Mobile laser scanning microscope and beam stabilizer of laser scanning microscope

Abstract

The invention relates to a mobile laser scanning microscope (100) comprising a chassis (10) with rollers (12), an optical table (30) connected to the chassis (10) via a vibration damper (20), an upper optical cassette (40) comprising a scanning head (42) with an acousto-optic deflector, a strut (60) connecting the upper optical cassette (40) to the optical table (30) and holding it at a distance above the optical table (30), the invention relates to an optical system (10) comprising an optical table (30), a stage (31) arranged on the optical table (30), a microscope objective (33) arranged above the stage (31) and comprising a scanning head (42) and at least one detector (75a, 75b), at least one laser (35) arranged on the optical table (30), an optical beam path (36) directing a beam of the at least one laser (35) from the at least one laser (35) through an upper optical cassette (40) to the objective (33), a compartment (17) connected to the chassis (10) below the optical table (30) and housing a power supply (15) and a control system (16) of the microscope (100), and a light shield system (50) having a cover (52) covering a unit arranged on the optical table (30).

Description

Technical Field

[0001] The invention relates to a mobile laser scanning microscope.

[0002] The invention further relates to a beam stabilizer for a scanning microscope comprising a first and a second laser, in particular for a mobile laser scanning microscope according to the invention. Background Art

[0003] Prior art two-dimensional or three-dimensional laser scanning microscopes with acousto-optic deflectors are usually assembled on large optical tables, where each optical and electro-optical element is individually mounted and aligned, which requires a lot of time (up to several weeks) and considerable expertise. Not only is the assembled microscope setup not transportable due to its size, but the adjustment of the position and angle of the elements, which requires considerable fine-tuning, can be damaged by moving the system. As a result, high-precision laser scanning microscopes cannot be transported once assembled, nor can they be moved from one laboratory room to another without disassembling and reassembling the microscope elements. Summary of the invention

[0004] The aim of the invention is to create a mobile laser scanning microscope which, due to its size and design, can be easily moved and transported in the assembled state.

[0005] The invention also aims to create a beam stabilizer for a laser scanning microscope with two lasers, in particular for a mobile scanning microscope, which can simultaneously stabilize two laser beams from different lasers for a common scanning head.

[0006] The inventors have realised that by properly stabilising the optical table and the light beam used for scanning, and by properly arranging the components of the laser scanning microscope and the auxiliary units in space, a compact design can be achieved which can be integrated into a frame with rollers and thus moved and transported in the assembled state. Due to the compact design, the microscope assembly can be moved through doors of standard dimensions (e.g. 70×200 cm).

[0007] Based on the above-mentioned recognition, the problem is solved by means of a mobile laser scanning microscope according to claim 1 and a beam stabilizer according to claim 26 .

[0008] Preferred embodiments of the invention are defined in the dependent claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Further details of the invention will be explained by means of exemplary embodiments with reference to the accompanying drawings, in which:

[0010] Figure 1is a front perspective view of a first embodiment of a microscope according to the present invention, with the cover removed;

[0011] Figure 2 yes Figure 1 a rear perspective view of the illustrated embodiment with the cover removed;

[0012] Figure 3 yes Figure 1 a side view of the illustrated embodiment;

[0013] Figure 4 yes Figure 1 a front view of the illustrated embodiment;

[0014] Figure 5 yes Figure 1 another side view of the illustrated embodiment;

[0015] Figure 6 yes Figure 1 an enlarged detail of the embodiment shown;

[0016] Figure 7 Has a cover Figure 1 an exploded view of the illustrated embodiment;

[0017] Figure 8 is connected to Figure 1 a perspective bottom view of the elements of the chassis of the illustrated embodiment;

[0018] Fig. 9 is a schematic diagram showing the arrangement of an optical bench of a microscope according to the present invention;

[0019] Fig.10 is a schematic diagram showing the arrangement of elements between the chassis and the optical bench;

[0020] Fig.11 is a schematic diagram showing the main units of a microscope according to the present invention;

[0021] Fig.12 is a schematic diagram showing a light shield surrounding a cable conduit;

[0022] Fig.13 is a block diagram of the optical arrangement;

[0023] Fig.14 is an enlarged block diagram of the reference beam path of the beam stabilizer. DETAILED DESCRIPTION

[0024] Figure 1A perspective front view of a first embodiment of a mobile laser scanning microscope 100 according to the present invention is shown, with the cover 52 removed. The microscope 100 can be mechanically and functionally divided into six main units, which are: a base frame 10, an optical bench 30, a support and support column 60, an upper optical box 40, a stage 31, and a microscope module 32.

[0025] The base frame 10 is the basis of the microscope 100 and has several functions. The base frame 10 is responsible for supporting the entire weight of the microscope 100 by means of support legs 14 standing on the ground, so that the microscope does not require a separate stand. In an advantageous embodiment, the legs 14 attached to the base frame 10 include two leg sections that can be moved relative to each other, thereby allowing the length of the legs 14 to be varied. For example, the leg section in contact with the ground can be telescopically screwed into the other leg section. The microscope 100 according to the invention also includes rollers 12 attached to the base frame 10, which are lifted from the ground in a default position by the legs 14 so that the rollers 12 are not in contact with the ground. For example, in Figure 1-5 and Figure 7-8 In the illustrated embodiment, such a chassis 10 is shown with adjustable support legs 14 and rollers 12. By shortening the legs 14 (e.g., by screwing one leg portion into the other leg portion), the rollers 12 can be brought into contact with the ground, so that the weight of the microscope 100 rests on the rollers 12, so that the microscope 100 can be moved on the rollers 12. In other words, the mobility and ease of movement of the entire system is preferably provided by the rollers 12, which support the weight of the structure and make it rollable when the adjustable legs 14 are adjusted to be shorter than the rollers 12. In the default arrangement, the two parts of the legs 14 are simply screwed together until they are shorter than the rollers 12. During operation, the rollers 12 do not contact the ground, and the microscope 100 stands on its extended legs 14, with the legs adjusted to the same height by leveling during adjustment.

[0026] An additional function of the chassis 10 is that it directly supports the mechanical vibration generating elements of the microscope 100, such as a power supply unit 15 with cooling elements, fans, etc., or a control system 16 with fans and other heavier elements. The central element of the chassis 10 is a solid welded frame 10', such as Figure 9-11 As shown, it is supported on the ground during operation of the microscope 100 by legs 14 preferably provided with rubber pads. The frame 10' supports the weight of the microscope 100 and provides mechanical stability. However, mechanical vibrations from the ground and vibrations generated in the equipment placed thereon are not absorbed by the base frame 10, but by the vibration damper 20 connecting the base frame 10 and the optical bench 30, and the vibration damper 20 is preferably configured as an active system, which includes, for example, air springs 22.

