Scanning microscope and use thereof

Abstract

The invention relates to a scanning microscope (1) having a first assembly (I) comprising a sample stage (2) for holding a sample carrier (4) and a sample (5) placed thereon in a sample space. In addition, a second assembly (II) is present, which is provided with an illumination optical unit (7, 8) for guiding and shaping an illumination radiation and for illuminating the sample (5); and comprising a detection optical unit (9) for collecting and guiding a detection radiation coming from the sample (5) along a detection beam path. The scanning microscope (1) is characterised in that the second assembly (II) has a first section (12.1) of the detection beam path; a third assembly (III) is present, which comprises a second section (12.2) of the detection beam path; and at least one drive (6) is present in order to move the second assembly (II) relative to the third assembly (III) in a controlled manner. The invention further relates to a use of the scanning microscope (1).

Description

[0001] Scanning microscope and its use

[0002] The invention relates to a microscope, in particular a scanning or scanning microscope, according to the preamble of the main claim, and its use.

[0003] Scanning microscopes are used to scan areas of a sample to be imaged with an illumination beam and / or with a focus of a detection beam path, in order to subsequently display the scanned area as an image.

[0004] The sample can be positioned on a sample carrier or sample holder, which in turn can be moved relative to the illumination beam path and / or the detection beam path by means of an adjustable sample table in order to perform the intended scanning of the sample.

[0005] Additionally or alternatively, the relative movement between the sample and the illumination or detection can be achieved by keeping the sample or sample holder stationary, while the illumination beam path and / or detection beam path are moved relative to it.

[0006] In the case of a moving sample, the disadvantage is that the sample is repeatedly set in motion and decelerated, which exposes it to mechanical stress and, due to its usual storage in a liquid medium, the position of the sample can change unpredictably, which can lead to significant losses in image quality.

[0007] If, on the other hand, the sample is held stationary and all the technical elements for illumination and detection are moved, the problems lie in accelerating and decelerating large masses while simultaneously placing very high demands on the accuracy of the positioning and scanning movements. Accuracies of several hundred nanometers are often required, which is hardly feasible with a technically acceptable level of effort and not economically practical when dealing with comparatively large masses.

[0008] The invention is based on the objective of proposing a scanning microscope that reduces the disadvantages of the prior art. Furthermore, a possible application of such a scanning microscope is to be proposed.

[0009] The task is accomplished using a scanning microscope comprising two modules. The first module includes a sample stage for holding a sample holder and a sample placed on it in a sample chamber. The second module features illumination optics for guiding and shaping the illumination beam and illuminating the sample. This second module also includes detection optics for collecting and guiding the detection beam emanating from the sample along a detection beam path.

[0010] A characteristic feature of a scanning microscope according to the invention is the presence of a third assembly. While the second assembly comprises a first section of the detection beam path, the third assembly comprises a second section of the detection beam path. Furthermore, actuators are provided to move the second assembly relative to the third assembly in a controlled manner.

[0011] The core of the invention is to divide the scanning microscope into at least three modules, with the second module performing a scanning or sampling movement on a sample that remains stationary during the scanning process. Since the second module comprises only a portion of the components required for illumination and detection, it has a low moving mass. This eliminates the need to move the sample during the scanning process, and at the same time, the mass of the second module, as well as its inertia and positioning accuracy, are significantly reduced compared to solutions known in the art.

[0012] For the purposes of this description, the term "assemblies" refers to technical units of the scanning microscope whose components are arranged on different supports and / or in individual housings, thus fundamentally allowing relative movement to one another. The first and third assemblies may be rigidly connected. For example, they may both be fixed, i.e., rigidly connected to each other, on a stand of the scanning microscope. This is not precluded if individual components of the first and / or third assemblies can be moved separately, as may optionally be the case with the sample stage (see below).

