Laser scanning microscope and method for adjusting the laser scanning microscope

Abstract

To improve a laser scanning microscope such that a fiber coupling laser and a fiber-free coupling laser can be aligned with each other at a lower cost than before in order to enable correct input coupling to an objective lens.SOLUTION: An optical system has a light guiding fiber between a first light source and a third beam deflection unit and has no light guiding fibers between a second light source and a third beam deflection unit. In this way, the second light source can be used as an adjustment reference for the first and second beam deflection units. The adjustment can be implemented using test images recorded by means of the third and fourth beam deflection units; additional sensors or internal calibration samples are not required.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The invention relates to a laser scanning microscope (LSM) with an optical system including two light sources, an optoelectronic detector, four movable beam deflection units, and a microscope objective having a pupil plane and a focal plane, wherein the third and fourth beam deflection units are arranged in or near a plane conjugate with the pupil plane, and the first and second beam deflection units are arranged upstream of the third beam deflection unit and upstream of the fourth beam deflection unit in the illumination direction, the optical system directing light from the first light source through the objective via the four beam deflection units to the focal plane and directing light from the second light source through the objective via the third and fourth beam deflection units to the focal plane without passing through the first and second beam deflection units, and imaging a point in the focal plane through the objective onto the detector, and a method for adjusting the LSM and the laser scanning microscope. [Background technology]

[0002] If multiphoton fluorescence excitation is to be implemented in an LSM, correspondingly long wavelength and high intensity (N)IR pulsed lasers are required. If such lasers are fed into the microscope via optical fibers, undesirable spectral and temporal broadening of the light pulses occurs. Therefore, they are usually coupled in as free beams without fibers. However, this increases the need for alignment, since the free beam introduces four additional degrees of freedom into the system. If the system includes other lasers, whether in the visible range (VIS) or other (N)IR lasers, it is absolutely necessary to align the lasers with each other to be able to record corresponding images across the entire spectrum.

[0003] In the prior art, a general-purpose microscope and a general-purpose method are disclosed in DE 10 2007 011 305 A1, the drawback of which is that commercially available microscopes must be equipped with an internal pivoting device for the calibration standard sample, which entails significant costs.

[0004] Other solutions, such as those known for example from DE 10111824, do not require an internal calibration standard but instead require two internal position-sensitive sensors to ascertain the relative beam positions, which again is a complex modification of the device.

[0005] It is also known, for example from EP 19592921, to stabilize the free beam outside the microscope. This requires an additional position-sensing sensor to determine the relative beam position, but outside the microscope. However, even if the relative beam position is constant upstream of the microscope, the correct in-coupling of the free beam to the objective lens is still not ensured. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] German Patent Application Publication No. 102007011305 [Patent Document 2] German Patent No. 10111824 [Patent Document 3] European Patent No. 19592921 Summary of the Invention [Problem to be solved by the invention]

[0007] The present invention is based on the object of improving a microscope of the type mentioned at the beginning in such a way that fiber-coupled lasers and fiber-free coupled lasers can be aligned with each other within the microscope at a lower cost than before in order to enable correct input coupling to the objective lens. [Means for solving the problem]

[0008] The object is achieved by a microscope having the features specified in claim 1 and by a method having the features specified in claim 11. To the extent that components are referred to in the claims as being of a particular type as "first," "second," "third," or "fourth," this designation does not imply any order of placement in the optical system, but is used merely for terminological distinction.

[0009] Advantageous refinements of the invention are specified in the dependent claims. According to the present invention, 2 a light guide fiber between the light source and the third beam deflection unit; 1 There is no optical fiber between the light source and the third beam deflection unit.

[0010] The discovery underlying the present invention is that a laser ("second light source") coupled in by an optical fiber can be used as an alignment reference since the optics are typically already aligned and kept aligned based on the beam, and a sample that is conventionally placed in front of the objective lens can be used to align the free beam laser ("first light source").