[0027] The frame 10' of the chassis 10 holds at least one laser 35 shown later, the control system 16 and power supply unit 15 required to operate the elements of the microscope 100, and most of the cables 83 required, as shown in FIG. Fig.10 and Fig.11 can be observed in . For the microscope 100 according to the invention, the spatial arrangement of the individual elements is essential because it ensures compactness (the ability to pass through a door of a given size without destroying the device) and thermal stability (i.e. the heat generated by each element does not overheat the individual units). The spatial arrangement of the elements on the chassis 10 is the result of targeted mechanical design work, with maximum housing size and thermal balance as target parameters. In one possible embodiment, the control system 16, which is preferably configured as a desktop computer, is located in a slide-out compartment 17. The compartment 17 is pushed in until it abuts when the microscope 100 is moved, in order to meet the need for minimum lateral dimensions to fit through the door. During operation, the compartment 17 of the control system 16 can be pulled out to facilitate access to the top-mounted sockets and standard control buttons.

[0028] The optical bench 30 attached to the base frame 10 serves as a base support for the sensitive optical components of the microscope 100. In a particularly preferred embodiment, the optical bench 30 achieves passive vibration damping by its structure, for example by the optical bench 30 comprising a plurality of layers, for example two thicker steel plates and thin-walled cells 30' arranged in a honeycomb pattern therebetween. This structure ensures passive vibration damping, i.e. the resonant frequency of the optical bench 30 falls outside the typical vibration frequency range of buildings and furniture (4-100 Hz), typically being much higher. Therefore, it does not transmit environmental vibrations to the optical elements and other components of the microscope 100. The composite structure not only does not resonate with environmental vibrations, but also damps (attenuates) vibrations generated and propagated on the optical bench 30.

[0029] Isolation from ambient vibrations is provided by active vibration dampers 20, which attach the optical bench 30 to the base frame 10 in addition to passive table damping, i.e. the optical bench 30 is connected to the base frame 10 via vibration dampers 20, such as the above-mentioned air springs 22. The vibration dampers 20 are preferably provided with air springs 22 configured as legs. Figure 9-11 In the embodiment shown, the active vibration damper 20 comprises four air springs 22, three of which have independently adjustable heights, i.e. three degrees of freedom, to adjust the plane of the optical bench 30. A control system (not shown in the figure) releases air into a small reservoir on each height-controlled air spring 22 via a three-stage valve (not shown in the figure), thus always aiming to maintain the set height. Preferably, the valve is fed by an air inlet pipe known to the skilled person, and preferably, the air inlet pipe has a variable degree of throttling. In the present embodiment, the air spring 22 has a support profile 22a, which has a lower support flange 22b (see FIG. 2 ) supporting the optical bench 30. Figure 7 ). In this way, the optical bench 30 can be recessed between the air springs 22, thereby further reducing the vertical size of the microscope 100. In addition, in a preferred embodiment, an additional spring damper 21 can be provided below the optical bench 30 (see Figure 8 ) to dampen sway or bumps during transportation, thereby reducing the load on the air spring 22.

[0030] The control frequency of the air spring 22 is much lower than the ambient frequency and is not comparable to the optical adjustment and measurement time. While the active vibration damper 20 system only allows very low frequency vibrations <1 Hz to pass, the passive damping of the optical bench 30 will resonate only at high frequencies >100 Hz, so the vibration bands are limited in both directions, amplifying the influence of each other. Effective vibration damping ensures the stability of the laser beam during focusing, by limiting the random displacement between the focal spot and the sample, with a mechanical precision ten times higher than the diffraction limit (50nm).

[0031] The amount and velocity of air required to control the height of the air spring 22 affects the stability and statics of the entire microscope 100. At lower air injection rates, the static center of gravity of the entire system can be placed higher because it will oscillate at a lower amplitude and speed during control. For example, the speed of air allowed into the control system can be adjusted by a throttle valve so that the entire microscope 100 remains stable and does not oscillate - the controller will then only make the necessary slowing corrections. The center of gravity of the microscope 100 is located above the optical table 30, but with the correct throttle valve, the oscillation caused by the high center of gravity can be completely eliminated.

[0032] One or more lasers 35, 35a, 35b are fixed to the optical bench 30. The design of the box of lasers 35a, 35b selected for the compact microscope 100 is preferably such that it also includes an externally controllable dispersion compensation element capable of emitting light at 50.000 to 80.000 fs. 2 The magnitude of compensates for the wavelength-dependent second-order group delay dispersion of the microscope 100. Dispersion compensation ensures that the shortest possible pulses that can optimally produce two-photon excitation are applied to the sample and that their dispersion-induced time extension in the sample plane is minimized.

[0033] Light from one or more lasers 35a, 35b is transmitted to the objective lens 33 through a beam path 36. In the context of the present invention, a beam path 36 is understood to be a path of a laser beam defined by one or more optical elements (e.g., a reflector T, a lens, a beam splitter, etc.). The beam path 36 includes a lower beam path section 36a, a middle beam path section 36b, and an upper beam path section 36c. The lower beam path section 36a is preferably in a shielded box to prevent scattered laser light from entering the measurement space. If desired, the elements of the lower beam path 36a can be connected to the optical bench 30, or can be arranged in a separate dedicated unit. The elements of the lower beam path 36a can include: an optical power regulator 92a, 92b for each laser 35a, 35b, and a main beam expander 93a, 93b for amplifying the beam of the laser 35a, 35b to a diameter of 4.5 to 5mm. A beam expander 93a, 93b is fitted to each of the lasers 35a, 35b, and the diameter of the laser beam is also different when leaving the lasers 35a, 35b. The optical power regulator 92a, 92b includes a combination of a motor-rotatable half-wave plate 92a', 92b' and a polarized light transmission beam splitter cube 92a", 92b", which together control how much light passes through the beam splitter cube 92a", 92b" and how much light is reflected. In particular, when aligning or servicing, it is necessary to accurately reduce the light output over the entire length of the laser beam. During normal operation, the optical power regulators 92a, 92b are set to maximum transmission, and power control is performed by the acousto-optic scanning head 42 by tuning the diffracted acoustic power, which will be discussed in detail later.

[0034] The optical bench 30 is mounted on a pillar 60 that supports the upper optical box 40. As one of the main functional elements of the microscope 100, the pillar 60 has a multifunctional role. On the one hand, it provides mechanical support and stability for the components placed above it, and on the other hand, its hollow interior is the optical path from the lasers 35a, 35b to the upper optical box 40, which performs stabilization, beam expansion, scanning and patterning. In order to provide mechanical stability, the wall of the pillar 60 on the side connected to the upper optical box 40 is preferably formed of a thick metal plate (such as steel or aluminum) with a hole whose size is designed to firmly support the heavy components attached thereto and allow minimal oscillation and vibration.

