Method and apparatus for illuminating a sample surface with wide-field illumination.
Patent Information
- Application Number
- JP2026030365
- Authority / Receiving Office
- JP · JP
- Patent Type
- Applications
- Current Assignee / Owner
- Priority Date
- 2025-02-27
- Filing Date
- 2026-02-27
- Publication Date
- 2026-09-08
Smart Images
Figure 2026143381000001_ABST
Abstract
Description
[[TECHNICAL FIELD]]
[0001] The present invention relates to a method and an apparatus for illuminating a sample surface with wide-field illumination according to the preamble of an independent claim. [[BACKGROUND ART]]
[0002] In the field of microscopes, various possibilities are known for illuminating a sample to be imaged. In addition to other possibilities, instead of point-shaped or linear scanning, a planar illumination or a planar illumination pattern within the sample plane can be used to irradiate the sample, which is referred to as wide-field illumination (wide field). In wide field, the illumination pattern is generated above or below the sample surface substantially transversely to the illumination axis (optical axis).
[0003] Here, wide-field illumination also refers to illumination patterns that do not extend transversely to the optical axis but are inclined with respect to the optical axis. Examples of such inclined illumination patterns include so-called HILO illumination (HILO = highly inclined and laminated optical sheet) or TIRF illumination (TIRF = total internal reflection fluorescence).
[0004] The use of illumination beams, in particular laser beams, with different characteristics is known from the prior art. When single-mode light is used, the beam has a Gaussian beam profile. This light exhibits high spatial coherence. Thereby, the single-mode light can be focused to the diffraction limit. However, under a microscope, the high spatial coherence of light causes non-uniformity in the beam profile, which can be caused, for example, by interference and / or existing contamination and / or multiple reflections that occur.
[0005] In contrast, light having multiple spatial modes (multimodes) can be used for illumination. Such multimode light is guided by a suitable waveguide, such as a multimode fiber, and is obtained, for example, by a multimode laser diode.
[0006] In a waveguide, its shape determines which modes can be guided within it. Different excitation modes of an optical fiber are added to the ends of the optical fiber. If the coherence length of the individual modes is sufficiently long, and based on the coherence of the modes, the light interferes at the ends of the waveguide, resulting in an undesirable non-uniform illumination pattern ("speckle pattern") during wide-field illumination. Therefore, to obtain uniform illumination of a sample, the light after a multimode fiber must be influenced so that the light distribution is as uniform as possible. Uniformization of the light distribution ("speckle removal") can be achieved, in particular, by averaging the illumination pattern, which is initially non-uniform and changes over time. Such temporal changes in the illumination pattern can be achieved, for example, by repeatedly moving the diffuser before coupling the illumination beam into the fiber (e.g., Optotune Switzerland AG, Schweiz, (https: / / www.optotune.com / laser-speckle-reducers) or generated by moving an optical conduction fiber (e.g., Molex LLC, USA, https: / / www.molex.com / en-us / products / optical-solutions / optical-fiber / de-speckler-system; and Schroeder, D.) et al., 2020, Cost-efficient open source laser engine for Microscopy. OPTICS EXPRESS 11 (2): 609 - 623; Kwakwa K., et al. 2016, easySTORM: a robust, lower-cost approach to localization and TIRF microscopy. J. Biophotonics 9 (9): 948-957).
[0007] For further possibilities regarding providing a uniformly illuminated field of view of objects, see Mau et al. 2021. Fast widefield scan provides tunable and uniform illumination optimizing super-resolution microscopy on large fields, Nature Communications 12.3077 This is described at doi.org / 10.1038 / s41467-021-23405-4). Using a scanner, the illumination beam is scanned along a predetermined pattern, and therefore, on the illuminated sample surface, the illumination spot is systematically guided across the sample surface, resulting in illumination with an approximate top-hat distribution. Mau et al. mimic wide-field illumination by rapidly scanning the sample surface.