[0011] In this way, successful adjustment can be performed at little cost, since no additional internal sensors are required, and no internal calibration standards are required. Preferably, the first light source is an ultrashort pulsed laser emitted in the (N)IR range.

[0012] Automatic adjustment of the free beam laser is possible if the microscope includes a control unit for recording a test image through the objective by means of a detector under illumination by a first light source for the purpose of adjusting the optical system, and the control unit scans the focal plane by means of the third and fourth beam deflection units and uses the test image to set the first and / or second beam deflection units, in particular in multiple repetitions of recording the test image and setting the associated beam deflection units. Scanning the focal plane means continuously moving the illumination spot to different positions in the focal plane, whereby samples, in particular calibration standard samples, located in the focal plane are also excited to fluoresce.

[0013] An LSM is advantageous in which the control unit determines a characteristic related to the illumination of the test image and sets a first beam deflection unit, which is located upstream or downstream of the second beam deflection unit in the illumination direction, based on the determined characteristic. This allows adjustment of two of the four degrees of freedom of the free beam with little cost. Advantageously, the uniformity of the illumination of the test image and / or the intensity of the illumination of the test image can be determined as a characteristic used by the control unit to set the first beam deflection unit. This facilitates high accuracy during adjustment. For example, the intensity can be determined by integrating the intensity of all pixels of the test image. Intensity can be a characteristic that is more prone to change, especially in the case of image enlargement or high zoom ratios.

[0014] Furthermore, an advantageous LSM has a control unit that records a test image through the objective lens with a detector illuminated by a first light source for the purpose of adjusting the optical system, and the control unit scans the focal plane with the third and fourth beam deflection units and records a reference image from an earlier or later focal plane under illumination by a second light source. The control unit scans the focal plane with the third and fourth beam deflection units, determines a geometric offset between the test image and the reference image, and sets a second beam deflection unit, located upstream or downstream of the first beam deflection unit in the illumination direction, based on the determined offset. This allows adjustment of the remaining two degrees of freedom of the free beam at little cost. In this case, the reference image can be recorded either before or after the test image is recorded.

[0015] Particularly preferred is an embodiment in which the control unit initially iteratively sets the first beam deflection unit based on a certain characteristic using a test image ("internal iteration"), and then, after the control unit has terminated the internal iteration, for example, when a threshold value for that characteristic is reached (undershoot or overshoot), sets the second beam deflection unit based on an offset using the test image and a reference image. Preferably, the control unit performs another ("external") iteration, which begins with iteratively setting the first beam deflection unit using at least one other test image. The offset between the test image and the reference image is then again confirmed, and the second beam deflection unit is set based on the offset. The iteration ends, for example, when the offset reaches another specified threshold value (undershoot). Advantageously, the control unit can confirm the offset using the last recorded test image during each iterative setting of the first beam deflection unit. In this way, there is no need to record images again, thereby accelerating the adjustment.

[0016] In such an arrangement, where the control unit initially sets the first beam deflection unit based on the illumination characteristics and only thereafter sets the second beam deflection unit based on the offset, the control unit preferably keeps the setting of the first beam deflection unit constant while the second beam deflection unit is set, thereby achieving adjustment of all four degrees of freedom in the shortest possible time.

[0017] In an alternative embodiment, a control unit is provided for recording a first test image by a detector through an objective lens for the purpose of adjusting the optical system, the control unit scans a focal plane by means of the third and fourth beam deflection units, then moves the focal plane by means of the adjustable focusing unit, and records a second image from the moved focal plane by means of the detector through the objective lens, the control unit scans the moved focal plane by means of the third and fourth beam deflection units, determines a geometric offset between the first test image and the second test image, and sets a second beam deflection unit arranged upstream or downstream of the first beam deflection unit in the illumination direction based on the determined offset, wherein the first test image is recorded under illumination by a different one of the light sources from the second test image, or both test images are recorded under illumination by the first light source. The offset between the images from different focal planes allows for determination of an angular deviation of the beam in a pupil plane, through which a normally telecentric objective lens focuses on an offset point.