[0035] In a particularly preferred embodiment, microscope 100 comprises a beam stabilizer 38 according to the invention for stabilizing the beam of at least one laser 35. Beam stabilizer 38 comprises a reference beam extraction element 70, such as a polarizing beam splitter cube, arranged in upper beam path section 36c and a downstream beam splitter 70'.

[0036] exist Fig.13In the illustrated embodiment, a first laser 35a and a second laser 35b are included, and a beam path 36 has a first branch 36′ and a second branch 36″ upstream of an upper beam path segment 36c, such that the first branch 36′ includes a first lower beam path segment 36a′ that directs the beam of the first laser 35a into the pillar 60 and a first intermediate beam path segment 36b′ that directs the first laser beam into the upper optical box 40 in the pillar 60. In the second branch 36″, there is a second lower beam path segment 36a″ that directs the beam of the second laser 35b into the pillar 60 and a second intermediate beam path segment 36b″ that directs the second laser beam into the upper optical box 40 in the pillar 60, and a dichroic coupling element 39, preferably a dichroic mirror, is arranged upstream of the upper beam path segment 36c that couples the first and second laser beams. The coupling element 39 transmits the wavelength of one laser 35a, 35b, and reflects the wavelength of the other laser 35b, 35a. In a possible embodiment, a half-wave plate 71 for adjusting the beam extraction amount of the extraction element 70 is arranged in the lower beam path segment 36a of the optical beam path 36 upstream of the extraction element 70. It should be noted that in an embodiment comprising two lasers 35b, 35a, the first lower beam path segment 36a' and the second lower beam path segment 36a" preferably each include a half-wave plate 71, such as Fig.13 shown.

[0037] The reference beam extraction element 70 defines a reference beam path 90a and a main beam path 90b. In a particularly preferred embodiment, the beam stabilizer 38 includes a first and a second reference beam 86', 88' obtained by splitting the reference beam path 90a into two parts, whose starting point is the reference beam splitter 70'. That is, the reference beam path 90a is split by the beam splitter 70' into a first reference beam path 86' and a second reference beam path 88'. The first reference beam path 86' is routed to at least one proximal first detector 87a, 87b that is sensitive to the beam position, and the second reference beam path 88' is routed to at least one distal second detector 89a, 89b that is sensitive to the beam position. The detectors 87a, 87b and the detectors 89a, 89b form a detector pair. In Fig.13 and Fig.14In the illustrated embodiment, at least one proximal first detector 87a, 87b is located at a distance from the reference beam splitter 70' along the first reference beam path 86' that is less than the distance from the reference beam splitter 70' along the second reference beam path 88' of at least one distal second detector 89a, 89b. In this way, the proximal first detector 87a, 87b can be used to detect beam position errors, while the distal second detector 89a, 89b can be used to detect beam direction errors. Obviously, instead of placing the distal detectors 89a, 89b further away, other known optical methods can be used to provide enhanced sensitivity of the detectors 89a, 89b to the beam direction, such as by placing a focusing lens in front of each detector 89a, 89b. In this case, it is not necessary that the detectors 89a, 89b are further away from the beam splitter 70' than the detectors 87a, 87b along the beam path, so physically we do not necessarily have to compare "distal" and "proximal" detectors. In the following, a "near" or "first" detector is understood to mean a detector 87a, 87b configured to detect beam position, while a "distal" or "second" detector is understood to mean a detector 89a, 89b configured to detect beam direction, whether or not the latter is located further along the second reference beam path 88'. Therefore, the arrangement (position, distance from the laser source) of the detectors 87a, 87b, 89a, 89b, their interaction with other optical elements and the use of the measurement signals they provide are fundamental to discussing the functionality of configuring each detector 87a, 87b, 89a, 89b. Note that each of the detectors 87a, 87b, 89a, 89b can be used for position detection or beam direction detection, and their arrangement (optionally using other optical elements, such as lenses) means that the "near" detectors 87a, 87b are more sensitive to position errors, while the "distal" detectors 89a, 89b are more sensitive to beam direction errors.

[0038] The extraction element 70 designed as a polarization beam splitter cube forms a power regulator with the half-wave plate 71 to accurately control the amount of low-intensity light extraction to the detectors 87a, 87b, 89a, 89b of the beam stabilizer 38.

[0039] exist Fig.14In the embodiment illustrated in FIG. 8 , a first dichroic mirror 86 is arranged in a first reference beam path 86′ to split the first and second laser beams, which further splits the first reference beam path 86′ into a first reference branch 86a for the first laser beam and a second reference branch 86b for the second laser beam. In this embodiment, beam position sensitive first detectors 87a, 87b are arranged at the ends of the first reference branch 86a and the second reference branch 86b. Similarly, in the second reference beam path 88′, a second dichroic mirror 88 is arranged to split the first and second laser beams, which further splits the second reference beam path 88′ into a third reference branch 88a formed for the first laser beam and a fourth reference branch 88b formed for the second laser beam. Beam direction sensitive second detectors 89a, 89b are arranged at the ends of the third and fourth reference branches 88a, 88b so that the beam path from the reference beam splitter 70' to the proximal first detectors 87a, 87b is shorter than the beam path from the reference beam splitter 70' to the distal second detectors 89a, 89b.

[0040] The beam stabilizer 38 preferably further comprises a movable lower reflector 37a and an upper reflector 37b, each reflector having a controllable moving mechanism arranged in at least one intermediate beam path section 36b, and a control unit (not shown), the control unit being used to move the movable lower reflector 37a and the movable upper reflector 37b via control of the moving mechanism based on signals detected by at least one proximal detector 87a, 87b and at least one distal detector 89a, 89b. In a preferred embodiment, the lower reflector 37a and the upper reflector 37b provided with the controllable moving mechanism are configured as controllable motorized reflectors, and the proximal detectors 87a, 87b and the distal detectors 89a, 89b are quadrant detectors known to those skilled in the art.

[0041] The design of the beam stabilizer 38 is optimized to compensate for as much mechanical instability as possible and to minimize displacement or angular rotation of the beam as close to the point of use as possible. To this end, a pair of position sensitive detectors 87a, 87b; 89a, 89b are mounted close to the scanning unit, and both pairs are arranged in the upper optical box 40 to compensate for mechanical displacement of the upper optical box 40 relative to the microscope 100, the support 60, the rest of the lower mounting plate, and the beam.