[0013] In an advantageous embodiment of the scanning microscope according to the invention, the illumination beam path and the first section of the detection beam path are aligned at a 90° angle to each other and separated from one another. The axes of the beam paths intersect in a region where the sample can be positioned on the sample holder. Furthermore, the beam paths are oriented at a non-zero angle to a normal on the sample stage. This results in the beam paths passing obliquely through the sample holder when the first and second assemblies are arranged in reverse. To achieve improved matching of the refractive indices of the sample and sample holder, a meniscus lens can be provided, through which both beam paths pass. A suitable immersion medium can be introduced or already present in a meniscus or cavity facing the sample holder.The meniscus lens is part of the second assembly and moves together with it. To prevent unwanted forces from being transmitted to the sample holder, a gap is advantageously present between the meniscus lens and the sample holder.

[0014] Since the second and third assemblies can be moved relative to each other, but also contain the first and second sections of the detection beam path respectively, a detection beam guided along the sections of the detection beam path must be able to travel from the first section to the second section despite a relative movement of the two sections relative to each other.

[0015] In an advantageous embodiment of the invention, the first and second sections of the detection beam path are connected via a free-jet link. As described, the detection radiation is collimated, for example, in the first section and then transmitted to the second section without additional optical guide elements. The advantage over, for example, coupling into a fiber optic cable or controlled tracking of the detection radiation lies in the small number of optical elements required and the low mass of the second assembly. To prevent unwanted interference signals from the environment of the scanning microscope, the free-jet link can be light-tightly encapsulated.

[0016] In a further advantageous embodiment of the invention, the third assembly comprises an optical deflection element by means of which the detection radiation received along the free-space path is directed along the further course of the second section. In this way, the detection radiation can, for example, be directed onto a detector without the detector having to be constantly moved along with it in accordance with the relative motion.

[0017] The detector preferably comprises a plurality of detector elements arranged in a detection plane and is, for example, a CCD, CMOS, sCMOS, or SPAD detector. The optical deflection element can be, for example, a flat mirror, a curved mirror, or an arrangement of several mirrors (mirror array, MMA, mirror staircase). The optical deflection element should be arranged and dimensioned such that a detection beam coming from the first section of the detection beam path strikes the optical deflection element and is deflected over at least a predefined range of possible positions of the second and third assemblies relative to each other.

[0018] Designing the deflection element as a curved mirror or as a controllable mirror array can reduce or correct angular deviations of the detection radiation that result from the different possible positioning of the assemblies relative to each other.

[0019] As a result of the movement of the second and third assemblies relative to each other, the angle at which the detected radiation reaches the detector changes (detection angle, tilt angle). This causes a tilting of the field of view. This tilting can be neglected if the axial extent of the field of view caused by the tilting is less than the image-side depth of field. It is possible to determine the currently existing tilt angles and take them into account when evaluating the detector measurements. Thus, based on knowledge of the current positioning of the assemblies relative to each other, and possibly also considering the current position of, for example, an adjustable sample stage, the magnitude and direction of one or more tilt angles can be determined.Due to the separate movements of the second and third assemblies, the resulting tilting of the field of view, and potential free-jet transmission, scanning movements can only be performed over short distances, typically a few millimeters. The achievable scan distance also depends on the magnification of the selected detection lens.

[0020] In addition to the mobility of the second assembly, the sample stage of the first assembly can have drives by means of which the sample stage, or parts thereof, and a sample holder located on the sample stage can be moved in a controlled manner. In further embodiments of the invention, the first assembly can also be moved relative to the second assembly. As will be explained below, a coarse adjustment of the sample holder or the sample can be made by adjusting the sample stage and / or the first assembly, while precise scanning of the sample is achieved by moving the second assembly. For this reason, the requirements for the positioning precision of the sample stage or the first assembly can be kept low. Only the drive or drives of the second assembly, by means of which the scanning movement is to be generated, must meet higher precision requirements.A rough adjustment of, for example, the sample stage and / or the first assembly may be necessary if samples extended in xy, such as wells of a microtiter plate, are to be imaged.