[0018] In a special configuration where only test images are recorded under illumination by the first light source, the second fiber-coupled light source can be omitted. In this embodiment, the control unit may also initially set the first beam deflection unit based on the illumination in an "internal" iteration, as described above, and then record first and second test images and set the second beam deflection unit using an offset. These steps may be repeated as an "external" iteration, whereby the iterative setting of the first beam deflection unit is initially repeated, terminated when, for example, a certain threshold is reached, and then the second beam deflection unit is set. This allows adjustment of all four degrees of freedom to be achieved in the shortest possible time.

[0019] Advantageously, a calibration standard with symmetrical arrangement of fluorescent emitters in the z-direction is placed in the region of the focal plane of this embodiment. Advantageously, the offset can then be determined using test images recorded by the control unit in the focal plane in which the fluorescent emitters are distributed in a congruent fashion. As a result, the offset can be determined with greater accuracy.

[0020] Setting different focal planes is realized using means already available in conventional laser scanning microscopes, based on an adjustable focusing unit including an objective lens, or based on a focusing unit including a collimation optical unit that is optically arranged between the second and third deflection units (in particular between the first and third beam deflection units), in particular in a state in which the collimation optical unit can be moved along the optical axis of the illumination in order to set different focal planes. In this way, no modifications of available LSMs are necessary.

[0021] Preferably, the first and second beam deflection units are each formed as a mirror rotatable about two different spatial axes. As a result, the space required for adjusting the four degrees of freedom of the free beam is minimized. Alternatively or additionally, the first and / or second beam deflection units can be displaceable, in particular linearly displaceable. All beam deflection units include motor drives, which are electrically or electronically connected to the control unit. In particular, a rotatable plane plate with multiple parallel planes can also be used instead of a rotatable or displaceable mirror. This allows for the generation of an adjustable beam offset.

[0022] Preferably, the third and fourth beam deflection units are each formed as a mirror rotatable about exactly one spatial axis, in particular each formed as a galvanometer mirror, the spatial axis being different between the two beam deflection units, or the third and fourth beam deflection units are jointly formed by one mirror rotatable about two different spatial axes, in this way high-quality and fast image recording is possible according to known laser scanning principles.

[0023] Advantageously, no calibration standard sample can be optically placed between the light source and the objective lens. Advantageously, the optical systems between the first beam deflection unit and the objective lens and between the second beam deflection unit and the objective lens do not branch out to sensors for ascertaining the beam position and / or beam direction. Advantageously, the first and second beam deflection units have a constant setting during each image recording (scanning the focal plane with the third and fourth beam deflection units).

[0024] The present invention also includes a method for adjusting a laser scanning microscope having an optical system including two light sources, an optoelectronic detector, four movable beam deflection units, and a microscope objective having a pupil plane and a focal plane, wherein third and fourth beam deflection units are arranged in or near a plane conjugate with the pupil plane, and first and second beam deflection units are arranged upstream of the third and fourth beam deflection units in the illumination direction, the optical system directs light from the first light source through the objective to the focal plane via the four beam deflection units and the beam splitter, and directs light from the second light source through the objective to the focal plane without passing through the first and second beam deflection units, and images points on the focal plane through the objective onto the detector, and the optical system 2 a light guide fiber between the light source and the third beam deflection unit; 1 and a third beam deflection unit, wherein the test image is recorded by the detector under illumination by the first light source through an objective lens, the focal plane of which is scanned by the third and fourth beam deflection units, and the first and / or second beam deflection units are set using the recorded test image, in particular with multiple iterations of recording the test image and setting the associated beam deflection units. In this way, the aforementioned advantages are realized. In general, the steps performed by the aforementioned control unit can be general steps of the method according to the invention and do not necessarily have to be performed by the control unit.

[0025] Preferably, the first beam deflection unit, which is arranged optically upstream or downstream of the second beam deflection unit, is in this case set based on the characteristics of the illumination of the test image, in particular based on the characteristics of the uniformity and / or intensity of the illumination of the test image.

