achieved using other image rotation optics, instead of a K-mirror 211 , for example, a Dove prism, a Schmidt-Pechan prism or an Abbe-Koenig prism.

[0117] In the absence of the optional image rotator section 210, the control signal of the laser scanner 100 may be recalculated based on the signal of the rotation sensor 287, so that the scanned beam 105 follows the rotation of the head of the experimental animal, and the objective 282 continues to focus the scanned beam 105 on the point to be examined. The new position of the target point and the corresponding scanner control functions can be obtained by simple mathematical calculation.

[0118] In the case of the laser scanner 100 comprising the previously described acousto-optic deflectors X1 , X2, Y1 , Y2, for example, the method disclosed in publication WO2018042214A3 may be applied for calculating the control signals, the content of which is incorporated herein by reference.

[0119] Image rotation relative to the examined sample may be caused not only by the rotation of the head bearing 286 (and with it the examined sample) between the first end 200a and the second end 200b of the optical arm 200, but also by the mechanical movement or torsion of the optical arm 200.

[0120] The image rotation optics described above (or image rotation compensation by modification of the scanner control) may also be used to compensate for image rotation resulting from displacements of the rotational degrees of freedom of the optical arm 200.

[0121] In the case where the optical arm 200 comprises mirrors arranged to bend the optical beam at right angles, mounted rotatably with bearings coaxial with the incident local optical axis, as in the embodiment described above, the image rotation angle caused by such rotatable mirrors is determined as the algebraic sum of the mechanical rotation angles of the individual mirrors (that is, the signed sum of the rotation angles). Thus, the image rotation angle to be compensated can be expressed as a simple linear combination (signed sum) of the rotations occurring at the respective rotational degrees of freedom (bearings). The rotations occurring at the respective rotational degrees of freedom can be measured directly by rotation sensors (encoders) arranged adjacent the bearings, or, if the mirrors are motorised, can be determined from the motor positions. Where the optical arm 200 is moved by a robot arm 300, the mechanical position of the optical arm 200, and thereby the rotations occurring at the respective rotational degrees of freedom, can also be calculated from the known (and queryable) position and orientation of the robot arm, which in this case corresponds to solving the inverse kinematic equation of the optical arm 200, or of the combined system of the optical arm 200 and the robot arm 300, as a linked mechanical system. (For carrying out such calculations, as is known to the skilled person, commercial and freely available software packages exist, such as MATLAB Robotics System Toolbox or Robotics Toolbox for Python. For robotic calculations see, for example, John J. Craig, Introduction to Robotics, Mechanics and Control, Third Edition, Pearson Education International, 2005.)

[0122] In more complex configurations of the optical arm 200 (for example where the optical beam is not bent at right angles by the rotatable mirrors, or the rotation axes are not coaxial with the incoming local optical axis), the image rotation angle may be determined by another method. For example, as a first step, during movement of the sample 60 the actual mechanical position of the optical arm 200 is determined, and from this the refractions, reflections, diffractions and so on — generally, optical deflections — are determined by optical ray tracing calculation for at least two beams passing through the known microscope system 10, preferably for a beam starting along the bent optical beam path 16, and for a beam deflected slightly along a first perpendicular axis relative to the bent optical beam path 16. Accuracy can be improved by applying three beams, for example: (1 ) a beam starting along the bent optical beam path 16, (2) a beam deflected slightly along a first perpendicular axis relative to the bent optical beam path 16, and (3) a beam deflected slightly along another axis perpendicular to both axes. From this it is determined with what displacement and with what rotation these beams reach the image plane, or the sample 60. From this the expected image displacement and rotation can be directly inferred for the given mechanical position of the optical arm 200. In this case real-time motion correction is carried out by modifying the position of the focal spot of the beam exiting through the second end of the optical arm 200 so as to compensate the image rotation, and, where appropriate, the image displacement. By this is meant that the change in the image position is described as an image rotation about the optical axis of the objective 282, and, where appropriate, an additional image displacement. The image rotation is preferably compensated by image rotation optics, for example the K-mirror 211 , while the image displacement is preferably compensated by suitable control of the scanner 100. A further possibility is to calibrate the optical microscope system 10 in such a way that the second end 200b of the optical arm 200 is mechanically connected to a static sample, the optical arm 200 is moved through its N-dimensional range of motion defined by its possible mechanical positions with the desired resolution (for example with a position resolution of 100 mm and an angular resolution of 5°), in each mechanical position an image of the static sample is taken, and the image rotation and image displacement on the acquired image are measured relative to a reference image. From this, by interpolation, the image rotation and image displacement corresponding to each mechanical position of the optical arm 200 are obtained. Thereafter, during measurement of the moving sample, the image rotation and image displacement corresponding to the actual mechanical position of the optical arm 200 are determined from the calibration measurements, and real-time motion correction is carried out by modifying the position of the focal spot of the beam exiting through the second end 200b of the optical arm 200 so as to compensate the image rotation and image displacement. The image rotation is preferably compensated by image rotation optics, for example the K-mirror 211 , while the image displacement is preferably compensated by suitable control of the scanner 100.

