Scanning device for confocal scanning imaging systems

Abstract

Scanning device for confocal scanning imaging systems, designed for X,Y,Z positioning of laser spots in a sample, comprising, - an X,Y scanner arranged in the illumination beam path (BS) for the lateral positioning of the laser spots with respect to the optical axis (OA) of a lens, - an optical element arranged in the illumination beam path (BS) and / or in the detection beam path (DS) and designed to influence the wavefront or the intensity of a light, - two beam deflection elements (U1, U2) arranged symmetrically to a pupil plane (P3), wherein - the illumination beam path (BS) is directed by means of a first beam deflection element (U1) via a first and a second lens optic (L1, L2) onto the optical element and from there via the second and a third lens optic (L2, L3) onto the second beam deflection element (U2), and / or - the detection beam path (DS) is directed by means of the second beam deflection element (U2) via the third and second lens optics (L2, L3) to the optical element and from there via the second and first lens optics (L1, L2) to the first beam deflection element (U1), - an intermediate image plane (ZB) is formed between the second lens optics (L2) on the one hand and the first and third lens optics (L1, L3) on the other hand, and - a further pupil plane (P2, P3) is formed between the first beam deflecting element (U1) and the first lens optics (L1) and between the second beam deflecting element (U2) and the third lens optics (L3), and the three pupil planes (P1, P2, P3) are conjugated to one another.

Description

[0001] The invention relates to a scanning device for confocal scanning imaging systems, in particular for X,Y,Z positioning of laser spots in a sample, equipped with optical means for correcting image errors.

[0002] The use of, for example, adaptive elements to compensate for aberrations and / or for focus manipulation in optical systems such as laser scanning microscopes, fluorescence laser scanning microscopes or laser scanning ophthalmoscopes is known per se.

[0003] In addition to transmissive adaptive elements based on liquid crystals, reflective adaptive elements are predominantly used due to their shorter response time when controlled, for example in the form of membrane mirrors, in which a highly flexible membrane is deformed by means of piezo elements or by electrostatic forces.

[0004] In confocal scanning imaging systems, the adaptive optical element is ideally positioned in a pupil plane, so that the beams of light coming from an object point at the X, Y, Z position and excited by a laser spot—depending on the system configuration—incide onto the membrane mirror in a collimated or diverging manner. Beams of light coming from object points at other X, Y, Z positions with the same divergence hit the membrane mirror at the same location but with different angles of incidence.

[0005] A disadvantage of such arrangements is that the angles formed by the incident beams with the optical axis of the adaptive elements are so large that deformations of the reflector surface of the membrane mirror result in additional undesirable wavefront changes. For example, a spherical shape of the reflector surface causes astigmatism at oblique incidence.

[0006] In addition, in sample scanning where no deformation of the reflector surface is intended, but where it is only to be used as a non-adaptable reflector, losses in terms of the intensity of the illumination or detection radiation arise simply due to the beam guidance via this element, for example due to a non-ideal reflectivity of the coating of the adaptive reflector surface.

[0007] JP 2006 - 106 336 A discloses a scanning microscope comprising a light source, an optical system having a wavefront conversion element that imparts an arbitrary wavefront conversion to the illuminating light emitted by the light source, a luminous flux scanning device for scanning two orthogonal directions with the illuminating light, an objective lens for concentrating the illuminating light onto an object, and a detector for detecting signal light emitted by the object.

[0008] DE 102 27 120 A1 describes a laser scanning microscope with an adaptive optical device in the microscope beam path, two of which are aligned with their reflector surfaces perpendicular to the optical axes of the microscope beam path. At the apex of two orthogonal arms of the microscope beam path is the splitter layer of a polarizing beam splitter, with one adaptive element assigned to one arm and the other to the second arm. Each arm contains a λ / 4 plate between the beam splitter and the adaptive element.

[0009] The λ / 4 plates serve to enable perpendicular light incidence onto the reflector surface at the adaptive elements. However, undesirable intensity losses also occur at the λ / 4 plates. Furthermore, the use of polarization beam splitters and second adaptive mirrors is relatively costly.

[0010] From WO 2013 / 010151 A1, a method for focusing a laser beam when scanning a sample in multiconfocal mode is known, in which the relative phases of the individual partial beams of the excitation beam are varied with a reflective adaptive optics in order to increase the emission of the detection radiation.

[0011] The associated arrangement has the disadvantage described above that the angles of incidence and reflection relative to the reflector surface of the adaptive optics are relatively large, causing undesirable wavefront changes that counteract the increase in the intensity of the detection radiation. Two different optics are used before and after a light modulator (SLM). Therefore, minimizing the angle of incidence at the adaptive optics is limited by the diameter of the optics, including their mounts, given the limited overall length. On the other hand, in multi-confocal arrangements, the adaptive optics must be arranged in a conjugate pupil plane in order to influence all partial beams simultaneously.