[0042] In the intermediate beam path section 36b, for guiding the light, mirrors 37a, 37b are positioned on the inside of the wall of the support 60 at an angle of 45° to the beams of the lasers 35a, 35b, which directs the light vertically upwards parallel to the axis of the support 60, deflected by 90°. These are motorized movable mirrors 37a, 37b for the beams of the two lasers 35a, 35b. The motorized mirrors 37a, 37b, in addition to directing the beams upwards, also serve to keep the center of the beams centered on the position-sensitive detectors 87a, 87b, 89a, 89b used as a reference, by means of motor control signals provided by the control circuit. In the beam stabilizer 38, two laser beams (of a given wavelength) pass through a beam stabilization control loop formed by two mirrors 37a, 37b, each of which is motorized to move (tilt) in two perpendicular directions, and two optically aligned detectors 87a, 87b, 89a, 89b that are position sensitive along two perpendicular axes.

[0043] The second detectors 89a, 89b of the two control loops are mounted directly in front of (next to) the entrance of the common scanner. The motorized lower reflector 37a is mounted on the inner wall of the lower part of the support 60, so that the support 60 provides a long beam path for fast iteration of the control loop between the lower reflector 37a and the position sensitive detectors 87a, 87b. The motorized upper reflector 37b is already located at the entrance of the upper optical box 40 and is used to adjust the position of the beam on the detectors 89a, 89b located close to the scanner. For each wavelength laser beam, the beam adjustment using the lower reflector 37a and the near-side detectors 87a, 87b and the beam adjustment using the upper reflector 37b and the far-side detectors 89a, 89b are iteratively performed respectively. The length of the support 60 (i.e. the distance between the motorized reflectors 37a, 37b) helps to ensure fast convergence of stable iterations. The large distance between the upper motorized reflectors 37b and 89a, 89b helps to keep the beam direction accurate. Again, it should be noted that instead of choosing this spacing to be large, other optical methods may be used to provide increased sensitivity of the detectors 89a, 89b to the beam direction, for example by placing a focusing lens in front of each of the detectors 89a, 89b as known to those skilled in the art. The motorized upper reflectors 37b are preferably arranged in or above the pillar 60 on a support extending from the upper optical box 40 along the pillar 60: they thus receive the beams from the pillar 60 and direct them into the upper beam path section 36c.

[0044] The compact and target-oriented solution is based on coupling two laser beams of different wavelengths by means of a wavelength-dependent reflective coupling element 39 (such as a dedicated mirror) after two beam stabilization control loops, and then passing them through a beam expander 72, an acousto-optic scanning system, imaging optics and a focusing objective 33 until they reach the sample. This arrangement allows simultaneous excitation of the sample with the laser beams and fast wavelength changes as required by the application, without moving mechanical elements. Note that the acousto-optic scanning head, common to both excitation beams, does not contain any moving mechanical elements, thus allowing scanning speeds at least an order of magnitude higher than mirror scanners and focal spot scanning in three dimensions (x, y, z).

[0045] In a possible embodiment, a shared triple-amplification beam expander 72 is arranged at the input of the upper optical box 40 downstream of the beam combining wavelength selective coupling element 39 to provide a beam diameter matched to the acousto-optic deflectors 73a-73d, preferably, for example, 15 mm. The beam expander 72 is arranged in the optical beam path 36 downstream of the reference beam extraction element 70 in the main beam path 90b as part of the beam stabilizer 38, and is capable of amplifying laser beams of different wavelengths along a precise common axis without chromatic aberration.

[0046] The optical functions of the upper optical box 40 include providing a position sensitive reference in the excitation beam stabilization circuit, beam expansion scanning of the excitation beam and directing the excitation beam to the sample, and directing the reflected light from the sample to an optional camera. The mechanical frame of the upper optical box 40 is provided by a base plate 44, which is preferably in the form of a high thickness metal plate (such as steel or aluminum plate), possibly in the form of an optical support plate with an internal vibration damping honeycomb structure, preferably with mechanical support elements mounted on both sides thereof for supporting and moving each optical element. Preferably, at least the optical elements of the beam stabilizer 38 other than the movable mirrors 37a, 37b are also mounted on one side of the base plate 44, and the optical elements of the scan head 42 are mounted on the other side of the base plate 44. The beam path 36 is directed from the beam splitter 70' into the scan head 42 through an aperture formed in the base plate 44.

[0047] The aforementioned scanning head 42 is arranged in the upper optical box 40, and includes a first acousto-optic deflector 73a and a second acousto-optic deflector 73b deflecting along an XZ plane, and a third acousto-optic deflector 73c and a fourth acousto-optic deflector 73d deflecting along a YZ plane, wherein the XZ plane is a first plane parallel to the optical axis of the objective lens 33, and the YZ plane is a second plane parallel to the optical axis of the objective lens 33 and perpendicular to the XZ plane. Preferably, the scanning head 42 includes a telecentric repeater 91, which is configured as an afocal telescope known to a skilled person, for example, so that along the beam path 36, the first acousto-optic deflector 73a and the third acousto-optic deflector 73c are arranged upstream of the telecentric repeater 91, and the second deflector 73b and the fourth deflector 73d are arranged downstream of the telecentric repeater 91. Preferably, an amplifier (not shown) for the RF control signals of the four AODs 73a-73d is also arranged in the middle of the upper optical box 40, so that a short and well-shielded cable is provided to the shielded AODs 73a-73d to deliver a signal with a power of several watts. This has the advantage of minimizing the antenna effect, the short cable does not resonate with the RF signal, and the loss is less.

[0048] In a preferred embodiment, the exit plane of the scanning head 42 is projected onto the rear aperture of the objective lens 33 by the achromatic telescope system 95, and the objective lens 33 is also focused on the sample without chromatic aberration. The first lens 95a (scanning lens) of the telescope system 95 is located in the upper optical box 40, and the second lens 95b (tube lens) and the objective lens 33 are located in the microscope module 32.

[0049] The microscope module 32 according to the present invention can be arranged on the optical bench 30, for example, but an embodiment can also be envisaged in which it is integrated with the upper optical box 40. The advantage of the separate design is that the upper optical box 40 mounted on the support column 60 can be placed on a conventional microscope used as the microscope module 32. Preferably, the microscope module 32 includes at least one detector 75 and at least one dichroic mirror 74, which transmits at least one laser beam and reflects visible light. In the microscope module 32, the scanning beam path 76a and the detection beam path 76b, which constitute part of the optical beam path 36, are configured so that the dichroic mirror 74 for at least one laser beam transmission and visible light reflection is arranged upstream of the objective lens 33 in the scanning beam path 76a, and the detection beam path 76b is formed between the at least one dichroic mirror 74 and the at least one detector 75. Fig.13 In the illustrated embodiment, the microscope module 32 includes a plurality of detectors 75a, 75b that are preferably arranged perpendicular to each other, and a detection beam path 76b is divided by a dichroic mirror 77 into a plurality of detection branches corresponding to the wavelengths to be detected, and a detector 75a, 75b from each of the plurality of detectors 75a, 75b is arranged at the end of the detection branch.