[0123] In another advantageous embodiment, the image rotation angle resulting from the position of the mechanical element providing rotation about at least one local optical axis is determined by reference measurements carried out on reference objects, as described below with reference to Figure 10. In the embodiment shown in Figure 5, a mirror 212 is arranged between the laser scanner 100 and the image rotator section 210, and a mirror 213 optically connects the image rotator section 210 to the output section 220; however, this may vary depending on the geometric arrangement, see for example the alternative embodiment shown in Figure 7.

[0124] It should be noted that among the rotation axes A, B, C, D, E, F, the rotation axis D may be omitted (fixed) without losing the free 3+3 dimensional (position and orientation) movement of the objective 282 (and thus of the experimental animal), since together with the head bearing 286, the rotation axes A, B, C, E, F are sufficient for this purpose. The significance of the D rotational degree of freedom is that it also provides the optical arm 200 with 3+3 dimensional freedom when rigidly coupled to the robot arm 300 (which is above the head bearing 286), so that the robot arm 300 can move completely freely along a continuous trajectory; its movement will be followed by the optical arm 200 without the occurrence of mechanical overconstraint (binding). As is known to the skilled person, mechanical overconstraint can also be avoided in alternative ways, for example by coupling the optical arm 200 and the robot arm 300 via a bearing coaxial with the objective 282 instead of rigidly fixing them together in 3+3 dimensions. In an embodiment without the robot arm 300, this extra rotational degree of freedom can be omitted.

[0125] The optical arm 200 contains an afocal lens system in which the rear focal plane of each lens coincides with the front focal plane of the next lens. The afocal lens system will be explained in more detail in connection with Figures 5 and 6A- 6D.

[0126] In the present embodiment, the afocal lens system comprises the following afocal relays and lenses.

[0127] The output section 220 and the upper arm 240 are optically connected by an afocal relay 234 around the shoulder, the lens element 234a before the shoulder unit 230 being arranged in the output section 220, and the lens element 234b after the shoulder unit 230 being arranged in the upper arm 240 in such a way that the rear focal plane 234a" of the lens element 234a before the shoulder unit 230 and the front focal plane 234b' of the lens element 234b after the shoulder unit 230 lie in a common plane between the first shoulder mirror 231 and the second shoulder mirror 232. The image of the aperture stop 100A lies in this plane. This is preferred because, along the optical path, the space occupied by the scanned beams 105 is minimal at the focal planes of the lenses, meaning they can pass through the smallest possible optics (e.g. the mirrors 231 , 232) at these points. The planes in which the scanned beam 105 is actually focused are less suitable for this purpose because at those points the small beam size results in increased intensity, which may burn the surfaces of the mirrors 231 , 232, and these planes are conjugate (object-image pairs) with the surface of the sample under examination, meaning that any contamination (or even damage, e.g. on the surface of a mirror) in these planes will be visible in the microscope image. Therefore, the optimal configuration is for the image of the aperture stop 100A to be in the vicinity of the mirrors 231 , 232, ideally falling between the mirrors 231 and 232.

[0128] The upper arm 240 and the forearm 260 are optically connected by an afocal relay 254 around the elbow, the lens element 254a before the elbow unit 250 being arranged in the upper arm 240, and the lens element 254b after the elbow unit 250 being arranged in the forearm 260 in such a way that the rear focal plane 254a" of the lens element 254a before the elbow unit 250 and the front focal plane 254b' of the lens element 254b after the elbow unit 250 lie in a common plane between the first elbow mirror 251 and the second elbow mirror 252. The image of the aperture stop 100A lies in this plane, for similar considerations as in the case of the mirrors 231 , 232.

[0129] The wrist joint unit 270 has a wrist joint section 273, which forms a 45- degree angle with each of the wrist mirrors 271 , 272 and in which a wrist joint lens 274, shown in Figure 6D, is arranged.

[0130] If there is no further lens between the lens element 234b after the shoulder unit 230 and the lens element 254a before the elbow unit 250, then the rear focal plane 234b" of the lens element 234b after the shoulder unit 230 coincides with the front focal plane 254a' of the lens element 254a before the elbow unit 250. Similarly, if there is no further lens, then the rear focal plane 254b" of the lens element 254b after the elbow unit 250 coincides with the front focal plane 274' of the wrist joint lens 274. The rear focal plane 274" of the wrist joint lens 274, if there is no further lens, coincides with the front focal plane of the objective 282, which, in the case of near-telecentric imaging, approximately coincides with the entrance pupil 282P of the objective 282, meaning that the wrist joint lens 274 images the aperture stop 100A onto the entrance pupil 282P of the objective 282. Ultimately, therefore, the aperture stop 100A is imaged at this location.

[0131] Preferably, the image rotator section 210 also contains an afocal relay 214, which, on its optical axis, consists of a lens element 214a before the K-mirror 211 and a lens element 214b after the K-mirror 211 . These are arranged such that the rear focal plane 214a" of the lens element 214a before the K-mirror 211 and the front focal plane 214b' of the lens element 214b after the K-mirror 211 coincide with the plane of the third mirror 211 c. The image of the aperture stop 100A is located here.