[0008] Non-uniform illumination patterns, particularly speckle, occur when using both single-mode and multi-mode light, despite known measures to reduce them. These undesirable effects on the illumination pattern impair the quality of the illumination without additional measures, and therefore, the quality of the acquired image data as well. [Overview of the initiative]
[0009] The object of the present invention is to propose at least one further possibility for reducing undesirable non-uniformity of illumination patterns in a wide field of view.
[0010] This problem is achieved by methods and apparatus as described in the independent and dependent claims. The advantageous development is the subject matter of the dependent claim.
[0011] A method for illuminating a sample surface with wide-field illumination involves the following steps: First, an illumination beam, at least partially coherent, in particular a laser beam, is provided. This is guided along the illumination beam path and focused onto the pupil of the illumination objective lens. As a result, a wide-field illumination pattern is generated on the sample surface. The illumination beam path contains an optical element that can move in a controlled manner. This controlled movement (Die gesteuerte Bewegung) serves to repeatedly change the state of the illumination pattern formation (die Ausbildung) on the sample surface in order to homogenize the wide-field illumination.
[0012] The method according to the present invention is characterized in that a scanner is used as an optical element to generate different formation states of illumination patterns, and the scanner swings or vibrates (schwingt) in at least one direction around a set position (um eine eingestellte Positionierung) to repeatedly change the position or illumination angle of the illumination pattern on the sample surface in at least one direction for the purpose of homogenization, also referred to here as speckle removal. The scanner may be a galvanometer scanner or a MEMS scanner. A mirror or glass plate that moves accordingly can be used.
[0013] Repeated changes in the lighting pattern (der wiederholten Veraenderung), or oscillation around a predetermined position (des The term Schwingens um eine Positionierung (periodischen) includes the term periodic oscillation. In addition to (swinging), random movements (zufaellige Bewegungen) and movements in only one direction should also be understood. Random movements can be caused, for example, by "shaking" the component used as the first scanner. This means that the lighting pattern is changed multiple times, and can also be said to change over time.
[0014] Within the framework of this invention, the entire illumination pattern is modified. For example, unlike generating an illumination pattern by scanning the sample surface to be illuminated, the framework of this invention generates and modifies the entire wide-field illumination pattern. In contrast to conventional methods that vibrate the optical fiber to reduce speckle, this invention causes movement of the illumination beam within the pupil of the objective lens, thereby generating a change in the illumination pattern on the sample surface. In this way, non-uniformity due to technical constraints that occur only after the fiber can also be reduced.
[0015] The core of this invention is to slightly alter the illumination pattern within the sample surface around a desired position while maintaining the average position of the illumination pattern. Unlike the prior art, the illumination beam reaching the pupil of the lens changes in a controlled manner. As briefly described below, altering the illumination pattern can be achieved by changing its position within the sample surface or by changing the illumination angle at which the illumination pattern is directed towards the sample surface.
[0016] In the first configuration of the present invention, the scanner is positioned in a plane optically conjugate to the pupil of the objective lens and receives a focused illumination beam. Next, a focal spot of the illumination beam is generated in the pupil of the lens. Here, if the scanner, hereinafter also referred to as the first scanner, performs an oscillating motion or swinging movement around a selected position, i.e., the center position of the scanner, the angle of incidence of the illumination beam to the pupil of the objective lens changes accordingly. In this case, the position of the focal spot in the pupil of the objective lens does not change. Because the angle of incidence changes, the illumination beam is projected onto the sample surface by the illumination objective lens at positions that are proportionally offset from each other.
[0017] In a further embodiment of the present invention, to change the incident position of the focal spot in the pupil of the objective lens as needed, a further optical element for controlling and changing the beam position of the illumination beam may be provided on an intermediate image plane, i.e., a plane conjugate to the focal plane or sample plane of the illumination objective lens. The further optical element can set or adjust the lateral position of the focal point of the illumination beam generated in the pupil of the objective lens.
[0018] A second method for changing the illumination pattern involves positioning the first scanner on a plane conjugate to the focal plane of the objective lens (the intermediate image plane) and directing the collimated illumination beam onto it. As the first scanner moves at a small deflection angle, the position where the focal spot is directed towards the pupil of the objective lens changes. This changes the illumination angle at which the illumination pattern is generated on the sample surface, depending on the incident position (Auftreffort) of the focal spot. In addition to oscillating around a selected position, the first scanner, thus positioned within the illumination beam path, can also be used to set the incident position of the focal spot in the pupil of the objective lens as desired.