[0012] DE 10 2006 045 839 A1 describes a laser scanning microscope with a scanning device that has two uniaxially deflecting scanning elements arranged one behind the other in the imaging direction. Furthermore, an element for pupil manipulation is present. The first scanning element is located in a first pupil, the pupil manipulation element is located in a second pupil, and the second scanning element is positioned in a third pupil. A relay optics system is arranged downstream of the first scanning element, which images the first pupil onto the second pupil, i.e., onto the pupil manipulation element. The pupil manipulation element is reflective, so that the relay optics are passed through again in the imaging direction. A beam deflector with two reflective surfaces ensures that the detection light from the third pupil does not reach the first pupil directly, but only via the pupil manipulation element.The illumination beam path passes through this scanning device in the opposite direction.

[0013] For example, an adaptive element connected to a control system is used for pupil manipulation. Depending on the radiation direction, a lens optic is arranged upstream or downstream of the adaptive element. This lens optic directs the radiation coming from an intermediate image onto the adaptive element and refocuses the radiation reflected from there into this intermediate image plane. The intermediate image is located in the area between the relay optics and the lens optics.

[0014] This arrangement has the disadvantage that the two pupil planes in which the scanners are positioned are at a predetermined distance from each other, requiring the use of an intermediate optic. This necessitates a frame-mounted arrangement of the adaptive optics in the illumination and detection beam paths.

[0015] Based on this, the object of the invention is to further develop a scanning device of the type mentioned at the outset in such a way that the disadvantages of the prior art are eliminated and the scanning of an object volume always ensures the achievement of a high image quality.

[0016] According to the invention, a scanning device for X,Y,Z positioning of laser spots in a sample, preferably for use in confocal or multi-confocal scanning imaging systems, - an X,Y scanner arranged in the illumination beam path for the lateral positioning of the laser spots with respect to the optical axis of a lens, - a preferably reflective optical element arranged in the illumination and / or detection beam path and designed to influence the wavefront or the intensity of the light, - two beam deflection elements arranged symmetrically to a pupil plane, whereby - the illumination beam path is directed by means of a first beam deflection element via a first and a second lens optic onto the optical element and from there via the second and a third lens optic onto the second beam deflection element, and / or - the detection beam path is directed by means of the second beam deflection element via the third and second lens optics to the optical element and from there via the second and first lens optics to the first beam deflection element, - an intermediate image plane is formed between the second lens optics on the one hand and the first and third lens optics on the other hand, and - a further pupil plane is formed between the first beam deflecting element and the first lens optics and between the second beam deflecting element and the third lens optics, and these three pupil planes are conjugated to one another.

[0017] In a preferred embodiment, the optical element is connected to a control device, which in turn is connected to a controller for specifying a temporally and spatially defined sequence of X,Y,Z positions for laser spots and - with a measuring device for detecting system-related and object-related image errors related to these X,Y,Z positions, or - is connected to an actual value memory for system-related image errors related to these X,Y,Z positions.

[0018] The optical element is arranged in a fixed pupil position of the illumination and / or detection beam path. In an alternative embodiment, the optical element can also be designed as a spatial light modulator (SLM) and used to generate partial beams for multi-confocal scanning imaging or to vary the relative phase or directions of the partial beams.

[0019] In a further preferred embodiment of the scanning device according to the invention, the two beam deflection elements can be pivoted out of the area of ​​the illumination or detection beam path around the pupil plane as needed. After pivoting out, the illumination and detection beam path is no longer guided via the lens optics and the adaptive optics, but passes through this stationary pupil plane directly. In this sense, it is also within the scope of the invention if not only the two beam deflection elements, but the entire assembly comprising beam deflection elements, lens optics, and optical element is not fixed to the frame, but is arranged so that it can be pivoted out or in relative to the system beam paths, thus fulfilling a switching function that enables variation in the beam guidance.

[0020] By pivoting the assembly according to the invention, relatively small angles of incidence and reflection of ≤ 10 degrees can be realized on the optical element for specific sample examinations, if necessary, without interfering with the basic function of the confocal scanning imaging system. For the duration of the inventive use, only an image inversion occurs. The basic function is restored after pivoting out. The assembly according to the invention can be implemented in a compact size, including an associated pivoting device.

[0021] The two beam-deflecting elements can be the reflecting surfaces of a 90° prism. However, the invention also expressly includes embodiments in which the angle formed by the reflecting surfaces deviates from a right angle.