[0026] Preferably, the second beam deflection unit is set based on an offset between the reference image recorded under illumination by the second light source and the test image recorded under illumination by the first light source, or based on an offset between the first test image recorded under illumination by the first light source and the second test image recorded under illumination by the first light source or by the second light source, and the focal plane is moved by an adjustable focusing unit, in particular the objective lens and / or the collimation optical unit, in particular between recording of the first and second test images.

[0027] In all described embodiments, a configuration in which the first beam deflection unit (which is essentially adjusted based on the illumination) is arranged upstream of the second beam changing unit (which is essentially adjusted based on the image offset) in the illumination direction is preferred, since in this configuration the free beam is adjustable over the largest spatial and angular range.

[0028] The invention is explained in more detail below on the basis of exemplary embodiments. [Brief explanation of the drawings]

[0029] [Figure 1] 1 shows a laser scanning microscope with an improved beam conditioning scheme. [Figure 2] Angular adjustment in the pupil plane is shown. [Figure 3] 10 illustrates the in-plane adjustment using test images from different focal planes. [Figure 4] A possible procedure for adjusting all degrees of freedom using a reference beam is presented. [Figure 5] A possible procedure for adjusting all degrees of freedom is shown using test images from different focal planes by adjusting the objective lens. [Figure 6] A possible procedure for adjusting all degrees of freedom is shown using test images from different focal planes by adjusting the collimator. DETAILED DESCRIPTION OF THE INVENTION

[0030] In all the drawings, corresponding elements are given the same reference numerals. 1 shows a laser scanning microscope 100 having a first light source 1, e.g., comprising a Ti:sapphire ultrashort pulse laser 1.1 emitting in the NIR range and a pre-chirp unit 1.2 for compensating group velocity dispersion, and a second light source 2 (e.g., a laser diode) emitting in the VIS range. An adjustable attenuator 3 (e.g., an AOM) allows the light from the first light source 1 to reach a periscope 4. The periscope 4 includes a first beam deflection unit 4.1 and a second beam deflection unit 4.2, each of which includes a mirror rotatable about two axes and with a motor drive. The light from the first light source 1 then reaches the scanning module 5, which includes a movable collimation optical unit 6, a connector 7 for a fiber plug 8, a dichroic beam splitter 9, a primary color splitter 10, and a detector 11 with a confocal diaphragm 12. The scanning module 5 also includes a third beam deflection unit 13 and a fourth beam deflection unit 14, each of which may be in the form of a galvanometer scanner, for example. The fiber plug 8 is part of an optical fiber 15 that connects the second light source 2 to the scanning module 5 via another adjustable attenuator 3. The light from the two light sources 1 and 2 is then passed through the dichroic beam splitter 9 before passing through a primary color splitter 10, whereby both beams are jointly affected by a third and a fourth beam deflection unit 13, 14, after which the beam leaves the scanning module 5 and passes through a microscope stand 16 and another dichroic beam splitter 17, which can for example be swiveled in and out, to an objective 18, where it is focused in the focal plane FE.

[0031] For example, the entire objective lens 18, or just the internal lens group, can be motorized along its optical axis to move the focal plane. Alternatively, or in addition, the collimation optical unit 6 can be motorized along its optical axis to compensate for longitudinal chromatic aberration on a wavelength-by-wavelength basis, thereby also effectively changing the focal plane.

[0032] In addition to the confocal detector 11, which enables descanned detection, the microscope 100 also includes two NDD detectors 19 (without a confocal aperture), which are coupled to the optical system by a beam splitter 17 and another dichroic beam splitter 20. These enable non-descanned detection. By appropriately selecting the dichroic beam splitter 20, the two NDD detectors 19 can simultaneously detect different wavelength ranges. In the illustrated configuration, the confocal detector 11 can only detect one wavelength range at a time. To enable this to be changed sequentially, the primary color splitter 10 can be alternatively placed on a motorized movable filter wheel (not shown), or appropriate filters can be alternatively placed upstream of the detector 11 on a motorized movable filter wheel (not shown). Alternatively, or in addition, one or more other detectors can be arranged for confocal, descanned, simultaneous detection of multiple wavelength ranges by one or more additional dichroic beam splitters. If only fluorescence from multiphoton excitation is measured, especially in optional embodiments without a second light source 2, the confocal aperture 12 is not required.