[0019] In a further embodiment of the present invention, the first scanner can be optically shifted from a plane optically conjugate to the pupil of the objective lens to a plane optically conjugate to the sample surface, which is achieved, for example, by inserting a Bertrand lens into the illumination beam path of a laser scanning microscope (LSM) mounted on a wide-field microscope stand, and in front of the illumination objective lens. In this way, the scanning mirror of the first scanner is optically conjugate to the sample surface. This creates a laser focus on the pupil of the objective lens, allowing the lens to be scanned through the LSM. The LSM remains as an unmodified module on the stand, and the confocal image and wide-field laser illumination can be switched by switching the Bertrand lens.
[0020] To adequately "blur" the non-uniformity of the illumination pattern, the illumination pattern within the sample plane is changed multiple times within the integration time of the detector used for image acquisition. If the changes are performed in steps, for example, at least three positional changes or at least three changes in illumination angle may occur within the integration time of the detector. Advantageously, continuous changes in the illumination pattern do not interrupt positional changes.
[0021] The positional shift of the generated illumination pattern on the sample surface is at most one-fifth, preferably at most one-tenth, and more preferably at most one-twentieth, in at least one of the multiple (lateral) spreads (Ausdehnungen) of the illumination pattern on the sample surface. The positional shift depends, for example, on the type and size of the optical fiber, the quality of the optical coupling, and the type and degree of contamination in the illumination beam path. Within the scope of the present invention, the illumination pattern is shifted by a distance smaller than the spread of the current field of view (FOV).
[0022] In further embodiments of the present invention, the position change or step size per integration time of the detector can be based on the average diameter of existing or expected speckles. For example, the step size may be 20% of the average diameter. Alternatively, the number of position changes can be specified for each integration time. Optionally, the value can be determined based on the average diameter of the speckles. For example, it can be set to cross at least one average speckle diameter within each integration time. In further embodiments, per integration time, a distance equivalent to at least 1.5 times, 2 times, 3 times, or more of the average speckle diameter can be crossed.
[0023] Especially when the illumination pattern changes continuously, such as when using a scanner, the position change, particularly the amplitude of the oscillation, and the frequency of the position change can be selected to achieve sufficient uniformity. For example, the frequency may be half the frame rate of the detector used, in which case the amplitude passes completely once within the integration time.
[0024] In a further embodiment of the present invention, in order to avoid acquiring the same position or illumination angle of the illumination pattern multiple times when the first scanner oscillates during the integration time of the detector, the movement performed to change the illumination pattern can be changed within the integration time of the detector. For example, the first scanner can be driven and controlled such that the oscillating motion within the integration time of the detector is not temporally linear, but correspondingly different positions or illumination angles are set. The oscillating motion can also be performed along a curve instead of a straight line within the integration time, for example, with deflection in the x direction and the y direction. To this end, the first scanner can be controlled in a first direction and a second direction transverse to the illumination beam path around a set position, wherein the first direction and the second direction are different from each other. The change of the illumination pattern can also be performed randomly, that is, the first scanner can be driven and controlled randomly with respect to the change of the incident angle or the change of the position of the focal spot caused thereby.
[0025] One possible embodiment of the method according to the present invention can be described as follows. For example, in order to draw a circle in the objective lens pupil by means of the illumination beam, a cosine waveform can be applied to one mirror of the scanning device, and a sine waveform can be applied to the second mirror. The angle difference in the sample plane is determined by the amplitude of the waveform. For example, if image acquisition is performed with an integration time of 10 ms, this corresponds to a frequency of 100 Hz. If the result of speckle removal is not satisfactory, this process can be repeated with two further amplitude values.
[0026] The change of the illumination pattern can also be performed only in one direction within one integration time, so it can advantageously avoid acquiring the same position or the same illumination angle multiple times. However, in this case, undesirable fringes may occur in the illumination pattern.