[0132] If there is no further lens, the front focal plane 214a' of the lens element 214a before the K-mirror 211 coincides with the rear focal plane 102" of the output lens 102 of the laser scanner 100. If there is no further lens, the rear focal plane of the lens element 214b after the K-mirror 211 coincides with the front focal plane of the lens element 234a before the shoulder unit 230.

[0133] The microscope system 10 may optionally comprise an examination table 500 arranged beneath the suspension point of the robot arm 300, or, in the absence thereof, another surface on which the observed experimental animal may move. On the examination table, an arena or maze is preferably formed, which the experimental animal, such as a mouse, may explore in a quasi-free manner. The wall height of the maze is preferably selected so that the mouse cannot climb over the walls, while at the same time preventing the focusing unit 280 from colliding with them. The robot arm 300 is configured so as to be capable of tracking the movement of the head of the experimental animal and moving the objective 282 accordingly, while also providing the force required to support and appropriately move the optical arm 200. For this purpose, the robot arm 300 is preferably equipped with a force and torque sensor 302 for measuring the force and torque acting at the head bearing, and the microscope system 10 comprises a control unit which moves the robot arm 300 on the basis of the signals from the force and torque sensor 302 in such a way as to continuously predict the movement of the animal from the measured forces and torques, and to move the focusing unit 280 to the position and desired orientation intended to be assumed by the animal. To predict the desired position and orientation, a mechanical model having parameters characteristic of the animal (inertia, damping coefficient, stiffness) and equations of motion are used. The resulting motion is the combination of three translational and three rotational movements, carried out with controlled velocity and acceleration. An alternative possibility is that the displacement of the head of the experimental animal is detected optically or with a magnetic-based sensor, or with another type of sensor suitable for continuous 3D position measurement, and the movement of the robot arm 300 is controlled on that basis so that the robot arm 300 follows as precisely as possible the movement or intended movement of the animal. When such position and orientation sensors are used, it is not necessary to mechanically connect the head of the experimental animal to the robot arm 300 via the force and torque sensor 302, which may increase the freedom of movement; however, tracking the freer movement without substantial phase delays may become more difficult. A suitable robot arm 300 is commercially available, for example the UR10e model manufactured by Universal Robots. A suitable force sensor is also commercially available, for example the NANO17-E model manufactured by ATI Industrial.

[0134] Figure 7 shows an alternative embodiment illustrating how the optical arm 200 according to the invention can be fitted to a conventional microscope 11 comprising a laser scanner 100. Similar elements are denoted by similar reference numerals. Compared with the previous embodiment, the difference is that the output lens 102 of the laser scanner 100 is followed by a tube lens 104a of the base microscope. In place of the objective of the base microscope, a mirror 106 is arranged, which is followed by a second tube lens 104b, the pair of the tube lens 104a, the two together likewise forming an afocal relay. In this embodiment, the image rotation section 210 and the output section 220 lie on a common axis, and only in front of the image rotation section 210 is a mirror 212 provided.

[0135] Microscopic movements of the animal (or, in general, the examined moving specimen) relative to the optical axis, as well as small displacements resulting from mechanical inaccuracies or uncertainties of the optical arm 200, and from residual errors in the optical alignment, can be eliminated in the image using motion correction methods - either in real time or post hoc. The effects of mechanical uncertainties arising during movement of the optical arm 200 can also be minimized by using a beam stabilizer, motorizing certain mirrors, or incorporating beam position detectors (using a wavelength for the reference beam that does not interfere with imaging and can be filtered out, which is coupled into the optical arm 200 bypassing the laser scanner 100 and independently thereof). A suitable motion correction method is disclosed, for example, in published application WO201 8042214A2, the contents of which are incorporated herein in their entirety by reference. A suitable beam stabilizer is disclosed, for example, in published application WO2024013529A2, the contents of which are incorporated herein in their entirety by reference.

[0136] In a particularly preferred embodiment of the present invention, a reference object 64 falling within the field of view 62 of the microscope system 10 of a moving sample 60, as illustrated in Figure 8, is designated, and by measuring the displacement of the reference object 64, small displacements arising from microscopic motion of the sample 60, from mechanical inaccuracies or uncertainties of the optical arm 200, and from residual errors of the optical adjustment, i.e. image displacement error, are corrected. It is noted that this is always applied together with image rotation correction, preferably first calculating the necessary image rotation correction, followed by the additional image displacement correction required.

[0137] In a particularly preferred embodiment, the microscope 11 is a multiphoton microscope, and the optical scanner 100 is a laser scanner in which acousto-optic deflectors serve for rapid beam deflection, as described with reference to Figure 2. In this case, designation and measurement of the reference objects 64 may be carried out as follows.

[0138] As a reference object 64, a naturally occurring bright object present in the sample 60 may be selected (that is, an object that gives a strong response signal when excited with a given intensity), or an artificially introduced reference object giving a strong response, such as a fluorescent bead, as is known to the skilled person, which may likewise serve as a reference object 64.