[0050] In conventional microscopy, the signal reflected from the sample passes through the objective lens 33 and the second lens 95b in the microscope module 32 to the upper optical box 40, where the first lens 95a and the camera-lens pair image it onto the camera. When switching between laser microscope mode and conventional microscope mode, a motor inserts a mirror into the beam path that blocks the excitation laser beam and transmits the beam from the first lens 95a to the camera lens and through it to the camera.

[0051] The microscope module 32 according to the present invention has the following functions:

[0052] - delivering the excitation laser beam to the sample via the objective lens 33, i.e. focusing it,

[0053] - separating the fluorescence beam from the excitation beam, dividing them into different wavelength ranges and detecting them by means of dichroic mirrors 74, 77 and detectors 75a, 75b,

[0054] - providing illumination for conventional microscopy, preferably by means of LED 96,

[0055] - and preferably provides a non-laser light source and beam path for optogenetics.

[0056] For two-photon neural activity measurements, the excitation laser is transmitted from the upper optical box 40 by the second lens 95b to the objective lens 33, which focuses the excitation laser on the sample. The fluorescence generated during the excitation is directed back by the objective lens 33 and separated from the excitation beam by the dichroic mirror 74 with wavelength-dependent reflection and directed toward the detectors 75a, 75b. The beam containing the fluorescence extracted from the central beam path is split into two wavelength bands by the dichroic mirror 77 with wavelength-dependent reflection and detected by two (or more, if necessary) detectors 75a, 75b (e.g., photomultiplier detectors).

[0057] Conventional microscope sample observation in the upper optical box 40 is mainly applicable to sample alignment, selection of the area to be measured and adjustment of the focal plane. The conventional microscopic beam path includes an LED illumination 96 mounted on an arm supporting the detectors 75a, 75b, whose light is coupled to the beam path defined by the axis of the objective lens 33 in a known manner (for example, by means of a sliding mirror) so that in this mode, a reflector (glass plate) inserted into the beam path by a motorized actuator reflects a portion of the collimated light of the LED 96 light onto the objective lens 33. The size of the illuminated sample area is set by the field of view of the objective lens 33. The light reflected from the sample passes through the objective lens 33 and the second lens 95b and returns to the upper optical box 40, where it is imaged onto the camera by the camera lens of an inappropriate size. The appropriate magnification between the field of view of the objective lens 33 and the camera sensor is accurately set by the imaging optical system.

[0058] In one possible embodiment, the camera image is displayed on a screen of the control system 16 (e.g., a desktop computer). For example, switching between conventional microscope and laser beam paths can be accomplished by simultaneously pulling out and pushing in a motorized beam path mirror in the upper optical box 40 and a motorized 45° mirror in the microscope module 32, respectively. By using a motor to push the mirror out of the beam path, light from the objective lens 33 enters the camera unimpeded. When light from the LED illumination 96 reaches the sample and is reflected back from it, conventional microscopic imaging is achieved. For example, light from the LED illumination 96 can be coupled to the sample by means of a mirror that reflects at an angle of 45° and can be mechanically removed from the beam path by hand or by a motor.

[0059] The stage 31, which serves as a user interface, provides the functionality of the microscope 100, namely the insertion, movement and optimal positioning of the sample under the objective lens 33. In a preferred embodiment, the stage 31 is motorized for movement and preferably rests on legs 31', such as Figure 6 As shown. Legs 31' allow various components to be placed under the stage 31. For example, in some embodiments, a detection unit 85 for detecting light passing under the platform 31 can be arranged on the optical platform 30. The microscope 100 according to the present invention can be used for in vivo and in vitro sample examinations. In the latter case, a sample holding chamber (not shown) is required to keep the sample alive. Similarly, for in vivo measurements, several types of animal holders can be used, as known to those skilled in the art. The mechanically required space is provided by the mechanical design of the microscope 100, in particular the relative positioning of the support column 60 and the upper optical box 40. The preferred arrangement and mechanical design of the support column 60 and the upper optical box 40 ensure optimal and convenient usability of the microscope 100 for the user.

[0060] The advantageous design of the microscope 100 according to the invention includes an optimal placement of the necessary cables 83 and tubes. A large amount of space is required to connect the power supply 15 and the cooling device 34 located on the base frame 10 to the acousto-optic amplifier and the device located in the upper optical box 40. In an exemplary embodiment, cooling water is effectively piped upstream and downstream to the cooling device 34 from the four acousto-optic deflectors 73a-73d connected in series by connecting the four acousto-optic deflectors 73a-73d in series; the pipe connects the connector arranged in the base frame 10 to the water connector located on the upper optical box 40. In the exemplary embodiment, the cooling water is effectively piped upstream and downstream to the cooling device 34 from the four acousto-optic deflectors 73a-73d connected in series; the pipe connects the connector arranged in the base frame 10 to the water connector located on the upper optical box 40. Figure 2 In the embodiment shown, the cooling device 34 is arranged in the chassis 10, but embodiments are also conceivable in which the cooling device 34 is arranged on a unit separate from the microscope 100 in order to reduce noise and resonance.

[0061] The short cables carrying the RF signals connecting the amplifiers to the acousto-optic deflectors 73a-73d are inside the upper optical box 40, while the cables 83 carrying the signals from the power supply 15 and the control system 16 extend outside the upper optical box 40 and enter the interior of the upper optical box 40 via dedicated connectors without interfering with the laser beam path anywhere. The electrical and optical cables 83 (and possible cooling tubes) on the chassis 10 connecting the power supply 15 for operating the lasers 35a, 35b to the lasers 35a, 35b on the optical bench 30 are routed through a dedicated, preferably U-shaped cutout 81 in the optical bench 30. This does not affect the vibration separation and attenuation parameters. The vibrations transmitted to the optical bench 30 by the cables 83 are attenuated by the optical bench 30 using the active and passive vibration attenuation mechanisms previously described.