[0027] The possibility disclosed by the present invention of controlling and changing the focus position in the objective lens pupil can advantageously be used to illuminate the sample surface with different types of illumination patterns.
[0028] When the focal point is substantially at the center of the objective lens pupil, the illumination objective lens produces a wide-field illumination directed planarly onto the sample surface. In contrast, when the focused illumination beam (focal spot) is directed to a region off-axis from the optical axis of the objective lens pupil, the illumination beam is incident on the sample surface as gradient illumination. This effect is used, for example, in HILO illumination. The gradient increases as the focal spot shifts toward the periphery of the objective lens pupil. Above or outside a certain radial position (Ab einer bestimmten radialen Position), the illumination angle of the illumination beam becomes large enough to cause total internal reflection of the illumination beam at the surface of the sample on the sample surface. This effect depends on the wavelength of the illumination beam and the optical properties of the illumination objective lens and the sample. The resulting total internal reflection can be used in TIRF imaging methods.
[0029] For focal spots located outside the optical axis, both the radial and azimuthal positions affect the specific formation of the illumination pattern. For example, if the focal spot is shifted along a circular path of the same radial distance from the optical axis, the illumination inclination does not change, but the direction of illumination changes depending on each position (azimuth angle) along the circular path. Therefore, a sample can be illuminated not only from different illumination angles but also from different directions. For example, by shifting the focal spot along a circular path at the periphery of the pupil of the objective lens and acquiring images of the sample at different azimuth angles, it is possible to obtain a so-called ring TIRF image of the sample.
[0030] The object of the present invention is further achieved by an apparatus for illuminating a sample surface with wide-field illumination. The apparatus comprises an illumination beam path for guiding an illumination beam and an illumination objective lens for generating an illumination pattern of wide-field illumination on the sample surface. At least one movable optical element is provided within the illumination beam path, i.e., it can be oscillated around a selected position. The movement of the optical element is used to repeatedly change the formation state of the illumination pattern on the sample surface in order to homogenize the wide-field illumination. The term oscillating around a position (Schwingens um eine Position) includes not only linear motion but also movement in directions that overlap each other, i.e., movement within a plane.
[0031] A feature of the apparatus according to the present invention is that the optical element is a first scanner, such as a galvanometer scanner, a resonant scanner, or a MEMS scanner. A correspondingly movable mirror or glass plate can also be used. Furthermore, a controller is provided, and the scanner is driven and controlled using its control commands. The controller may be, for example, a computer, a microcontroller, or an FPGA (field-programmable gate array). The first scanner oscillates around a set position in at least one direction during the execution of the control command. This movement serves to repeatedly change the position or illumination angle of the illumination pattern on the sample surface in at least one direction.
[0032] In embodiments of the apparatus according to the present invention, the first scanner is positioned in a plane substantially conjugate to the pupil of the objective lens, and a slight deviation in the precise axial position may be tolerated. As already described above, the angle of incidence of the illumination beam to the pupil of the objective lens can be changed by moving the first scanner.
[0033] In a further embodiment of the present invention, an additional optical element for controlling and changing the beam position is positioned downstream of the scanner in the illumination beam path, on a plane (intermediate image plane) conjugate to the sample plane (focal plane of the objective lens). The position of the focal spot of the illumination beam generated in the pupil of the objective lens can be set by the action of the additional optical element. Advantageously, the position of the focal spot can be set by the additional optical element in the x and y directions, both of which are perpendicular to the optical axis (z direction) and perpendicular to each other.
[0034] Further optical elements may include, for example, further scanners, mirrors, or devices that generate a parallel offset of the illumination beam, such as a planar plate that is transparent to the wavelength of the illumination beam but has a refractive effect. If the position of the focal spot can be dynamically adjusted, further optical elements may be connected to a drive that can be driven.