[0022] The optical axes of the two lens optics are preferably arranged parallel, but can also be aligned non-parallel.

[0023] It is within the scope of the invention if the optical element - is designed as an adaptive optical element and is intended to influence the wavefront, or - designed as a spatial light modulator and intended to influence the intensity.

[0024] If an adaptive optical element is used, it comprises a membrane with a deformable, preferably radially symmetrical, mirror surface, and actuators for deforming the mirror surface. A measuring device with a wavefront sensor can be provided. The deformation occurs either by closed-loop or open-loop control depending on the respective X, Y, Z position of a laser spot and thus depending on the image aberrations related to this X, Y, Z position, so that the image aberrations are corrected for each X, Y, Z position.

[0025] Of course, the use of adaptive optical elements with a torically deformable mirror surface is also included in the inventive concept, but with a mirror surface that can only be deformed radially symmetrically, but is more cost-effective, the astigmatism that occurs during defocusing is kept small due to the smaller angle of incidence.

[0026] On the other hand, spherical errors, such as third-order or higher-order spherical aberrations, remain correctable with the radially symmetric design.

[0027] In this way, the radially symmetric deformation of the mirror surface can be used to position the spot position or to defocus without causing additional astigmatism.

[0028] If, for example, a light modulator is applied with a line structure, several outgoing beams can be generated from one incident beam for the purpose of multi-confocal scanning of a sample, while simultaneously correcting image errors.

[0029] A key advantage of the arrangement according to the invention is that the position of the system-specific pupil remains unchanged in the basic structure of a confocal scanning imaging system, thus ensuring that its area in the illumination and detection beam path remains accessible for other manipulations or switching operations. Another advantage is that no optical deflection elements are present near an intermediate image, thus preventing contamination of beam deflection elements from becoming visible in the image.

[0030] The scanning device according to the invention is particularly advantageous for use in the scanned beam path of the confocal scanning imaging system. It is also advantageous in conjunction with multi-spot scanning of the sample, in which multiple laser spots image a line or even a field.

[0031] The invention will be explained in more detail below using exemplary embodiments. The accompanying drawings show Fig. 1 an optical assembly with an adaptive optical element in a confocal scanning imaging system, Fig. 2 an optical assembly with a spatial light modulator in a multiconfocal scanning imaging system, Fig. 3 an optical assembly corresponding Fig. 1, but with color splitter plates instead of a prism to deflect the illumination beam path.

[0032] In Fig. 1 shows an optical assembly arranged in the region of a pupil plane P3 of the illumination and detection beam path BS, DS of a confocal scanning imaging system designed for 3D scanning of a sample. The position of the pupil plane P3 is - as shown by way of example in Fig. 2 - defined by a scanning mirror SP3.

[0033] The optical assembly comprises a 90° prism with two beam deflection elements U1 and U2, three lens optics L1, L2, and L3, and an adaptive optical element AOE. The illumination beam path BS is directed by the first beam deflection element U1 via the lens optics L1 and L2 to the adaptive optical element AOE, and from there via the lens optics L2 and L3 to the second beam deflection element U2. After this second deflection, the illumination beam path BS returns to its original direction.

[0034] The adaptive optical element (AOE) is designed as a reflective element and serves for the axial or Z-positioning of the laser spots relative to the optical axis (OA) and for compensating for image aberrations at the respective X, Y, Z positions of the laser spots in the sample. For this purpose, it has a membrane with, for example, a radially symmetrically deformable mirror surface and is connected to a control device (not shown in the drawing), which in turn is connected to a controller for specifying a temporally and spatially defined sequence of X, Y, Z positions of the laser spots.

[0035] For the purpose of regulating the image error compensation, the control device is preferably coupled to a measuring device equipped with a wavefront sensor for detecting system- and object-related image errors at the respective X, Y, Z positions. In an alternative embodiment, in which the image error compensation is controlled based on pre-stored system-related error data, the control device is connected to an actual value memory for image errors at specific X, Y, Z positions (not shown in the drawing).

[0036] The detection beam path DS, which runs opposite to the illumination beam path BS, is directed by the beam deflection element U2 via the lens optics L3 and L2 to the adaptive optical element AOE, and from there via the lens optics L2 and L1 to the beam deflection element U1. The detection beam path DS then also returns to its original direction.

[0037] An intermediate image plane ZB1 is formed between the second lens optic L2 on the one hand and the two adjacent lens optics L1 and L3 on the other. Furthermore, the lens optic L1 creates a pupil plane P1, and the lens optic L3 creates a pupil plane P2.