[0021] The movements of the second assembly can be generated, for example, by a traversing stage that allows position changes in the x, y, and optionally the z directions. In a further embodiment of the invention, an xy traversing stage can be combined with a z traversing stage to move the second assembly. The same applies to the first assembly. Adjustability in the z direction, i.e., for example, a change in the vertical distances between the second and third assemblies, is optional.

[0022] All movements can be coordinated using a control system for the scanning microscope.

[0023] In a further embodiment of the invention, the scanning microscope can include an evaluation unit configured to determine, based on current position data of the sample carrier and the relative positions of the assemblies, the resulting detection angle (tilt angle of the field of view) of the detection radiation of the detector located in the second section of the detection beam path. This determination of the detection angle can be performed computationally or by using pre-stored data from a database or a look-up table (LUT). The respective resulting detection angles can then be assigned to the measured values ​​acquired by the detector. This assignment of the detection angles allows the acquired measured values ​​to be converted into image values, taking into account the respective assigned detection angle, and thereby correcting errors caused by the different detection angles.A detection angle is understood to be an angle at which an imaginary main beam of a detected beam of detection radiation strikes the detection surface relative to an axis perpendicular to a detection surface of the detector.

[0024] The invention can be used for scanning microscopes in which the illumination radiation is directed onto the sample in the form of a light spot and / or the detection radiation is captured as a point or line.

[0025] In further advantageous embodiments of the invention, the illumination radiation in the scanning microscope can be shaped into a light sheet. A light sheet can be generated by means of a cylindrical lens (static light sheet) and / or by means of a scanning motion of the illumination radiation (dynamic light sheet). A beam-guiding device (scanner) can be provided to generate the scanning motion.

[0026] In addition to generating a light sheet by shaping and / or scanning a Gaussian beam, advantageous embodiments of the invention allow the illumination radiation used to generate the light sheet to be in the form of a Bessel beam, a Mathieu beam, or a Sinc beam. 3 - The beam must be provided and scanned. In this way, unwanted side maxima of the light sheet are reduced or completely avoided.

[0027] The control system mentioned earlier can also be designed to control the relative movement between the second and third assemblies by means of control commands based on an evaluation of the characteristics of the illumination radiation. A controlled relative movement for the purpose of area-by-area scanning of the sample, particularly using a light sheet, is therefore advantageously controlled depending on the illumination radiation (beam type, intensity, wavelength) as well as the current and, if applicable, the future position of the assemblies and components involved in the relative movement.

[0028] The scanning microscope according to the invention enables very high-quality resulting images while simultaneously minimizing mechanical stress on the sample and requiring minimal movement of the microscope itself. Therefore, the scanning microscope is suitable for processing large quantities of samples per unit of time. For use as a high-throughput scanner, the combination of coarse positioning and subsequent precise scanning is advantageous.

[0029] The invention is described in more detail below with reference to exemplary embodiments. These show:

[0030] Fig. 1 shows an embodiment of a scanning microscope according to the invention in a starting position;

[0031] Fig. 2 shows the embodiment of the scanning microscope according to the invention in a first lateral scanning position;

[0032] Fig. 3 shows the embodiment of the scanning microscope according to the invention in a second lateral scanning position;

[0033] Fig. 4 shows the embodiment of the scanning microscope according to the invention in a first vertical scan position; and Fig. 5 shows the embodiment of the scanning microscope according to the invention in a second vertical scan position.

[0034] The following examples of the invention are shown schematically and not to scale. Identical reference numerals refer to identical technical elements.

[0035] A scanning microscope 1 according to the invention comprises a first assembly I with a sample stage 1, wherein the sample stage 1 can accommodate a sample holder 4 on which a sample 5 to be imaged can be placed (Fig. 1). The sample stage 2 is controlled by a drive 3 in an xy-plane and optionally also in the direction of a z-axis of a given Cartesian coordinate system.