[0033] The moving components of the microscope 100 are controlled by a control unit 21, which for this purpose is electrically connected to the components and to the detector (possibly also to a filter wheel which may be present). The periscope 4 allows setting four degrees of freedom of the light L1 from the first light source, which is coupled into the scanning module 5 as a free beam.

[0034] The control unit 21 can have a user interface, by means of which the first beam deflection unit (4.1) and the second beam deflection unit (4.2) can be adjusted by the user, for example in different increments. To this end, the user interface can indicate a respective pointer for each rotation axis, which informs the user of the total number of steps in each direction. As a result, a previous state can be restored at little cost.

[0035] Preferably, the control unit is configured to make the adjustments automatically, either electronically or by programming. In FIG. 2A, part a) shows the beam path (main beam axis) of the NIR light L1, which is not adjusted with respect to the angle at the pupil plane PE. The VIS light L2 (main beam axis) from the second light source extends along the optical axis OA of the objective lens 18 because the scanning module 5 is already adjusted using this beam. If an image is recorded using the VIS illumination L2 and an image is recorded using the NIR illumination L1, and these images are superimposed, they will not correspond, but instead will have an offset, as shown in part b). After adjustment, both beams are on the optical axis, as shown in part a) of FIG. 2B. As mentioned above, if the two images are recorded with different illumination, they will now correspond, as shown in part b).

[0036] In FIG. 3A, part a) also shows the beam path (main beam axis) of the NIR light L1, which is not adjusted with respect to angle in the focal plane FE. A calibration standard sample, for example in the form of one or more latex beads, with a fluorescent emitter arranged with spherical or cylindrical symmetry is placed in the focal plane FE. The VIS light L2 (main beam axis) from the second light source extends along the optical axis OA of the objective lens 18, because the manipulation module 5 has already been adjusted using this beam. If an image is recorded in the focal plane FE using the NIR illumination L1 and an image is recorded in the shifted focal plane FE' using the NIR illumination L1, and if these images are superimposed, they will not correspond, as shown in part b), but will instead have an offset. After adjustment, both beams are positioned on the optical axis, as shown in part a) of FIG. 3B. As mentioned above, if the two images are recorded at different focal planes FE, FE', they will now correspond, as shown in part b).

[0037] For example, the adjustment can proceed according to the diagram shown in FIG. 4. For this purpose, a thin sample is preferably used that can be imaged by the light source and includes structures arranged in a plane extending at an angle to the optical axis. The sample can be fluorescent and reflective. For example, this can involve a chrome grid in front of a uniform fluorescent material. The sample is preferably recorded in an image-filling manner when the test and / or reference images are recorded. The reliability of the image evaluation can be improved by measures such as averaging if the adjustment must be performed on dark samples or with little excitation light, for example in the case of real biological samples.

[0038] First, a reference image is recorded and saved using the second light source 2. Next, test images are repeatedly recorded under illumination by the first light source 1 and evaluated based on whether the illumination reaches or exceeds a specified criterion, such as a specified threshold for uniformity. For example, the control unit 12 can determine the ratio of the grayscale value at the image edge to the grayscale value at the center of the relevant image as a quantitative characteristic of the illumination and compare it to a threshold. Alternatively, a contour line can be created across the entire object, and the resulting ratio of the peak grayscale values ​​can be determined as a characteristic. Alternatively, a linear function from curve fitting can be fitted to the peak, and its slope can be compared to a threshold as a characteristic of the illumination. If the illumination is not sufficiently uniform, regardless of the type of characteristic and the method of determination, the first deflection unit 4.1 is adjusted, and the test image is recorded and evaluated again. If the illumination is sufficiently uniform according to the specified criterion, the first ("internal") iteration ends. The offset between the reference image and the test image is then checked and evaluated using another specified criterion (e.g., whether a specified threshold for the offset has been nearly reached or even undershot). If the criterion is not met, the second beam deflection unit 4.2 is adjusted and the iterative setting of the first beam deflection unit 4.1 is repeated. This represents an "outer" iteration. If the offset is small enough according to the specified criterion, the second ("outer") iteration ends and the adjustment is thus completed.