[0035] In a further embodiment of the apparatus, a Bertrand lens can be optionally inserted in front of the illumination objective lens. This positions the first scanner optically in an intermediate image plane conjugate to the focal plane of the illumination objective lens, i.e., the sample plane. For example, if the illumination beam path is that of a laser scanning microscope (LSM), a Bertrand lens inserted into the illumination beam path as needed allows switching between confocal imaging and (laser) wide-field illumination. The LSM can be maintained without any other technical modifications. In a configuration where the scanner is positioned in the intermediate image plane, the first scanner can position the focal spot within the pupil of the objective lens.
[0036] A detector, which plays a role in capturing the detection beam generated on the sample surface, can be connected to a controller in a manner suitable for data transmission. The controller, or an evaluation unit connected to it, such as a computer, can be configured to evaluate the measurements acquired by the detector as image data. In addition to image acquisition, the measurements can be evaluated to, as a result, initiate or adjust the drive control of the driver or further drivers as needed.
[0037] The apparatus according to the present invention may be part of a microscope such as an LSM. In advantageous embodiments, the position of the illumination pattern within the sample surface is changed multiple times within the integration time of the detector used for image acquisition. In particular, an illumination beam provided in the form of a laser beam can generate a detection beam within the sample, for example by exciting a suitable fluorescent marker in the sample to emit a fluorescent beam. Additionally or alternatively, the autofluorescence of components of the sample can also be stimulated and / or used. [Brief explanation of the drawing]
[0038] The present invention will be described in more detail below with reference to the drawings. [Figure 1] Figure 1 is a schematic diagram of a first embodiment of the apparatus according to the present invention, a top view of the pupil of the objective lens, and a schematic diagram of the beam path of the illumination beam within the pupil of the objective lens and within the sample surface. [Figure 2] Figure 2 is a schematic diagram of a second embodiment of the apparatus according to the present invention having a further beam deflection optical element in the first operating mode, a top view of the objective lens pupil, and a schematic diagram of the beam path of the illumination beam in the objective lens pupil and on the sample surface. [Figure 3] Figure 3 is a schematic diagram of a second embodiment of the apparatus according to the present invention having a further beam deflection optical element in the second operating mode, a top view of the objective lens pupil, and a schematic diagram of the beam path of the illumination beam in the objective lens pupil and on the sample surface. [Figure 4] Figure 4 shows a schematic diagram of one embodiment of a conventional laser scanning microscope, a top view of the objective lens pupil, and a schematic diagram of the beam path of the illumination beam within the objective lens pupil and on the sample surface. [Figure 5] Figure 5 shows a schematic diagram of a third embodiment of the apparatus according to the present invention, which includes a movable Bertrand lens that is not currently positioned in the illumination light path, a top view of the objective lens pupil, and a schematic diagram of the beam path of the illumination beam within the objective lens pupil and the sample surface. [Figure 6]Figure 6 is a schematic diagram of a third embodiment of the apparatus according to the present invention, which includes a movable Bertrand lens currently positioned in the illumination light path, a top view of the objective lens pupil, and a schematic diagram of the beam path of the illumination beam within the objective lens pupil and the sample surface.
[0039] The drawings illustrate the present invention in a simplified form. The same reference numerals indicate the same technical elements. [Modes for carrying out the invention]
[0040] In a first embodiment of the apparatus 2 according to the present invention, apparatus 2 is a component of a microscope 1, such as a laser wide-field microscope. Apparatus 2 may also be a component of a unit (module, apparatus) for laser wide-field illumination. An illumination beam provided by a light source (not shown) is coupled to an optical fiber 5, which can be designed as a single-mode fiber or a multimode fiber. The end of the fiber 5 is located on a planar PB that is optically conjugate to the pupil plane PB (objective lens pupil 13, back focal plane) of the illumination objective lens 10. The illumination beam emitted from the fiber 5 is guided or directed along the optical axis 4 of the illumination beam path 3. In doing so, the illumination beam reaches and is focused on a first scanner 7 located on the planar PB via an optical lens 6. The (pupil) planar PB of the first scanner 7 is optically conjugate to the objective lens pupil 13 of the illumination objective lens 10. The first scanner 7 is connected to a driver 12 and can be moved in a controlled manner. The illumination beam path 3 is followed by the first scanner 7, comprising a scanning lens 8, a cylindrical lens 9, and an illumination objective lens 10. Further designs of the present invention may optionally include additional optical elements. The driver 12 of the first scanner 7 is controlled by control commands from the controller 11.