[0062] In order to shield the measurement space, the microscope 10 according to the invention is equipped with a light shield system 50. The light shield system 50 comprises an external cover 52 housing, which can be attached externally to the base frame 10 (optionally to the optical bench 30), comprising side panels 54 and a cover 55, as for example in Figure 7. The cover 52 of the light shield system 50 can be attached directly or indirectly to the lower frame 10, so that it is only indirectly connected to the optical bench 30 via the vibration damper 20. In order to provide access to the measurement space, at least one of the side panels 54 is preferably configured to be easily removable or displaceable, for example in a retractable manner. In a possible embodiment, the light shield system 50 includes a lower light blocking foil 56, preferably made of plastic, which is arranged between the lower frame 10 and the optical bench 30 to block light from the lower frame 10. In order to prevent light interference from leaking when passing through the cables 83 required for the operation of the microscope 100, in a preferred embodiment, the cables 83 are wrapped in a cable guide 80 that connects the upper side of the optical bench 30 to the lower side of the optical bench 30, and the cable guide 80 is arranged in the cutout 81 of the optical bench 30. In the cable guide 80, a light sealing material 84 (e.g., a foam) is arranged between the cables 83. A gap is formed between the wall of the cable duct 80 and the cutout 81 of the optical bench 30, and a flexible light sealing collar 82 is formed between the wall of the cable duct 80 and the edge of the cutout 81 on at least one side of the optical bench 30, the collar folding back onto the wall of the cable duct 80 and the side of the optical bench 30 and covering the gap, such as in Fig.10 or Fig.12 In a preferred embodiment, the light shield can be further enhanced by using a light shielding cap 53 arranged on the air spring 22 and the support profile 22a of the optical bench 30 (see Figure 7 ).

[0063] The electronics box 94 on the side of the support 60 contains most of the electronic control card (not shown in the figure), which controls, for example, the acousto-optic deflectors 73a-73d and the detectors 75a, 75b. The cable that links the signals from the detectors 75a, 75b to the card connects the microscope module 32 and the electronics box 94 via a connector. Similarly, the cable is also connected from the card to the objective motor via a connector in an ergonomic design, which drives and controls the various motors that drive the Z-axis motor of the objective lens 33, the motor that drives the reflector that reflects the excitation laser beam into the beam path of the objective lens 33, the motor that drives the beam stabilization reflectors 37a, 37b, and the motor that drives the reflector of the conventional light source.

[0064] An acousto-optic scanner that can operate simultaneously in two wavelength ranges is one of the most important innovations of the microscope 100 according to the present invention. It has the advantage of allowing simultaneous or sequential optical excitation and examination of two different biological processes on a sample. The combination of optical stimulation or inhibition of neural function (optogenetics) and neural function measurement based on two-photon fluorescence is the most widespread and useful application of two lasers of different wavelengths. The acousto-optic deflectors 73a-73d keep the two laser beams of different wavelengths in different acoustic frequency ranges on the scanning axis. Therefore, the two optical wavelength ranges can be selected so that the corresponding acoustic frequency ranges do not overlap, and only one wavelength at a given acoustic frequency is deflected at the correct angle and reaches the correct target area. This is an important engineering design task to ensure that crosstalk is completely eliminated.

[0065] The switching between the two functions (stimulation and inhibition) is realized by the scanner simply by switching the acoustic frequencies, in a completely electronic way, by generating the corresponding electronic control functions. This means that the switching is performed at high speed and does not generate mechanical vibrations that could interfere with the sample or the measurement, but by applying the corresponding acoustic frequencies simultaneously also simultaneous excitation is possible, since crosstalk is minimized. In this case, the total acoustic power that can be tolerated by the acousto-optic deflectors 73a-73d is divided between the two frequency ranges, so that the efficiency of the two optical wavelengths will be lower.

[0066] Note that in point scanning mode, the switching time between any two points in the 3D range scanned on the sample is the acoustic wave transit time through the aperture of the AOD 73a, 73d (typically 21 μs), but in continuous motion along any 3D straight or curved line, the focal spot can be moved continuously at a much higher speed. Thus, the focal spot can be moved across the sample at least an order of magnitude faster than with a conventional mirror scanner.

[0067] The scanning head 42 of the microscope 100 according to the present invention can move the focus along all three spatial coordinates with equal speed and efficiency within the 3D range to be aimed - the edge of the 3D range is determined by the angular range of the scanning acousto-optic deflectors 73a, 73d, that is, the maximum acoustic bandwidth. From an optical point of view, the scanning head 42 focuses two light beams of different wavelengths into the same three-dimensional domain, where the minimum aiming inaccuracy error is only one-tenth of the diffraction limit between the two light wavelengths, that is, it can focus the two beams on the same sample set with an accuracy of 50nm. This is particularly important for optogenetic experiments, where the position of the beam stimulating neural activity relative to the dendrites needs to be adjusted more accurately than the diffraction limit to optimize the excitation signal, so the position of the readout beam relative to the excitation beam must be more accurate and stable than the diffraction limit. In addition to minimizing optical errors, beam stabilization and vibration isolation, this is also achieved by generating electronic control functions with 16-bit depth and very low digitization errors.

[0068] The mobile laser scanning microscope 100 according to the present invention has the following advantages. The built-in beam stabilizer 38 allows fast post-transport setup of optical subsystems such as the acousto-optic scanning head 42, which is highly sensitive to the position of the input light. This allows the optical path to be self-adjusted so that imaging is performed with a spatial resolution of up to 50 nm. The optical and electro-optical elements of the microscope 100 assembly are fixed in preset positions so that the microscope 100 assembly can be disassembled, packed and operated in tens of minutes. The simplicity of installation means that it can be operated by one person. The light blocking foil 56 below the optical table 30 provides light blocking and, together with the cover 52, can form an airtight light blocking housing for high-sensitivity measurements (e.g., photon counting). After rolling, the optical table 30 can be raised to the desired height (e.g., about 30 cm) with the aid of the legs 14. Due to its compact size, the microscope 100 of the present invention can be fitted into an elevator and pass through a standard door, which typically has a door opening greater than 70×200 cm. Despite its high superstructure, it does not resonate due to the throttled air technology.

[0069] Obviously, the practitioner may conceive alternative embodiments to those disclosed herein, but within the scope of protection defined by the claims.

Claims

1. A mobile laser scanning microscope (100), characterized in that: include: A chassis (10) having rollers (12), an optical bench (30) connected to the base frame (10) via a vibration damper (20), an upper optical box (40) comprising a scanning head (42) having an acousto-optic deflector, a support (60) connecting the upper optical box (40) to the optical bench (30) and holding the upper optical box (40) at a distance above the optical bench (30), a stage (31) arranged on the optical table (30), a microscope module (32) arranged above the stage (31) and comprising at least one detector (75a, 75b) and a microscope objective (33) optically coupled to the scanning head (42), at least one laser (35) arranged on the optical table (30), an optical beam path (36) which directs the light beam of the at least one laser (35) from the at least one laser (35) through the upper optical box (40) to the objective lens (33), a compartment (17) connected to the chassis (10) below the optical bench (30) and housing a power supply (15) and a control system (16) for the microscope (100), and A light shield system (50) has a cover (52) covering a unit arranged on the optical bench (30).