[0139] During measurement, three mutually perpendicular straight lines A, B, C passing through the selected reference object 64 are designated. These straight lines A, B, C are preferably taken parallel to the x, y, z axes of the coordinate system fixed to the sample 60 (that is, measurement is made in the reference frame of the sample). Naturally, continuous scanning may also be carried out along three mutually perpendicular, intersecting straight lines A, B, C of any other orientation fixed relative to the coordinate system of the sample 60, and the displacement of the reference object 64 can also be determined therefrom.

[0140] During scanning of the moving sample 60, the straight lines A, B, C passing through the reference object 64 are scanned in continuous mode, that is, by continuous movement of the focal spot 19, in such a way that the laser beam 18 is focused to one end of the given straight line A, B, C, and acoustic frequency signals are applied as linear chirp signals in the case of lines lying in the X-Y plane, or as non-linear chirp signals in the case of lines having a component in the Z direction, to the deflectors deflecting in the x-z plane and in the y-z plane, thereby continuously moving the focal point 19 along the given straight line A, B, C. A continuous measurement along one straight line will hereinafter be referred to as a “drift”. The method of determining acousto-optic deflector control functions usable for continuous scanning along the straight lines A, B, C can be found, for example, in publication WO / 2018 / 042214 A2, the content of which is incorporated herein by reference.

[0141] Thereafter, the displacement (translation) of the reference object 64 along the x, y and z axes fixed to the sample 60 is determined, for example by obtaining, from the three line scans A, B, C, detector signal versus position coordinate functions, from which a centroid is calculated, which is taken as the centroid of the reference object 64. The change of the centroid between successive reference measurements is the displacement of the reference object 64 along the respective X, Y, Z axes.

[0142] During scanning of the moving sample 60, the position of the focal spot 19 is modified so as to compensate the linear displacement of the reference object 64 determined in this way.

[0143] It is preferred to carry out the measurement in such a way that the scanning of the sample 60 (that is, the actual measurement) is frequently interrupted to measure again on the designated reference object 64, either along all three selected lines A, B, C, or along one line in each such reference measurement. In this way, the displacement of the reference object 64 can be quasi-continuously tracked, and thus essentially in real time compensated during the actual measurement of the sample 60. Figures 8 and 9 illustrate the course and the results of such a measurement interrupted by reference measurements and essentially real-time motion corrected. It is noted that in the context of the present description, essentially (quasi) real-time motion correction is simply referred to as real-time motion correction.

[0144] Figure 9 shows a schematic diagram of a measurement loop. The sample 60 was excited with a single excitation wavelength (e.g. 920 nm). In each loop, displacements in the x, y and z directions were measured at predetermined intervals on the previously selected fluorescent reference object 64 for the purpose of reference measurement. For displacement correction, during the reference measurement drifts along the x, y and z axes fixed to the sample 60 are applied. From any starting point, three-dimensional drifts can be generated in any direction and at any desired speed, whereby a reference object 64 located at any position can be measured. In the embodiment presented, between the regular measurements, measurements along the x, y and z axes were alternately inserted in such a way that the drift lines passed through the most recently measured centroid of the reference object 64

[0145] From the measurement, the translation (displacement) of the centroid of the reference object 64 along the x, y and z axes is determined, on the basis of which the control signals of the laser scanner 100 are corrected so that during the measurement the focal spot 19 is shifted in the direction and by the magnitude of the displacement, thereby enabling measurement at the desired point (or region) of the sample 60 despite the displacement. The motion correction is likewise applied at the next reference measurement, so that the reference object 64 is always measured taking into account the previously registered displacement.

[0146] The scanning of the reference object 64 and the modification of the focal spot 19 are repeated during the scanning of the moving sample at such a frequency that essentially real-time motion correction is provided. For example, the drifts required for the measurement may run consecutively at about 33 kHz. If reference drifts are inserted in such a way that they constitute 10-20% of the total number of drifts (the sum of the drifts used for normal measurement and for reference measurement), this means that reference measurement is carried out at least at 3.3 kHz, which, divided among the three directions, provides stabilisation at a frequency of at least about 1 kHz along each axis. This, in principle, allows biological / mechanical vibrations at 500 Hz to be largely compensated, while in practice biological / mechanical vibrations occurring at about 100 Hz can be excellently handled, as illustrated with reference to Figure 10.

[0147] The modification of the position of the focal spot 19 compensating the linear displacement of the reference object 64 is preferably corrected partly or wholly by controlling the acousto-optic deflectors of the laser scanner 100. However, compensation on other principles is also conceivable. For example, the position of the focal spot 19 along the Z optical axis can also be corrected by an optical element with adjustable focal length, such as a liquid lens or a zoom lens. The position of the focal spot 19 perpendicular to the Z optical axis can also be corrected by a motorised mirror (typically a galvanometer mirror) tiltable about axes perpendicular to the Z optical axis. Such a mirror may be inserted into essentially any section of the optical system, provided that each deflecting element is imaged onto one another and finally onto the pupil 282P of the objective 282, preferably by afocal relays.