[0041] The effects of the present invention are shown in the first partial image on the right side of the illumination beam path 3. The image shows a top view of the entrance pupil of the illumination objective lens 10, i.e., the objective lens 13, along the optical axis 4. Due to the action of the optically effective elements in the illumination beam path 3, the illumination beam is directed towards a focal spot 14 at the center of the objective lens pupil 13. According to the present invention, at least one of the incident angles of the illumination beam to the fixed focal spot 14 is changed (indicated by a double arrow).
[0042] The second partial image shows the illumination objective lens 10 that receives the illumination beam (incident illumination beam 15.1) along the optical axis 4. The illumination beam is directed planarly (flaechig) to the sample surface 17 as an outgoing illumination beam 15.2 by the action of the illumination objective lens 10. Optionally, the sample 16 to be imaged is placed within this beam. Image data of the illuminated sample 16 can be acquired using a detector 18 positioned in the detection beam path, shown merely as an example, which can be associated with the microscope 1 or apparatus 2 in various configurations.
[0043] For example, the detection beam emitted from the sample 16 can be collected by the illumination objective lens 10. For example, by using a (primary) color separator (Farbteiler) (not shown), the illumination beam and the detection beam can be separated based on their different wavelengths and then guided to the detector 18. The detection beam from a sample receiving particularly wide-field illumination can also be collected using a separate detection objective lens (not shown). The controlled movement of the first scanner 7 is advantageously sized so that the illumination pattern on the sample surface 17 changes multiple times during the integration time of the detector 18.
[0044] The detector 18 can optionally be connected to the controller 11. Measurement values acquired by the detector 18 can be evaluated as image data by an evaluation unit (not shown). In addition to image acquisition, the measurement values can be evaluated to initiate or adapt the drive control of the drive 12 or any additional driver 12 (see also Figures 2, 3, 5, and 6) as needed.
[0045] In a second embodiment of the apparatus 2 according to the present invention, a further scanner, particularly a further beam deflection optical element 19 in the form of an xy scanner, is positioned in a plane conjugate to the intermediate image plane ZB, i.e., the sample plane 17. The further optical element 19 is adjustable via a driver 12 and a controller 11. While the first scanner 7 generates the movement of the illumination beam described above, the position of the focal spot 14 in the objective lens pupil 13 can be set by the further optical element 19 cooperating with the cylindrical lens 9.
[0046] In a further design of the present invention, the additional optical element 19 can be positioned and designed such that the corresponding parallel offset of the illumination beam is brought into the pupil 13 of the objective lens.
[0047] In Figure 2, a partial view shows a focal spot 14 (e.g., azimuth angle 270°) or incident illumination beam 15.1 shifted to the left from the observer's viewpoint. The position away from the optical axis 4 results in an inclination of the exit illumination beam 15.2. This configuration of the illumination beam path 3 can generate so-called HILO illumination. In the first partial view, the area inside the objective lens pupil 13 is exemplified by a dashed circle, within which the incident illumination light 15.1 either does not produce an exit illumination light 15.2 (along the optical axis 4) or only a slightly inclined exit illumination light 15.2, neither of which causes total internal reflection in the sample 16.
[0048] On the other hand, as shown in Figure 3, when the focal spot 14 is located within the outer ring, the emitted illumination beam 15.2 strikes the sample 16 at a shallow angle, as measured between the emitted illumination beam 15.2 and the sample surface 17, and is therefore totally internally reflected. As known from the prior art, a so-called evanescent field is generated within the sample 16. In this way, thin regions of the sample 16 can be illuminated or excited by a detection beam, particularly a fluorescence beam, despite the total internal reflection.
[0049] In the modified illumination configurations shown in Figures 2 and 3, such as HILO or TIRF, the radial distance of the focal spot 14 to the optical axis 4 determines the illumination angle, i.e., the inclination of the emitted illumination beam 15.2, and the azimuth angle of the focal spot 14 determines the direction of the emitted illumination beam 15.2.