[0039] The VIS reference image does not have to be recorded at the start, but can also be recorded at a later point in time (e.g., after the first internal iteration for setting the first beam deflection unit 4.1 has finished). If the adjustment is performed automatically by the control unit 21, the offset can be ascertained, for example, by two-dimensional cross-correlation between the relevant images. The setting of the first beam deflection unit 4.1 in the "internal" iteration can be performed using any known optimization method. In addition to, or as an alternative to, uniformity, the intensity of the illumination, i.e., the sum of the intensity values ​​of all pixels, can be used as a simple illumination characteristic. An explicit illumination criterion checked during the internal iteration is whether or not a maximum value of the integrated intensity exists.

[0040] As an alternative to the procedure shown in FIG. 4, the adjustment can proceed, for example, according to one of the diagrams shown in FIG. 5 or FIG. 6. In FIGS. 5 and 6, the offset between a first test image recorded at focal plane FE under illumination by the first light source 1 and a second test image similarly recorded under illumination by the first light source 1 but at a different focal plane FE' is used to set the second beam deflection unit 4.2 instead of the offset between the first test image and the second test image under illumination by the second light source 2. For this, the second light source 2 is not required. However, a reference image can be recorded under illumination by the second light source 2 instead of the first test image or instead of the second test image. What is important is that the images are recorded at different focal planes FE, FE'. The different focal planes FE, FE' can be obtained by adjusting the focusing unit, for example by adjusting the objective lens 18 (as in FIG. 5) or the collimation optical unit 6 (as in FIG. 6).

[0041] It is possible to couple several light sources into the microscope 100 for each free beam. For this purpose, each of these light sources requires a respective first and second beam deflection unit, i.e., a separate periscope 4 for each free beam into which each light source is coupled in the example according to Fig. 1. Each pair of first and second beam deflection units should be adjusted separately with respect to the second light source 2 (for example as in Fig. 4) or with respect to the light source coupled per free beam, either by adjusting the first and second beam deflection units as described above (for example again as in Fig. 4) or by an image offset between recordings from different focal planes FE, FE' (for example again as in Fig. 5 or Fig. 6). [Explanation of symbols]

[0042] 1...First light source 1.1…Ti:sapphire ultrashort pulse laser 1.2…Pre-chirp unit 2...Second light source 3...Attenuator 4...Periscope 4.1...First beam deflection unit 4.2...Second beam deflection unit 5...Scanning module 6...Collimation optical unit 7...Connector 8...Fiber plug 9...Dichroic beam splitter 10...Primary color splitter 11...Detector 12...Confocal aperture 13...Third beam deflection unit 14...Fourth beam deflection unit 15...guiding optical fiber 16...Microscope stand 17...Dichroic beam splitter 18...Objective lens 19...NDD detector 20...Dichroic beam splitter 21...Control unit 100...Laser scanning microscope FE(')…focal plane PE…pupil surface L1: Light from the first light source L2: Light from the second light source OA...Optical axis S: Calibration standard sample