[0038] Due to the reflections at the beam-deflecting elements U1 and U2, the pupil planes P1, P2, and P3 are in conjugated positions. At the same time, the pupil plane P1 is imaged by the lenses L1 and L2 onto the adaptive optical element AOE, and the adaptive optical element AOE is in turn imaged by the lenses L2 and L3 into position P2. The adaptive optical element AOE is thus again in a pupil position. The joint use of the lens L2 according to the invention enables almost perpendicular illumination of the adaptive optical element AOE, whereas the use of two optics L1 and L2 allows the separation of the pupils P1 and P2.

[0039] Due to the almost perpendicular incidence on the adaptive element, even when it is deformed, the astigmatism remains small.

[0040] Fig. Figure 1 shows a beam of rays coming from a first object point of the illumination beam path BS, which runs centrally to the axis OA. The outer edges of this beam run parallel to the axis OA on both sides and are indicated by directional arrows. To achieve multi-confocal scanning imaging, at least one further object point is illuminated in addition to the first object point. If the optical assembly were not present at this position or were temporarily pivoted out, the beam of rays coming from this further object point would run along the axis S0. However, due to the effect of the optical assembly, this beam of rays runs along the axis S0'. The outer edges of this beam of rays run parallel to the axes S0 and S0', respectively; they are not shown for the sake of clarity.

[0041] The optical assembly is optionally either - fixed in the position shown here relative to the pupil plane P3, so that the beam guidance along the axis S0' is permanently specified, or - connected to a pivoting device with which at least the prism can be pivoted into or out of the beam path in order to enable the beam to be guided alternatively along the axis S0 or axis S0'.

[0042] In a Fig. In the embodiment shown in Figure 2, the optical assembly comprises a spatial light modulator SLM instead of the adaptive optical element AOE.

[0043] If the light modulator SLM is subjected to a line structure, several outgoing beams can be generated from a beam incident on the OA axis, for example for the purpose of multi-confocal scanning of a sample.

[0044] As from Fig. As can be seen in Figure 2, the illumination beam path BS coming from an intermediate image ZB2 or a light source is collimated by a lens optic L4. The illumination light is directed via the beam deflection element U1 and the lens optics L1 and L2 to the light modulator SLM and subsequently via the lens optics L2 and L3 to the beam deflection element U2. After this second deflection, the illumination beam path BS returns to its original optical axis, with several beam bundles S0 and S1 generated at the light modulator SLM now available for multi-confocal illumination of the sample.

[0045] The beam deflection elements U1, U2 are arranged symmetrically to the pupil plane P3, whose position is defined by the scanning mirror SP3. The pupil planes P1, P2, and P3 are also in conjugated positions.

[0046] The inventive concept includes a variant in which the Fig. The object plane shown in Figure 2 is designed as an intermediate image plane, which is imaged in a greatly reduced size into an object using further lens optics and a microscope objective, as shown, for example, in Naumann / Schroeder et al. "Handbook of Optical Components: Fundamentals, Materials, Devices, Measurement Technology", Carl Hanser Verlag, 7th completely revised edition, 2014, page 335. Accordingly, the arrangement according to the invention can be part of a confocal laser scanning microscope, wherein it is arranged between the scanning device on the one hand and the main color splitters, laser source and pinhole on the other.

[0047] Instead of a single scanning mirror SP3, several scanning mirrors can be arranged in conjugate planes and determine a position-position, which is then imaged into the pupil plane P3 by a lens optic L5. The optical assembly according to the invention is symmetrical to this pupil position P3. Fig. 1 or Fig. 3 arranged.

[0048] If a radially symmetrically deformable adaptive element is used instead of the light modulator SLM as in Fig. 1 or Fig. 2 is used, there is the advantage that due to the almost perpendicular incidence on the adaptive element, the astigmatism remains small even when it is deformed.

[0049] In Fig. Figure 3 shows an embodiment of the optical assembly, again with an adaptive optical element (AOE). However, instead of the 90° prism with the beam deflection elements U1, U2, two separate color splitter plates (HFT1 and HFT2) are provided. For clarity, only the optical axis of a confocal and the principal rays of a multi-confocal scanning imaging system are shown, emanating from an edge point.

[0050] While in Fig. 2 the marginal rays are drawn as they run from a point in the intermediate image ZB2 to two points S0 and S1 in the object plane OE or a microscope intermediate image plane, is in Fig. 3 the optical axis is drawn as it runs between the point S1 in the object plane OE to ZB2.