[0148] The results achieved by motion correction are illustrated in Figure 10. Figure 10A, left: schematic illustration of sparse neuron-level labelling in the CA1 region of the hippocampus, using a mixture of four different rAAV vectors to deliver the JEDI- 2P voltage sensor and the tdTomato anatomical marker into the cells (SO: stratum oriens, SP: stratum pyramidale, SR: stratum radiatum, SLM: stratum lacunosum- moleculare). Right: example of a maximum intensity z-projection of an imaging field of view (FOV), showing one reference (tdTomato+) and one functional (JEDI-2P+) CA1 pyramidal cell (PC). Scale bar: 50 pm.

[0149] Figure 10B shows fluorescent signals detected using JEDI-2P from a region of interest (ROI) selected on dendrites in a CA1 pyramidal cell (CA1 PC) of an awake mouse, integrated with and without the 3D-RTMC (real-time motion correction) system (black, w / RTMC; grey, w / o RTMC). The (w / o RTMC) traces were obtained by back-projecting the compensated 3D motion, shown in the lower part of the figure, onto the image sequence and extracting the signals accordingly. Bottom: the motion corrected by 3D-RTMC along the x-, y- and z-axes.

[0150] Figure 10C shows the power spectrum of motions corrected by 3D-RTMC along the x (w / o RTMC) and y (w / o RTMC) axes, as well as the power spectrum of residual motion after 3D-RTMC correction (x, w / RTMC; y, w / RTMC; n = 7 measurements from 7 different mice). It can be seen that motion correction reduced the amplitude of displacement by two orders of magnitude over a wide frequency range. The residual displacement was measured by cross-correlation.

[0151] Figure 10D shows the cumulative density distribution of soma displacements in datasets acquired with (w / RTMC) and without (w / o RTMC) the 3D-RTMC technique, demonstrating a significant difference (Kolmogorov-Smirnov test, p = 1 .78x1 O’39in the x-direction, 8.90x10’11in the y-direction). The insets show time-averaged images of somatic recordings with correction after 3D-RTMC (w / RTMC) and with back-projected 3D motion in front of the image sequence (w / o RTMC).

[0152] Figure 10E shows fluorescent activity transients measured from CA1 pyramidal cells (n = 3 cells) during experiments on awake mice, using high-speed sampling (frequency: 1.51-1.66 kHz) and high-power laser excitation (160 mW average power). Vertical ticks indicate action potentials detected by VolPy (Cai, Changjia et al. “VolPy: Automated and scalable analysis pipelines for voltage imaging datasets.” PLoS Computational Biology vol. 17,4 e1008806. 14 Apr. 2021 , doi: 10.1371 / journal. pcbi.1008806), while arrows indicate plateau-burst depolarisations.

[0153] The measurement on the reference object 64 is not only suitable for correcting errors along the X, Y and Z axes caused by mechanical and biological vibrations or displacements, as also illustrated in Figure 8. As described above, the image rotation about the Z axis resulting from movement of the optical arm 200 and from rotation of the head bearing fixed to the sample (for example, to the head of a mouse) can likewise be determined by similar reference measurements. In this case, in addition to the reference object 64, a secondary reference object 65 is also selected, preferably at a distance within the field of view 62 from the primary reference object 64.

[0154] During the reference measurements, the positions of at least the two reference objects 64, 65 are measured, and an image rotation is determined such that the relative positions of the reference objects 64, 65 remain unchanged. The primary reference object 64 is preferably measured again with three drifts along local axes corresponding to the x, y, z axes of the deflectors, while for the secondary reference object 65 it is sufficient to scan with a single drift along an axis corresponding to either x or y. Preferably, the drift direction is chosen so that the distance is maximised between the scanning line D traversing the secondary reference object 65 and the parallel line A or B traversing the primary reference object 64. From the change in the positions of the two reference objects 64 and 65, the overall rotation of the field of view 62 about the optical Z axis of the objective 282 can be readily determined, which corresponds to the desired image rotation to be determined.

[0155] For example, it may be carried out such that first the reference object 64, designated preferably in the central region of the field of view, is measured with three drifts passing through the reference object 64, and the measured translation is compensated. In this model, this means that only a rotation can remain in the image at that moment. Since compensating the displacement precisely means keeping the reference object 64 fixed in the image throughout the entire measurement, the rotation manifests itself in that everything else - including the reference object 65 - shifts slightly along an arc around the reference object 64.

[0156] Thereafter, the reference object 65 is measured with a drift passing through the reference object 65. The deviation of the centroid from the expected position is caused by the rotation, that is, by the displacement along the arc described above. It may be assumed that the rotation between two reference measurements is at most a few degrees, so that the small-angle approximation is valid, from which the rotation angle can be determined by a simple geometrical calculation.

[0157] The position of the focal spot 19 of the beam 18 exiting through the second end 200b of the optical arm 200 is then modified so as to compensate the image rotation angle thus determined, preferably by image rotation optics, for example by the K-mirror 211 . Another possibility is to modify the control signals of the acoustooptic deflectors so that the focal spot 19 compensates the image rotation, thereby directing the focal spot 19 to the point (or region) actually to be scanned.