2. The mobile laser scanning microscope (100) according to claim 1, characterized in that: The vibration damper (20) is an actively controlled air spring system comprising at least three air springs (22) connected to independently controllable valves and air inlet pipes supplying the valves, and preferably the air inlet pipes have variable throttle valves.

3. The mobile laser scanning microscope (100) according to claim 1, characterized in that: The optical beam path (36) includes at least one lower optical beam path segment (36a) that directs the at least one laser beam into the pillar (60), at least one intermediate optical beam path segment (36b) that directs the at least one laser beam into an upper optical box (40) within the pillar (60), and an upper optical beam path segment (36c) that directs the at least one laser beam through the upper optical box (40) into the microscope module (32).

4. The mobile laser scanning microscope (100) according to any one of claims 1 to 3, characterized in that: The lower optical beam path section (36a) includes a beam expander (72) and / or a power regulator corresponding to the at least one laser (35).

5. The mobile laser scanning microscope (100) according to any one of claims 1 to 4, characterized in that: The invention relates to a method for stabilizing the optical beam of the at least one laser (35), wherein the at least one optical beam stabilizer (38) comprises a movable lower mirror (37a) and an upper mirror (37b) arranged at the at least one intermediate beam path section (36b) and each having a controllable movement mechanism, and a reference beam extraction element (70) and a subsequent reference beam splitter (70') arranged in the upper optical beam path section (36c), and a first reference beam path (86') and a second reference beam path (88'), wherein the first reference beam path (86') leads to at least one first detector (87a, 87b) configured to detect a beam position. 87b), and the second reference beam path (88') leads to at least one second detector (89a, 89b) configured to detect the beam direction, and the second reference beam path (88') leads to at least one second detector (89a, 89b) configured to detect the beam direction, and the at least one beam stabilizer (38) further includes a control unit, which is used to move the movable lower reflector (37a) and the movable upper reflector (37b) via control of the moving mechanism based on signals detected by the at least one first detector (87a, 87b) and the at least one second detector (89a, 89b).

6. The mobile laser scanning microscope (100) according to any one of claims 1 to 4, characterized in that: The optical beam path (36) comprises a first laser (35a) and a second laser (35b), and the optical beam path (36) has a first branch (36') and a second branch (36") upstream of the upper optical beam path segment (36c), so that the first branch (36') comprises a first lower optical beam path segment (36a') that guides the beam of the first laser (35a) into the pillar (60) and a first middle optical beam path segment (36b') that guides the first laser beam into the upper optical box (40) in the pillar (60). , and in the second branch (36"), there is a second lower optical beam path segment (36a") that guides the light beam of the second laser (35b) to the pillar (60) and a second middle optical beam path segment (36b") that guides the second laser beam to the upper optical box (40) in the pillar (60), and a dichroic coupling element (39) is arranged upstream of the upper optical beam path segment (36c) to couple the first laser beam and the second laser beam, and the dichroic coupling element (39) is preferably a dichroic mirror.

7. The mobile laser scanning microscope (100) according to claim 6, characterized in that: A beam stabilizer (38) for stabilizing the beams of the first laser (35a) and the second laser (35b) comprises: a lower reflector (37a) and an upper reflector (37b), each reflector having a controllable movement mechanism, arranged in the first middle beam path section (36b') and the second middle beam path section (36b"), a reference beam extraction element (70) arranged in said upper beam path section (36c), a reference beam splitter (70') following the reference beam extraction element (70) and defining a first reference beam path (86') and a second reference beam path (88') starting at the reference beam splitter (70'), and A first dichroic mirror (86) is arranged in the first reference beam path (86') separating the first laser beam and the second laser beam, the first dichroic mirror (86) divides the first reference beam path (86') into a first reference branch (86a) for the first laser beam and a second reference branch (86b) for the second laser beam, and a first detector (87a, 87b) configured for beam position detection is arranged at the end of each of the first reference branch (86a) and the second reference branch (86b), a second dichroic mirror (88) is arranged in the second reference beam path (88') separating the first laser beam and the second laser beam, the second dichroic mirror divides the second reference beam path (88') into a third reference branch (88a) configured for the first laser beam and a fourth reference branch (88b) configured for the second laser beam, and a second detector (89a, 89b) configured for beam direction detection is arranged at the end of each of the third reference branch (88a) and the fourth reference branch (88a, 88b), A control unit is also included for moving the movable lower reflecting mirror (37a) and the movable upper reflecting mirror (37b) by controlling the moving mechanism based on the signals detected by the first detector (87a, 87b) and the second detector (89a, 89b).

8. The mobile laser scanning microscope (100) according to claim 5 or 6, characterized in that: The lower reflector (37a) and the upper reflector (37b) with controllable movement mechanism are controllable motorized reflectors and / or the detectors (87a, 87b, 89a, 89b) are quadrant detectors.

9. The mobile laser scanning microscope (100) according to claim 5, 7 or 8, characterized in that: The reference beam extraction element (70) is a polarization beam splitter cube, and a half-wave plate (71) is arranged in the at least one optical beam path (36) upstream of the polarization beam splitter cube for adjusting the beam extraction amount of the polarization beam splitter cube.

10. The mobile laser scanning microscope (100) according to claim 5 or 7 or 8 or 9, characterized in that: The beam stabilizer (38) includes a beam expander (72) arranged in the optical beam path (36) downstream of the reference beam extraction element (70).

11. The mobile laser scanning microscope (100) according to any one of claims 1 to 10, characterized in that: The scanning head (42) includes a first acousto-optic deflector (73a) and a second acousto-optic deflector (73b) deflected along an XZ plane, and a third acousto-optic deflector (73a) and a fourth acousto-optic deflector (73b) deflected along a YZ plane (73c, 73d), wherein the XZ plane is a first plane parallel to the optical axis of the objective lens, and the YZ plane is a second plane parallel to the optical axis of the objective lens and perpendicular to the XZ plane.

12. The mobile laser scanning microscope (100) according to claim 11, characterized in that: The scanning head (42) includes a telecentric repeater (91), and along the optical beam path (36), the first deflector (73a) and the third deflector (73c) are arranged upstream of the telecentric repeater (91), and the second deflector (73b) and the fourth deflector (73d) are arranged downstream of the telecentric repeater (91).