[0036] A second assembly II comprises an illumination optic for guiding and shaping an illumination beam and for illuminating the sample 5. The illumination beam is provided by a light source 10, guided into the second assembly II by means of a light-conducting fiber 11, and directed into an illumination beam path along a first optical axis A1. The illumination beam is guided by at least one optical lens 8 onto an illumination objective 7 and directed into the sample 5 by means of the illumination objective 7. The optical lens 8 may also include a beam-shaping optic. Detection radiation coming from the sample 5 is collected by means of a detection objective 9 and guided along a first section 12.1 of a detection beam path (second optical axis A2). The first and second optical axes A1 and A2 intersect at an angle of 90° in a region above the sample holder 4 in the sample 5.Furthermore, the first and second optical axes Al, A2 each run at a non-zero angle to a virtual normal N located on the sample holder 4. The detection objective 9 and optionally other optical elements present in the first section 12.1 (not shown) form a detection optic.

[0037] The first axis Al and the second axis A2 can be directed by a meniscus lens 19 to reduce imaging errors that occur as a result of oblique transmission of illumination and detection radiation through the sample holder 4. Optionally, a medium can be present in a cavity 20 between the meniscus lens 19 and the sample holder 4. The refractive power of this medium is, or can be, adapted to the existing or expected aberrations or optical properties of the sample 5 to be imaged. For this purpose, the medium can be exchanged at intervals or dynamically, for example, by providing a conduit (not shown) for the controlled supply and discharge of the medium. In other embodiments, the meniscus lens 19 can be configured as a spherical segment without a cavity (not shown).

[0038] In a third assembly III, a first deflecting element 13 in the form of a mirror is arranged, onto which the first section 12.1 of the detection beam path is directed. Detection radiation reflected by the first deflecting element 13 travels along a second section 12.2 of the detection beam path and a third optical axis A3 to a second deflecting element 14, from which the detection radiation is directed to an optional filter 15 and a subsequent detector 16. At least one optical lens 8, for example a tube lens 8, is arranged upstream of the second deflecting element 14. The first section 12.1 of the detection beam path is guided from the second assembly II to the third assembly III as a free jet.

[0039] The detector 16 can optionally be connected to an evaluation unit 17, for example a computer, graphics card, or frame grabber, which collects, processes, and optionally stores the measured values ​​acquired by the detector 16. Of importance for the scanning microscope 1 according to the invention is that the second assembly II can be moved relative to the third assembly III in a controlled manner. Furthermore, the first assembly I and / or the sample stage 2 can optionally be moved relative to the second assembly II and optionally relative to the third assembly III, for example, to achieve a rough alignment of the sample 5.

[0040] The second assembly II can be moved by means of a drive 6. Advantageously, drives 3 and 6 are connected to a drive controller 18 in a manner suitable for data transmission. As a result of the execution of control commands from the drive controller 18 by drives 3 and / or 6, the sample stage 2 and / or the second assembly II are moved accordingly, positioned at a specific location, or held in their current position. Movement of the second assembly II can occur in the xy-plane and optionally in the direction of the z-axis (z-direction).

[0041] To control the scanning microscope 1, a further control unit 21 can be provided, whose control commands can be used to control not only the drive control 18, but also, for example, the light source 10 and / or the filter 15 (indicated). The control unit 21 can also be connected to the evaluation unit 17 and generate control commands based on the evaluation results. For example, the control unit 21 can react to the image quality determined during the evaluation of the measurement data by issuing appropriately modified control commands.

[0042] While the first assembly I may optionally be surrounded by a housing (broken solid line), at least the beam paths of the second and third assemblies II and III respectively are surrounded by a preferably light-tight housing (simplified with a frame).

[0043] The illumination radiation can be shaped into a light sheet by arranging a beam-shaping optic, in particular a cylindrical lens, a Powell lens, and / or a beam-directing device (scanner), in the illumination beam path. The illumination radiation is compressed in a direction transverse to the first optical axis Al (cylindrical lens, Powell lens), or the illumination radiation is provided in the form of at least one light beam and scanned in a plane transverse to the first optical axis Al. A cylindrical lens, a Powell lens, and a beam-directing device can also be combined to shape the illumination radiation. In this way, a light sheet can be generated in the region of sample 5 in a plane perpendicular to the drawing plane.In other variations, the shaping of the illumination radiation into a light sheet can also be achieved using optics, which are, for example, arranged at the exit of a light-conducting fiber. Such optics can, for instance, be printed on or attached to a fiber exit point.