[0158] The optical arm 200 according to the invention is also suitable for connecting microscopes or laser scanning units to an endoscope, so that the former can be made flexibly usable in medical practice. In these cases, the aim is to connect the imaging system and the surgical or diagnostic area optically in a manner that is quasi freely movable according to the physician’s needs.

[0159] Various modifications and variations of the embodiments described herein will be apparent to those skilled in the art without departing from the scope of the invention as defined in the appended claims.

Claims

Claims1. A method for real-time motion correction during scanning of a spatially moving sample, characterized by providing an optical microscope system comprising an optical scanner and a spatially movable optical arm for guiding and focusing an optical beam, the optical arm having a first end and a second end, wherein the first end is optically connected to the optical scanner, a focusing unit comprising at least one objective is arranged at the second end, and the optical arm comprises optical elements having local optical axes defining a spatially bent optical axis, and a mechanical element providing rotation about at least one local optical axis, generating an optical scanning beam with the optical scanner and introducing it into the optical arm through the first end of the optical arm, mechanically connecting the second end of the optical arm to the spatially moving sample and tracking the motion of the sample by moving the optical arm, determining an image rotation angle relative to the sample resulting from the position of the mechanical element providing rotation about at least one local optical axis, and performing real-time motion correction by modifying the position of the focal spot of the scanning beam exiting through the objective so as to compensate for the image rotation angle.

2. The method according to claim 1 , characterized in that the optical scanner is a laser scanner with acousto-optic deflectors, comprising a first pair of acoustooptic deflectors for deflecting the focal point of the scanning beam exiting the objective in an X-Z plane defined by an optical Z axis of the objective and an X axis perpendicular thereto, and a second pair of acousto-optic deflectors for deflecting the focal point of the scanning beam in a Y-Z plane defined by a Y axis perpendicular to the X and Z axes and the Z axis.

3. The method according to claim 2, characterized in that within the moving sample a reference object is designated, and three mutually perpendicular straight lines passing through the reference object are designated, and during scanning ofthe moving sample the straight lines passing through the reference object are scanned such that the laser beam is focused to one end of the straight line, and linear or non-linear chirp signals are provided as acoustic frequency signals to the deflectors deflecting in the X-Z plane and to the deflectors deflecting in the Y-Z plane, thereby continuously moving the focal point along the straight line, determining the translation of the reference object along the mutually perpendicular x, y and z axes fixed to the sample, wherein the z axis fixed to the sample preferably coincides with the optical Z axis of the objective, and during scanning of the moving sample the position of the focal spot is further modified to compensate the translation of the reference object.

4. The method according to claim 3, characterized in that scanning of the reference object and modification of the position of the focal spot are repeated with such frequency during scanning of the moving sample as to provide essentially realtime motion correction.

5. The method according to claim 3 or 4, characterized in that the modification of the position of the focal spot compensating the linear displacement of the reference object is corrected at least partially by controlling the acousto-optic deflectors of the laser scanner.

6. The method according to any one of claims 1-5, characterized in that the image rotation angle is compensated with an image rotation optics arranged rotatably about the optical axis.

7. The method according to claim 6, characterized in that the image rotation optics comprises a K-mirror, the K-mirror comprising a first and a second mirror arranged at an obtuse angle relative to each other and facing in the direction of the obtuse angle, and a third mirror facing the first and the second mirrors and arranged parallel to the line of intersection of the planes of the first and the second mirrors, and further comprising an afocal relay arranged on the optical axis of the image rotation optics, the afocal relay comprising a lens element before the K-mirror and a lens element after the K-mirror, arranged such that the rear focal plane of the lenselement before the K-mirror and the front focal plane of the lens element after the K-mirror substantially coincide with the plane of the third mirror.

8. The method according to any one of claims 2-5, characterized in that the image rotation angle is compensated by controlling the acousto-optic deflectors of the laser scanner.

9. The method according to any one of claims 1-8, characterized in that the mechanical element providing rotation about at least one local optical axis comprises a plurality of mirrors arranged in the optical arm to bend the optical beam at right angles and mounted rotatably with bearings coaxial with the incoming local optical axis, and that the image rotation angle caused by the rotatable mirrors is determined as the algebraic sum of the mechanical rotation angles of the individual mirrors.

10. The method according to claim 9, characterized in that the rotatable mirrors are motorized or moved by an external motorized unit, for example a robot arm, and the mechanical position of the optical arm is at least partly determined from the motor positions.11 . The method according to any one of claims 1-10, characterized in that an objective is arranged at the second end of the optical arm, and the mechanical element providing rotation about at least one local optical axis is a head bearing arranged at the second end of the optical arm, rotatable about the axis of the objective while leaving the axis of the objective free, the sample being fixed to the head bearing, and during movement of the sample the rotation of the head bearing about the axis of the objective, as a local optical axis, is determined, and the resulting image rotation is compensated.