13. The mobile laser scanning microscope (100) according to any one of claims 1 to 12, characterized in that: The microscope module (32) includes a dichroic mirror (74) that transmits the at least one laser beam and reflects visible light and at least one detector (75), and the microscope module (32) is configured to have a scanning beam path (76a) and a detection beam path (76b) that form a part of the optical beam path (36), so that the at least one dichroic mirror (74) that transmits the laser beam and reflects visible light is arranged in the scanning beam path (76a) upstream of the objective lens (33), and the detection beam path (76b) is formed between the dichroic mirror (74) and the at least one detector (75).

14. The mobile laser scanning microscope according to claim 13, characterized in that: The microscope module (32) includes a plurality of detectors (75a, 75b), and the detection beam path (76b) is divided into a plurality of detection branches corresponding to the wavelength to be detected by a dichroic mirror (77), and detectors (75a, 75b) from the plurality of detectors are arranged at the ends of the plurality of detection branches.

15. The mobile laser scanning microscope (100) according to any one of claims 1 to 14, characterized in that: The light shield system (50) has a cover (52) secured to the optical bench (30) and includes at least partially removable side panels (54) and a lid (55).

16. The mobile laser scanning microscope (100) according to any one of claims 1 to 14, characterized in that: The cover (52) of the light shield system (50) is fixed directly or indirectly to the base frame (10) so that the cover (52) is only indirectly in contact with the optical bench (30) via the vibration damper (20).

17. The mobile laser scanning microscope (100) according to any one of claims 1 to 16, characterized in that: A cable duct (80) connecting the upper side of the optical bench (30) and the lower side of the optical bench (30) is arranged in the cutout (81) of the optical bench (30) so that a gap is provided between the cable duct (80) and the cutout (81) of the optical bench (30), and at least on one side of the optical bench (30) between the wall of the cable duct (80) and the edge of the cutout (81), the light shield system (50) comprising a flexible light shield collar (82) which folds back onto the wall of the cable duct (80) and the side of the optical bench (30) and covers the gap.

18. The mobile laser scanning microscope (100) according to claim 17, characterized in that: It includes a cable duct (80) connecting the upper side of the optical platform (30) and the lower side of the optical platform (30), and the light shield system (50) in the cable duct (80) includes a light sealing material (84), such as a light sealing foam, for filling a gap between the cable duct (80) and a cable (83) extending in the cable duct (80).

19. The mobile laser scanning microscope (100) according to any one of claims 1 to 18, characterized in that: A light blocking foil (56) is arranged below the optical bench (30).

20. The mobile laser scanning microscope (100) according to any one of claims 1 to 19, characterized in that: The optical bench (30) has a passive vibration damping structure.

21. The mobile laser scanning microscope (100) according to claim 20, characterized in that: The passive vibration damping structure of the optical table (30) comprises thin-walled units (30') arranged between two steel plates in a honeycomb configuration.

22. The mobile laser scanning microscope (100) according to any one of claims 1 to 21, characterized in that: The base frame (10) is provided with a height-adjustable support leg (14), and the support leg (14) lifts the roller (12) off the ground when raised.

23. The mobile laser scanning microscope (100) according to any one of claims 1 to 22, characterized in that: The stage (31) is motorized for movement.

24. The mobile laser scanning microscope (100) according to any one of claims 1 to 23, characterized in that: A detection unit (85) for detecting light passing under the stage (31) is arranged on the optical table (30).

25. The mobile laser scanning microscope (100) according to any one of claims 5 to 10, characterized in that: At least the optical elements of the beam stabilizer (38) except the movable mirrors (37a, 37b) are mounted on one side of a substrate (44), and the optical elements of the scanning head (42) are mounted on the other side of the substrate (44), and the optical beam path (36) is guided from the beam splitter (70') to the scanning head (42) through a hole formed in the substrate (44).

26. A beam stabilizer (38) for a scanning microscope (100), the scanning microscope (100) comprising a first laser (35a) for generating a first beam having a first wavelength and a second laser (35b) for generating a second beam having a second wavelength, characterized in that: The beam stabilizer comprises: Movable reflectors (37a, 37b) are arranged in the paths of the first laser beam and the second laser beam, each of the movable reflectors being provided with a pair of controllable moving mechanisms, a dichroic coupling element (39), preferably a dichroic mirror, arranged downstream of the pair of movable mirrors (37a, 37b) for coupling the first laser beam and the second laser beam, a reference beam extraction element (70) arranged in the optical beam path of the coupled laser beam, a reference beam splitter (70') arranged downstream of the reference beam extraction element (70), defining a first reference beam path (86') and a second reference beam path (88'), wherein the starting point of the first reference beam path (86') and the second reference beam path (88') is the reference beam splitter (70'), and a first dichroic mirror (86) is arranged in the first reference beam path (86') separating the first laser beam and the second laser beam, the first dichroic mirror (86) divides the first reference beam path (86') into a first reference branch (86a) for the first laser beam and a second reference branch (86b) for the second laser beam, and a first detector (87a, 87b) configured for beam position detection is arranged at an end of each of the first reference branch (86a) and the second reference branch (86b), a second dichroic mirror (88) is arranged in the second reference beam path (88') separating the first laser beam and the second laser beam, the second dichroic mirror divides the second reference beam path (88') into a third reference branch (88a) for the first laser beam and a fourth reference branch (88b) for the second laser beam, and a second detector (89a, 89b) configured to detect a beam direction is arranged at an end of each of the third reference branch (88a) and the fourth reference branch (88b), A control unit is also included for moving the lower movable reflector (37a) and the upper movable reflector (37b) by controlling the moving mechanism based on the signals detected by the first detector (87a, 87b) and the second detector (89a, 89b).

27. The beam stabilizer (38) according to claim 26, characterized in that The lower movable reflector (37a) and the upper movable reflector (37b) having a controllable moving mechanism are controllable motorized reflectors.

28. The beam stabilizer (38) according to claim 26 or 27, characterized in that The reference beam extraction element (70) is a polarization beam splitter cube, and a half-wave plate (71) for adjusting the beam extraction amount of the polarization beam splitter cube is arranged in the beam path of the laser (35a, 35b) upstream of the polarization beam splitter cube.

29. The optical beam stabilizer (38) according to any one of claims 26 to 28, characterized in that The reference beam extraction element (70) defines a reference beam path (90a) and a main beam path (90b), and the beam stabilizer (38) includes a beam expander (72) arranged downstream of the reference beam extraction element (70) in the main beam path (90b).

30. The beam stabilizer (38) according to any one of claims 26 to 29, characterized in that The optical path from the reference beam splitter (70') to the first detector (87a, 87b) is shorter than the optical path from the reference beam splitter (70') to the second detector (89a, 89b).

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