Claims

1. The optical system includes two light sources (1, 2), a photoelectric detector (11, 19), four movable beam deflection units (4.1, 4.2, 13, 14), and a microscope objective (18) having a pupil plane (PE) and a focal plane (FE), wherein a third beam deflection unit (13) and a fourth beam deflection unit (14) are arranged in a plane conjugate with the pupil plane (PE) or in the vicinity of the plane, and a first beam deflection unit (4.1) and a second beam deflection unit (4.2) are arranged upstream of the third beam deflection unit (13) and upstream of the fourth beam deflection unit (14) in the illumination direction, and the optical system directs light from a first light source (1) through the objective (18) and via the four beam deflection units (4.1, 4.2, 13, 14). and guides light from a second light source (2) through the objective lens (18) to the focal plane (FE) via the third beam deflection unit (13) and the fourth beam deflection unit (14), but not through the first beam deflection unit (4.1) and the second beam deflection unit (4.2), and forms an image of a point on the focal plane (FE) on a detector (11, 19) through the objective lens (18). In this laser scanning microscope (100), the optical system has an optical fiber guiding (15) between the second light source (2) and the third beam deflection unit (13), but does not have an optical fiber guiding between the first light source (1) and the third beam deflection unit (13).

2. 2. The microscope (100) according to claim 1, characterized by a control unit (21) for recording a test image through the objective (18) by the detector (11, 19) under illumination by the first light source (1) for the purpose of adjusting the optical system, the control unit (21) scanning the focal plane (FE) by the third and fourth beam deflection units (13, 14) and setting the first beam deflection unit (4.1) and / or the second beam deflection unit (4.2) using the test image, in particular during multiple repetitions of recording the test image and setting the associated beam deflection units (4.1, 4.2).

3. The microscope (100) of claim 2, wherein the control unit (21) checks the illumination characteristics of the test image and sets the first beam deflection unit (4.1), which is located upstream or downstream of the second beam deflection unit (4.2) in the illumination direction, based on the checked characteristics, and the control unit (21) uses, in particular, the uniformity and / or intensity of the illumination of the test image as the characteristics used to set the first beam deflection unit (4.1).

4. The optical system is characterized by a control unit (21) for recording a test image through the objective lens (18) by the detector (11, 19) under illumination by the first light source (1) for the purpose of adjusting the optical system, the control unit (21) scans the focal plane (FE) by the third and fourth beam deflection units (13, 14) and records a reference image from the focal plane (FE) before or after recording under illumination by the second light source (2), and the control unit (21) scans the focal plane (FE) by the third and fourth beam deflection units (13, 14) and records a reference image from the focal plane (FE) before or after recording under illumination by the second light source (2).

4. The microscope (100) according to claim 1, further comprising: scanning a focal plane (FE) to determine a geometric offset between the test image and the reference image; and setting the second beam deflection unit (4.2), which is arranged upstream or downstream of the first beam deflection unit (4.1) in the illumination direction, based on the determined offset, in particular based on multiple repetitions of repeatedly setting the first beam deflection unit (4.1), subsequently recording the test image, and setting the second beam deflection unit (4.2).

5. 5. The microscope (100) of claim 4, wherein the control unit (21) initially sets the first beam deflection unit (4.1) based on illumination characteristics and sets the second beam deflection unit (4.2) based on an offset, in particular, the setting of the first beam deflection unit (4.1) remaining constant.

6. The optical system is characterized by a control unit (21) for recording a first test image through the objective lens (18) by the detector (11, 19) for the purpose of adjusting the optical system, the control unit (21) scans the focal plane (FE) by the third and fourth beam deflection units (13, 14), and then moves the focal plane (FE) by an adjustable focusing unit, and records a second test image from the moved focal plane (FE') by the detector (11, 19) through the objective lens (18), and the control unit (21) scans the moved focal plane (FE') by the third and fourth beam deflection units (13, 14), and records the first test image and the second test image.

4. A microscope (100) according to any one of claims 1 to 3, wherein a geometric offset between the first and second test images is ascertained, and the second beam deflection unit (4.2), arranged upstream or downstream of the first beam detection unit (4.1) in the illumination direction, is set based on the ascertained offset, and the first test image is recorded under illumination by a light source different from the light source of the second test image, or both test images are recorded under illumination by the first light source (1), in each case in particular by repeatedly setting the first beam deflection unit (4.1), and then recording the first and second test images and setting the second beam deflection unit (4.2).