[0044] The following describes the relative movements of assemblies I, II, and / or III with reference to the Cartesian coordinate system. For the sake of clarity, directions in the drawing plane to the right, upwards, and into the drawing plane (corresponding to the arrow directions of the x, y, and z axes, respectively) are referred to as positive directions. Opposite directions are referred to as negative directions.

[0045] To image different areas of sample 5, the second assembly II is shifted relative to the first assembly I and the third assembly III. In Fig. 2, the second assembly II is shown in a position that is shifted in the negative direction of the x-axis of a Cartesian coordinate system compared to the initial position (see Fig. 1). The light sheet, symbolized by the intersection of the first optical axis Al and the second optical axis A2, now illuminates a different area of ​​sample 5 than in the initial position. As a result of the offset of the second assembly II relative to the third assembly III, the first section 12.1 of the detection beam path intersects the first deflecting element 13 at a different point in the z-direction than in the initial position.The actual beam path resulting from the offset is shown as a solid line, while the original path of the third optical axis A3 is still shown as a dash-dot line for illustrative purposes.

[0046] The detection radiation reflected at the first deflecting element 13 passes through the tube lens 8, reaches the second deflecting element 14, and is directed by this element at an angle (tilt angle, detection angle) onto the filter 15 and the detector 16. The tilt angle is measured between the third optical axis A3 (dash-dot line) in its original position and the path (center of the beam) of the detection radiation currently incident on the detector 16, and in this case lies in the xz-plane spanned by the x- and z-axes.

[0047] Fig. 3 shows the second assembly II at a lateral position shifted in the positive x-direction. The first section 12.1 now strikes the first deflecting element 13 in a positive z-direction, i.e., higher, so that the path of the reflected detection radiation is located above the original third optical axis A3. The tilt angle of the detection radiation incident on the detector 16 has the opposite sign to the tilt angle in Fig. 2.

[0048] Accordingly, the second assembly II can be shifted in the positive or negative y-direction. This changes, for example, the position of the light sheet in sample 5 in the y-axis direction. Any resulting tilt angle then lies in an xy-plane.

[0049] In a further embodiment of the invention, the distance in the z-direction between the third assembly III and the two assemblies I and II can be increased compared to the initial position (Fig. 4). In this example, no lateral displacement occurs between assemblies I, II, and III. Due to the larger distance in the (positive) z-direction, the first section 12.1 of the detection beam path strikes the first deflecting element 13 in a different direction in the z-direction, corresponding to the direction of displacement. The tilt angle is changed in the xz-plane. Simultaneously, the light sheet is positioned within the sample 5 in the direction of displacement.

[0050] If, however, as shown in Fig. 5, the distance between the second assembly II and the third assembly III is reduced, the light sheet is also displaced in the negative z-direction. The first section 12.1 therefore strikes the first deflecting element 13 in a negative z-direction, is reflected, and strikes the detector 16 at a corresponding tilt angle.

[0051] When using a scanning microscope 1 according to the invention, the relative movements described above can each be generated individually, thus scanning the sample 5 in one direction at a time. In further applications, it is possible to superimpose movements of the second assembly II. For example, a displacement in the positive x-direction and a simultaneous increase in the distance in the positive z-direction could occur. In addition to an oblique scan trajectory in the sample 5, such a superposition of movements also results in the detected radiation striking the detector 16 simultaneously at tilt angles in the xz-plane and the xy-plane.