[0051] Additionally, the principal ray is shown, which in a multi-confocal scanning system originates from an off-axis object point S0 in the object plane OE and arrives at an off-axis point (not shown in ZB2). Accordingly, this principal ray intersects the optical axis at the pupil planes, here at the pupil plane P3, as well as at the mirrors SP1, SP2, and the adaptive optical element AOE, and runs in the intermediate image planes, here ZB5 and ZB4, separated from the optical axis.

[0052] The color splitter plates HFT1 and HFT2 are arranged symmetrically to the pupil plane P3.

[0053] The color splitter plate HFT1 couples the illumination light from the shared illumination and detection beam path, directs it via the mirror SP1, the lens optics L1, the intermediate image ZB4, and the lens optics L2 onto the adaptive optical element AOE, and influences it as described above. The illumination light then travels via the lens optics L2, the intermediate image ZB5, the lens optics L3, and the mirror SP2 to the color splitter plate HFT2, where it is redirected to the original optical axis or recoupled into the illumination and detection beam path.

[0054] The detection light coming from the object plane OE or from the object passes both color splitter plates in the order HFT2, HFT1 and reaches a detection device (not shown) without the detour via the lens optics L3, L2, L1 and the adaptive optical element AOE.

[0055] The scope of the invention also expressly includes the decoupling and deflection of the detection beam path DS instead of the illumination beam path BS and its influence by the adaptive optical element AOE. List of reference symbols AOE adaptive optical element BS illumination beam path DS detection beam path HFT1, HFT2 color splitter plate L1, L2 ... L5 lens optics OA axis P, P1, P2, P3 pupil plane S0, S0',S1 axis SLM light modulator SP1, SP2, mirror SP3 scan mirror U1, U2 beam deflection element ZB1, ZS2 ... ZB5 intermediate image plane

Claims

[1] Scanning device for confocal scanning imaging systems, designed for X,Y,Z positioning of laser spots in a sample, comprising, - an X,Y scanner arranged in the illumination beam path (BS) for the lateral positioning of the laser spots with respect to the optical axis (OA) of a lens, - an optical element arranged in the illumination beam path (BS) and / or in the detection beam path (DS) and designed to influence the wavefront or the intensity of a light, - two beam deflection elements (U1, U2) arranged symmetrically to a pupil plane (P3), wherein - the illumination beam path (BS) is directed by means of a first beam deflection element (U1) via a first and a second lens optic (L1, L2) onto the optical element and from there via the second and a third lens optic (L2, L3) onto the second beam deflection element (U2), and / or - the detection beam path (DS) is directed by means of the second beam deflection element (U2) via the third and second lens optics (L2, L3) to the optical element and from there via the second and first lens optics (L1, L2) to the first beam deflection element (U1), - an intermediate image plane (ZB) is formed between the second lens optics (L2) on the one hand and the first and third lens optics (L1, L3) on the other hand, and - a further pupil plane (P2, P3) is formed between the first beam deflecting element (U1) and the first lens optics (L1) and between the second beam deflecting element (U2) and the third lens optics (L3), and the three pupil planes (P1, P2, P3) are conjugated to one another. [2] Scanning device according to claim 1, equipped with a control device connected to the optical element, which in turn - with a controller for specifying a temporally and spatially defined sequence of X,Y,Z positions for laser spots and - with a measuring device for detecting system-related and object-related image errors related to these X,Y,Z positions, or - is connected to an actual value memory for system-related image errors related to these X,Y,Z positions. [3] Scanning device according to claim 1 or 2, wherein the optical element is arranged in a pupil position (P3) in the illumination beam path (BS) and / or detection beam path (DS). [4] Scanning device according to one of the preceding claims, in which the two beam deflection elements (U1, U2) can be pivoted out of the illumination beam path (BS) and / or detection beam path (DS) as required, while the pupil plane (P3) remains in its position. [5] Scanning device according to one of the preceding claims, in which the two beam deflecting elements (U1, U2) are reflecting surfaces of a 90° prism, and the two lens optics (L1, L3) including their beam paths are arranged parallel to each other. [6] Scanning device according to one of the preceding claims, in which the optical element - is designed as an adaptive optical element (AOE) and is intended to influence the wavefront, or - designed as a spatial light modulator (SLM) and intended to influence the intensity. [7] Scanning device according to claim 6, wherein the adaptive optical element (AOE) has a membrane with a deformable, preferably radially symmetrically deformable mirror surface and actuators are provided for the regulated or controlled deformation of the mirror surface. [8] Scanning device according to claim 7, wherein the measuring device is equipped with a wavefront sensor. [9] Scanning device according to one of the preceding claims, designed for confocal or multi-confocal scanning of the sample, wherein the multi-confocal scanning provides for the simultaneous X, Y, Z positioning of several laser spots with simultaneous correction of image errors.

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