7. 7. The microscope (100) of claim 6, wherein the adjustable focusing unit includes an objective lens (18), and the focusing unit includes a collimation optical unit (6) optically arranged between the second beam deflection unit (4.2) and the third beam deflection unit (13), in particular, the collimation optical unit (6) being movable along the optical axis (OA) of illumination to set different focal planes (FE, FE').

8. 8. The microscope (100) according to any one of claims 1 to 7, wherein the first and second beam deflection units (4.1, 4.2) are each formed as mirrors rotatable about two different spatial axes.

9. 9. The microscope (100) according to claim 1, wherein the third and fourth beam deflection units (13, 14) are each formed as a mirror rotatable about exactly one spatial axis, in particular each formed as a galvanometer mirror, the spatial axis being different between the two beam deflection units (13, 14), or the third beam deflection unit (13) and the fourth beam deflection unit (14) are jointly formed by one mirror rotatable about two different spatial axes.

10. A microscope (100) as described in any one of claims 1 to 9, wherein in the microscope (100), a calibration standard sample cannot be optically placed between the two light sources (1, 2) and the objective lens (18), and / or the optical system between the first beam deflection unit (4.1) and the objective lens (18) and between the second beam deflection unit (4.2) and the objective lens (18) does not branch to sensors for checking the beam position and / or beam direction, and / or the first and second beam deflection units (4.1, 4.2) have constant settings during each image recording.

11. A method for adjusting a laser scanning microscope (100) having an optical system including two light sources (1, 2), a photoelectric detector (11, 19), four movable beam deflection units (4.1, 4.2, 13, 14), and a microscope objective (18) having a pupil plane (PE) and a focal plane (FE), wherein the third and fourth beam deflection units (13, 14) are arranged in or near a plane conjugate with the pupil plane (PE), and the first and second beam deflection units (11, 19) are arranged in or near a plane conjugate with the pupil plane (PE). The units (4.1, 4.2) are arranged upstream of the third and fourth beam deflection units (13, 14) in the illumination direction, and the optical system guides light from a first light source (1) through an objective lens (18) via the four beam deflection units (4.1, 4.2, 13, 14) to the focal plane (FE), and guides light from a second light source (2) through the objective lens (18) to the focal plane (FE) via the first and second beam deflection units (4.1, 4.2, 13, 14). a beam deflection unit (4.1, 4.2) for guiding the beam of light without the aid of a beam deflection unit (4.1, 4.2) and imaging a point of the focal plane (FE) through the objective lens (18) onto a detector (11, 19), the optical system having an optical fiber (15) between the second light source (2) and the third beam deflection unit (13) and having no optical fiber between the first light source (1) and the third beam deflection unit (13), a test image is recorded by the detector (11, 19) under illumination by the first light source (1) through the objective lens (18) by scanning the focal plane (FE) with the third and fourth beam deflection units (13, 14), the first and / or second beam deflection units (4.1, 4.2) being configured using the recorded test image, in particular during multiple repetitions of recording the test image and setting the associated beam deflection units (4.1, 4.2).

12. 12. The method according to claim 11, wherein the first beam deflection unit (4.1), which is arranged optically upstream or downstream of the second beam deflection unit (4.2), is set based on characteristics relating to the illumination of the test image, in particular the uniformity and / or intensity of the illumination of the test image as characteristics.

13. 13. The method according to claim 11 or 12, wherein the second beam deflection unit (4.2) is set based on an offset between a reference image recorded under illumination by the second light source (2) and a test image recorded under illumination by the first light source (1), or based on an offset between a first test image recorded under illumination by the first light source (1) and a second test image recorded under illumination by the first light source (1) or by the second light source (2), and the focal plane (FE) is moved by an adjustable focusing unit, in particular the objective lens (18) and / or a collimation optical unit (6), between recording of the first and second test images.

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