[0052] For all embodiments of the invention, the evaluation unit 17 and / or the control unit 21 can be configured to check the currently existing tilt angle at which the detection radiation is recorded and / or to take it into account during the evaluation of the measured values ​​and any subsequent image generation. For this purpose, the effects of one or more tilt angles on the measured values ​​and the image data derived therefrom can be computationally compensated. Reference numeral

[0053] 1 scanning microscope

[0054] 1 first assembly

[0055] II second assembly

[0056] III third assembly group

[0057] 2 Sample table

[0058] 3 Drive Sample table

[0059] 4 sample holders

[0060] 5 Sample

[0061] 6 Drive second assembly

[0062] 7 Lighting lens

[0063] 8 optical lens, tube lens

[0064] 9 Detection lens

[0065] 10 light sources

[0066] 11 light-conducting fibers

[0067] 12 Detection beam path

[0068] 12.1 first section

[0069] 12.2 second section

[0070] 13 first deflection element

[0071] 14 second deflection element

[0072] 15 filters

[0073] 16 Detector

[0074] 17 evaluation units

[0075] 18 Drive control

[0076] 19 Meniscus

[0077] 20 cavities, optionally filled with a medium

[0078] 21 Control

[0079] First optical axis

[0080] A2 second optical axis

[0081] A3 third optical axis

[0082] N Normal

Claims

Patent claims 1. Scanning microscope (1) comprising a first assembly (I) comprising a sample stage (2) for holding a sample carrier (4) and a sample (5) placed thereon in a sample chamber; and a second assembly (II) with illumination optics (7, 8) for guiding and shaping illumination radiation and for illuminating the sample (5); with detection optics (9) for collecting and guiding detection radiation coming from the sample (5) along a detection beam path; characterized in that the second assembly (II) has a first section (12.1) of the detection beam path; a third assembly (III) is provided which comprises a second section (12.2) of the detection beam path; and at least one drive (6) is provided to move the second assembly (II) relative to the third assembly (III) in a controlled manner.

2. Scanning microscope (1) according to claim 1, characterized in that the first section (12.1) and the second section (12.2) of the detection beam path are connected to each other via a free jet connection.

3. Scanning microscope (1) according to claim 2, characterized in that the third assembly (III) has a first optical deflecting element (13) by means of which the detection radiation received along the free jet connection is directed along the further course of the second section (12.2).

4. Scanning microscope (1) according to claim 3, characterized in that the first optical deflection element (13) is a flat mirror, a curved mirror or a mirror arrangement of several mirrors.

5. Scanning microscope (1) according to one of the preceding claims, characterized in that the sample stage (2) has at least one drive (3) by means of which the sample carrier (4) can be moved in a controlled manner.

6. Scanning microscope (1) according to one of the preceding claims, characterized by an evaluation unit (17) configured to to determine, based on current position data of the sample carrier (4) and the second and third assembly (II, III) relative to each other, a resulting detection angle of the detection radiation of a detector (16) arranged in the second section (12.2) of the detection beam path, and to assign the respective resulting detection angle to the measured values ​​acquired by means of the detector (16).

7. Scanning microscope (1) according to claim 6, characterized in that the evaluation unit (17) is configured to convert the measured values ​​into image values ​​taking into account the respective associated detection angle.

8. Scanning microscope (1) according to one of the preceding claims, characterized in that the illumination radiation is shaped into a light sheet.

9. Scanning microscope (1) according to claim 8, characterized in that the light sheet is generated by means of a beam-directing device with a scanning movement of the illumination radiation.

10. Scanning microscope (1) according to claim 9, characterized in that the illumination radiation to be scanned for generating the light sheet is in the form of a Bessel beam, a Mathieu beam or a Sinc beam. 3 - Beams are provided.

11. Scanning microscope (1) according to one of the preceding claims, characterized in that a control unit (21) is provided, by whose control commands the relative movement between the second assembly (II) and the third assembly (III) as well as between the second assembly (II) and the first assembly (I) is controlled by an evaluation of the shape of the illumination radiation.

12. Use of a scanning microscope (1) according to any of the preceding claims as a high-throughput scanner.

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