[0050] Therefore, by carrying out the present invention according to any example, it is possible to generate HILO illumination or TIRF illumination from different directions. For example, so-called ring TIRF illumination can be generated by guiding the focal spot 14 along the outer annular ring at least once.
[0051] When illumination is implemented using the TIRF-SIM (TIRF with structured illumination) principle, the structure of the illumination pattern can be generated, for example, by using a static grid or a spatial light modulator (SLM). In this case, the diffraction order of both the static grid and the grid generated by the SLM must be located at the edge of the objective lens pupil 13 of the illumination objective lens 10 having a high numerical aperture NA > 1.4.
[0052] To describe a third embodiment of the present invention, Figure 4 first briefly illustrates a prior art LSM in confocal operation. The end of the optical fiber 5 lies in a planar ZB conjugate to the intermediate image plane and guides the illumination beam into the illumination beam path 3. The fiber 5 is preferably implemented as a single-mode fiber so that its beam can generate a diffraction-limited focal spot 14. The illumination beam reaches the objective lens pupil 13 as described in relation to Figure 1, but the objective lens pupil does not move around a position (um eine Position) in order to reduce non-uniformity of the illumination pattern. The illumination beam irradiates the center of the objective lens pupil 13 and over most of the objective lens pupil 13. The emitted illumination beam 15.2 is focused to a point in the sample surface 17. In an LSM, a single-mode fiber is usually used because its light can be focused to the sample surface 17 in a diffraction-limited spot. The scanner 7 plays a role in generating the scanning motion (Scanbewegung) of the point in the sample surface 17.
[0053] In the LSM1 according to the present invention, a Bertrand lens 20 is provided between the intermediate image plane ZB and the illumination objective lens objective 10, and the Bertrand lens 20 is advantageously operable both inside and outside the illumination beam path 3 (Figure 5). To produce this feed movement, the Bertrand lens 20 is connected to a driver 12, which is further controlled and driven by a control device 11. When the Bertrand lens 20 is located outside the illumination beam path 3, the LSM can be used as a confocal microscope, as described with respect to Figure 4. A color separator (Farbteiler) can be placed downstream of the objective lens 10 and before the intermediate image plane ZB. Additionally or alternatively, a color separator can be provided upstream of the beam of the first scanner 7 to separate the collected detection beam from the illumination beam and send it to the detector 18.
[0054] On the other hand, when the Bertrand lens 20 is positioned in the illumination beam path 3 (Figure 6), the first scanner 7 is positioned within the intermediate image plane ZB. In this configuration, the focal spot 14 generated within the objective lens pupil 13 generates both wide-field illumination as the emitted illumination beam 15.2 and a wide-field ("planar") illumination pattern within the sample surface 17. The first scanner 7 is controlled to cause movement around the position of the focal spot 14, thereby changing the illumination angle on the sample surface 17. Furthermore, the position of the focal spot 14 can also be set using the first scanner 7. Thus, this configuration can be used to generate the same functionality as the second scanner 19 of the present invention described in relation to Figures 3 and 4. A third embodiment of the present invention allows the LSM to be switched between confocal illumination and laser wide-field illumination. [Explanation of symbols]
[0055] 1 LSM 2 equipment 3. Illumination beam path 4 Optical axis 5 Optical Fiber 6 Optical Lenses 7. First Scanner 8 scanning lenses 9. Cylindrical lens 10 Illumination objective lens 11 Controllers 12 drivers 13 Objective lens pupil 14 Focus Spots 15.1 Incident illumination beam 15.2 Emitting illumination beam, illumination pattern 16 samples 17. Sample surface, focal plane of illumination objective lens 10. 18 detectors 19. Optical elements, second scanner 20 Bertrand Lens ZB Intermediate Image Plane PB Pupil plane
Claims
1. A method for illuminating a sample surface (17) with wide-field illumination, Steps include providing a lighting beam, The steps include guiding the illumination beam along the illumination beam path (3), A step of focusing the illumination beam onto the pupil (13) of an illumination objective lens (10) to generate a wide-field illumination pattern on the sample surface (17), comprising the step of repeatedly changing the formation state of the illumination pattern on the sample surface (17) by moving at least one optical element of the illumination beam path for the purpose of homogenizing the wide-field illumination, A method wherein a first scanner (7) is used as an optical element to generate different formation states of the illumination pattern, and the first scanner (7) swings in at least one direction around a set position in order to repeatedly change the position or illumination angle of the illumination pattern within the sample surface (17) in at least one direction for the purpose of homogenization.