12. The method according to claim 11 , characterized in that the rotation of the head bearing is determined by a rotation sensor arranged at the head bearing13. The method according to any one of claims 1-12, characterized in thatduring movement of the sample the actual mechanical position of the optical arm is determined, and by optical ray tracing calculation at least two beams passing through the microscope system are determined, preferably a beam starting along the bent optical beam path and a beam deflected slightly along a first perpendicular axis relative to the bent optical beam path, and it is determined with what rotation and displacement these beams reach the sample, and real-time motion correction is carried out by modifying the position of the focal spot of the beam exiting through the second end of the optical arm so as to compensate for the image rotation and displacement.

14. The method according to any one of claims 1-12, characterized in that the optical microscope system is calibrated by mechanically connecting the second end of the optical arm to a static sample, moving the optical arm through its possible mechanical positions defined by its N-dimensional motion space with a given resolution, acquiring an image of the static sample at each mechanical position, and measuring image rotation and displacement on the acquired image relative to a reference image, and during measurement of the moving sample determining from the calibration measurements the image rotation and displacement corresponding to the current mechanical position of the optical arm, and performing real-time motion correction by modifying the position of the focal spot of the beam exiting through the second end of the optical arm so as to compensate for the image rotation and displacement.

15. The method according to any one of claims 1-12, characterized in that the image rotation angle resulting from the position of the mechanical element providing rotation about at least one local optical axis is determined by designating a plurality of reference objects within the moving sample, measuring their positions, and determining such an image rotation with which the relative positions of the reference objects remain unchanged, and modifying the position of the focal spot of the beam exiting through the second end of the optical arm so as to compensate for the thus determined image rotation angle.

16. An optical microscope system for scanning a spatially moving sample,comprising an optical scanner, characterized in that it comprises a spatially movable optical arm for guiding and focusing an optical beam, the optical arm having a first end and a second end, wherein the first end is optically connected to the optical scanner, a focusing unit comprising at least one objective is arranged at the second end, and the optical arm comprises optical elements having local optical axes defining a spatially bent optical axis, and a mechanical element providing rotation about at least one local optical axis, and the optical microscope system is configured to perform the following steps: generating an optical scanning beam with the optical scanner and introducing it into the optical arm through the first end, tracking the spatially moving sample mechanically connected to the second end of the optical arm by moving the optical arm, determining the image rotation angle resulting from the position of the mechanical element providing rotation about at least one local optical axis between the first and second ends of the optical arm, and performing real-time motion correction by modifying the position of the focal spot of the beam exiting through the second end of the optical arm so as to compensate for the image rotation angle.

17. The optical microscope system according to claim 16, characterized in that the optical scanner is a laser scanner with acousto-optic deflectors, comprising a first pair of acousto-optic deflectors for deflecting the focal point of the scanning beam exiting the objective in an X-Z plane defined by an optical Z axis of the objective and an X axis perpendicular thereto, and a second pair of acousto-optic deflectors for deflecting the focal point of the scanning beam in a Y-Z plane defined by a Y axis perpendicular to the X and Z axes and the Z axis.

18. The optical microscope system according to claim 17, characterized in that the optical microscope system is further configured to perform the following steps: designating a reference object within the moving sample and three mutually perpendicular straight lines passing through the reference object, scanning the straight lines passing through the reference object duringscanning of the moving sample by focusing the laser beam to one end of the straight line, and providing linear or non-linear chirp signals as acoustic frequency signals in the x-axis deflectors and in the y-axis deflectors, thereby continuously moving the focal point along the straight line, determining the linear displacement of the reference object along the local optical axis corresponding to the optical axis of the laser scanner and along the local axes corresponding to the x and y axes of the laser scanner, and modifying the position of the focal spot during scanning of the moving sample so as to additionally compensate the linear displacement of the reference object.

19. The optical microscope system according to claim 18, characterized in that the modification of the position of the focal spot compensating the linear displacement of the reference object is configured to be corrected at least partially by controlling the acousto-optic deflectors of the laser scanner.

20. The optical microscope system according to any one of claims 16-19, characterized in that it comprises image rotation optics arranged rotatably about the optical axis, and is configured to compensate the image rotation angle by the image rotation optics.

21. The optical microscope system according to claim 20, characterized in that the image rotation optics comprises a K-mirror, the K-mirror comprising a first and a second mirror arranged at an obtuse angle relative to each other and facing in the direction of the obtuse angle, and a third mirror facing the first and the second mirrors and arranged parallel to the line of intersection of the planes of the first and the second mirrors, and further comprising an afocal relay arranged on the optical axis of the image rotation optics, the afocal relay comprising a lens element before the K-mirror and a lens element after the K-mirror, arranged such that the rear focal plane of the lens element before the K-mirror and the front focal plane of the lens element after the K-mirror substantially coincide with the plane of the third mirror.

22. The optical microscope system according to any one of claims 16-19,characterized in that it is configured to compensate the image rotation angle by controlling the acousto-optic deflectors of the laser scanner.

23. The optical microscope system according to any one of claims 16-22, characterized in that the mechanical element providing rotation about at least one local optical axis is a head bearing arranged at the second end of the optical arm, rotatable about the axis of the objective while leaving the axis of the objective free.

24. The optical microscope system according to claim 23, characterized in that a rotation sensor for measuring the rotation of the head bearing is arranged at the head bearing.

25. The optical microscope system according to any one of claims 16-24, characterized in that the mechanical element providing rotation about at least one local optical axis comprises a plurality of mirrors arranged in the optical arm to bend the optical beam at right angles and mounted rotatably with bearings coaxial with the incoming local optical axis.

26. The optical microscope system according to claim 25, characterized in that the rotatable mirrors are motorized or movable by an external motorized device, for example a robot arm.

27. The optical microscope system according to any one of claims 16-26, characterized in that the optical arm comprises, arranged in succession for guiding the optical beam, an output section, a shoulder unit, an upper arm, an elbow unit, a forearm and a wrist joint unit with an optical output, wherein the shoulder unit mechanically connects the output section and the upper arm rotatably relative to each other about at least a first and a second perpendicular axis of rotation, the elbow unit mechanically connects the upper arm and the forearm rotatably relative to each other about at least a third axis of rotation, and the wrist joint unit mechanically connects the forearm and the optical output of the wrist joint unit rotatably relative to each other about at least one further axis of rotation, wherein the output section, the upper arm, the forearm and the opticaloutput of the wrist joint unit are optically connected by mirrors arranged in the shoulder unit, the elbow unit and the wrist joint unit, and wherein an afocal lens system is arranged in the optical arm such that a rear focal plane of one lens coincides with a front focal plane of a subsequent lens.

28. The optical microscope system according to claim 27, characterized in that each mirror is arranged at an angle of 45° to the axis of rotation passing therethrough, the shoulder unit comprising a first shoulder mirror arranged at 45° relative to the output section and rotatable relative thereto, and a second shoulder mirror arranged at 45° relative to the upper arm and rotatable relative to the first shoulder mirror, and the output section and the upper arm are optically connected by a shoulder afocal relay comprising a lens element arranged before the shoulder unit in the output section and a lens element arranged after the shoulder unit in the upper arm, such that a rear focal plane of the lens element before the shoulder unit coincides with a front focal plane of the lens element after the shoulder unit in a common plane near the first and second shoulder mirrors.

29. The optical microscope system according to claim 27 or 28, characterized in that the elbow unit comprises a first elbow mirror arranged at 45° relative to the upper arm and preferably fixedly connected thereto, and a second elbow mirror arranged at 45° relative to the forearm and rotatable relative to the first elbow mirror, and the upper arm and the forearm are optically connected by an elbow afocal relay comprising a lens element arranged before the elbow unit in the upper arm and a lens element arranged after the elbow unit in the forearm, such that a rear focal plane of the lens element before the elbow unit coincides with a front focal plane of the lens element after the elbow unit in a common plane near the first and second elbow mirrors.

30. The optical microscope system according to any one of claims 27-29, characterized in that the wrist joint unit comprises a first wrist mirror arranged at 45° relative to the forearm and rotatable relative thereto, and a second wrist mirror arranged rotatably relative to the first wrist mirror, and the wrist joint unit comprisesa wrist section connecting the first and second wrist mirrors, the wrist section being arranged at 45° to both wrist mirrors, and a wrist lens is arranged in the vicinity of the wrist section, preferably in the wrist section itself.

31. The optical microscope system according to any one of claims 27-30, characterized in that a rear focal plane of the lens element after the shoulder unit coincides with a front focal plane of the lens element before the elbow unit, and a rear focal plane of the lens element after the elbow unit coincides with a front focal plane of the wrist lens.

32. The optical microscope system according to any one of claims 27-31 , characterized in that the focusing unit is connected to the optical output of the wrist joint unit and comprises an objective, at least a first detector, and a beam splitter unit, wherein a main dichroic mirror is arranged so as to optically connect the objective with the second wrist mirror and with the first detector, and preferably the focusing unit is rotatable about the optical axis of the optical output of the wrist joint unit, and the dichroic mirror is arranged at 45° to the optical axis of the optical output of the wrist joint unit.

33. The optical microscope system according to claim 32, characterized in that the focusing unit comprises a second detector, and a secondary dichroic mirror is arranged in the beam splitter unit between the dichroic mirror and the second detector.

34. The optical microscope system according to any one of claims 16-33, characterized in that it comprises a robot arm with at least three degrees of freedom, and the optical arm is movably mounted on the robot arm.

35. The optical microscope system according to claim 34, characterized in that the mechanical element providing rotation about at least one local optical axis is a head bearing arranged at the second end of the optical arm, rotatable about the axis of the objective while leaving the axis of the objective free, and the robot armis provided with a force and torque sensor for measuring force and torque occurring at the head bearing, and the system is configured to move the robot arm according to the signals of the force and torque sensor such that it continuously predicts the movement of an experimental animal connected to the head bearing on the basis of the measured forces and torques, and moves the focusing unit to the desired position and orientation to be assumed by the animal.

36. The optical microscope system according to any one of claims 16-35, characterized in that the optical microscope system is a laser scanning microscope system comprising a laser scanning microscope, preferably a multiphoton microscope, and a laser scanner, and comprising a microscope objective arranged at the second end of the optical arm.

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