2. The method according to claim 1, wherein the first scanner (7) is positioned in a plane (PB) conjugate to the pupil (13) of the objective lens and is illuminated by a focused illumination beam.
3. The method according to claim 1, wherein the first scanner (7) is positioned in a plane (ZB) conjugate to the sample surface (17) of the illumination objective lens, and is illuminated with a collimated illumination beam.
4. The method according to any one of claims 1 to 3, wherein the illumination pattern within the sample surface (17) is changed multiple times within the integration time of the detector (18) used for image acquisition.
5. The method according to claim 4, wherein the movement performed to change the illumination pattern is not repeated identically within the integration time of the detector (18).
6. The method according to any one of claims 1 to 5, wherein the change in the position where the illumination pattern is generated on the sample surface (17) is one-fifth or less, one-tenth or less, more preferably one-twentieth or less, in at least one of the plurality of lateral spreads of the illumination pattern on the sample surface (17).
7. The method according to any one of claims 1 to 6, wherein the first scanner (7) is controlled in a first and second direction that crosses the illumination beam path (3) around a set position, and the first and second directions are different from each other.
8. The method according to any one of claims 1 to 7, wherein the position of the generated focal spot (14) of the illumination beam in the pupil (13) is set by a further optical element (19) that controls and influences the beam position.
9. The method according to claim 8, wherein the position of the focal spot (14) within the objective lens pupil (13) is set to the central region, the inner ring region, or the outer ring region of the objective lens pupil, thereby selectively setting the illumination pattern to wide-field illumination directed planarly to the sample surface (17), inclined illumination, or illumination based on the principle of internal total internal reflection, and the illumination of individual regions of the objective lens pupil (13) results in differences in the angular position of the illumination beam radiated onto the sample surface (17).
10. The method according to any one of claims 1 to 9, wherein a sample (16) is placed within the sample surface (17), a detection beam generated within the sample (16) by the illumination beam is acquired as a measurement value by a detector (18), the acquired measurement value is evaluated, and drive control of at least the driver (12) of the first scanner (7) is started or applied.
11. A device (2) for illuminating a sample surface (17) with wide-field illumination, A lighting beam path (3) that guides the lighting beam, An illumination objective lens (10) that shapes the illumination beam and generates an illumination pattern for wide-field illumination on the sample surface (17), For the purpose of homogenizing wide-field illumination, the system includes at least one optical element configured to be movable so as to repeatedly change the state of the illumination pattern formation on the sample surface (17), The optical element is a first scanner (7), in particular a galvanometer scanner, a MEMS scanner, a resonant scanner, a movable mirror, or a movable glass plate. The apparatus includes a controller (11) that drives and controls the first scanner (7) by control commands, wherein the first scanner (7) swings in at least one direction around a set position to repeatedly change the position or illumination angle of the illumination pattern on the sample surface (17) in at least one direction during the execution of the control commands.
12. The apparatus according to claim 11, wherein a further optical element (19) that controls the beam position is positioned downstream of the first scanner (7) in the illumination beam path (3), on a conjugate plane ZB with respect to the sample surface (17), and by its action, the position of the focal spot (14) generated by the illumination beam in the pupil (13) can be set.
13. The apparatus according to claim 12, wherein the further apparatus is a further scanner, mirror, or apparatus for parallel offsetting the illumination beam.
14. The apparatus according to any one of claims 11 to 13, wherein a Bertrand lens can be introduced into the illumination beam path upstream of the illumination objective lens, and as a result, the first scanner (7) is positioned in a plane optically conjugate to the sample surface (17).
15. A microscope (1) comprising the apparatus according to any one of claims 11 to 14.