Method and microscope for sim microscopy

Abstract

The invention relates to a method for high-resolution SIM microscopic imaging of a sample (4), comprising illuminating the sample (4) with a periodic illumination pattern (32) designed as a grating structure composed of bright spots and corresponding to a diffraction order, imaging the sample (4) along an optical axis onto a detector receiving radiation, wherein n original images of the sample (4) are taken by repeated illumination and imaging, wherein for each of the n original images a specific lateral position of the illumination pattern (32) is used in a sample plane perpendicular to the optical axis, and generating a high-resolution wide-field image from the n original images, wherein for each of the n original images (32) during the receiving of radiation the illumination pattern (32) is moved along the optical axis by an axial period length of the illumination pattern or a multiple of this axial period length relative to the sample (4).

Description

Technical Field

[0001] This invention relates to a method for high-resolution SIM microscopy imaging of a sample, wherein the method comprises: illuminating the sample with a three-dimensional periodic illumination pattern, the illumination pattern being designed as a grating structure composed of bright spots and corresponding to diffraction orders; imaging the sample along an optical axis onto a detector that receives radiation, wherein n raw images of the sample are captured by repeated illumination and imaging, wherein for each of the n raw images, the illumination pattern is used at a specific position in a sample plane perpendicular to the optical axis; and generating a high-resolution wide-field image from these n raw images.

[0002] The present invention also relates to a microscope for high-resolution sample SIM microscopy imaging, comprising: an illumination device for illuminating the sample with a three-dimensional periodic illumination pattern designed as a grating structure composed of bright spots and corresponding to diffraction orders; an imaging device for imaging the sample along an optical axis onto a detector receiving radiation; and a control device that manipulates the illumination device and the detector to acquire n original images of the sample by repeated illumination and imaging, wherein a specific location of the illumination pattern is used for each of the n original images in a sample plane perpendicular to the optical axis; and an evaluation device optionally designed as part of the control device for generating a high-resolution wide-field image from the n original images. Background Technology

[0003] This method or microscope uses the technique described below in lattice SIM, a design known as "Structured Illumination Microscopy," or SIM for short. SIM technology has become a mature microscopic imaging method. This microscopic imaging method is primarily based on R. Heintzmann's paper "Saturated patterned excitation microscopy with two-dimensional excitation patterns" (Micron 34 (6-7), 283-291 (2003)) and M. Gustafsson et al.'s paper "Three-Dimensional Resolution Doubling in Wide-Field Fluorescence Microscopy by Structured Illumination" (Biophysical Journal 94, 4957–4970 (2008)), and utilizes SIM illumination patterns to illuminate the sample. The sample is repeatedly imaged, with the illumination pattern being positioned at different lateral distances from the sample, and a so-called raw image is acquired for each such illumination state. These original images are then combined during image processing to obtain an improved image of the sample, with a higher resolution, particularly in depth, than a standard wide-field image taken using the same microscope. This improvement is called "high resolution" because it surpasses the resolution achievable with conventional wide-field microscopy.

[0004] The illumination patterns in SIM (Signal Imaging System) are regular and periodic. Each illumination pattern at a specific location is identical in structure, differing only in its position relative to the sample. Initially, periodic stripe patterns were used for illumination, which were then translated and rotated. This technique is now known as 2D SIM. Improvements have been made to increase depth resolution, hence the term 3D SIM.

[0005] By using a multidimensional periodic SIM illumination pattern composed of bright spots (typically in the form of square or hexagonal gratings), the acquisition speed of raw images can be significantly accelerated. This pattern can be generated by multiple interfering beams arranged in a two-dimensional point-symmetric manner within the illumination pupil along the illumination optical path. This technique is called LatticeSIM. The SIM illumination pattern here is generated from the interference pattern produced by the beam splitting. The SIM illumination pattern contains discrete illumination pattern frequencies, which originate from the interference probabilities of the beam splitting. These discrete spatial illumination pattern frequencies correspond to the interference between the grating diffraction orders (zeroth order and higher) and themselves, as well as with each other. Lattice SIM is achieved using the methods and microscopes described above, as detailed in the article "Introduction to Lattice SIM for Structured Illumination Microscopy for ZEISS Elyra 7 for Live Cell Imaging" by J. Siebenmorgen et al., published in the December 2018 ZEISS Technology Note, available at https: / / asset-downloads.zeiss.com / catalogs / download / mic / b05157e2-bcd6-4c82-b9f9-7639b446a74a / EN_WP_Introducing_Lattice_SIM_for_Elyra-7.pdf (Introducing Lattice SIM for ZEISS Elyra 7 - Structured Illumination Microscopy with a 3D Lattice for Live Cell Imaging, ZEISS Technology note, Dezember 2018). (https: / / asset-downloads.zeiss.com / catalogs / download / mic / b05157e2-bcd6-4c82-b9f9-7639b446a74a / EN_WP_Introducing_Lattice_SIM_for_Elyra-7.pdf).

[0006] In a lattice SIM, a single output beam can generate multiple beams. This output beam is diffracted into multiple diffraction orders by a two-dimensional grating located in the image plane at the midpoint of the illumination optical path. These diffraction orders then appear as interfering beams within the illumination pupil of the illumination beam. The concept of a diffraction order refers to the process of generating an illumination pattern through the interference of the beams within the pupil. This term is unrelated to diffraction at the grating that can be used to provide the beams. Therefore, the diffraction order corresponds to the illumination pattern itself, rather than to the grating that may be structurally illuminated.

[0007] Multiple raw images are captured within a specifically positioned SIM illumination pattern. These raw images contain moiré fringes with high-frequency components that provide information about the sample. For SIM microscopy, specialized image analysis is required to obtain high-resolution images of the sample from the moiré fringes. Such analysis is described in detail in US11867894B1, which is incorporated herein by reference in its entirety regarding image processing.

[0008] For further discussion of SIM technology, please see: Neil MAA et al., "Method of obtaining optical sectioning by using structured light in a conventional microscope." OpticsLetters 22, 1905–1907 (1997); Heintzmann R. and Huuser T., Super-resolution structured illumination microscopy. Chemical Reviews 117 (23), 13890-13908 (2017) (Super-Resolution Structured Illumination Microscopy. Chemical Reviews 117 (23), 13890-13908 (2017)); Ströhl F. and Kaminski C.: Frontiers in structured illumination microscopy, Optica 3, 667-677 (2016) (Frontiers in structured illumination microscopy, Optica 3, 667-677 (2016)); Schermelleh et al.: Super-resolution microscopy demystified, Nature Cell Biology 21, 72-84 (2019) (Super-resolution microscopy demystified, Nature Cell Biology 21, 72-84 (2019)); Schropp M. and Uhl R.: Two-dimensional structured illumination microscopy, Journal of Microscopy 256, 23-36 (2014) (Two-dimensional structured illumination microscopy. illuminationmicroscopy, Journal of Microscopy 256, 23-36 (2014)); Ingerman EAGuo, Y, et al.: Signal, noise and resolution in linear and nonlinear structured‐illuminationmicroscopy, Journal of Microscopy 273, 3-25 (2019); Guo, Y, et al.: Visualizing Intracellular Organelle and Cytoskeletal Interactions at Nanoscale Resolution on Millisecond Timescales, Cell 175, 1430-1442 (2018); Frohn JT, et al.: True optical resolution beyond the Rayleigh limit achieved bystanding wave illumination, Proceedings of the National Academy of Sciences 97, 7232-7236 (2000). Sciences of the United States of America 97, 7232–7236 (2000)); Beck M. et al.: Sub-100-nanometre resolution in total internal reflection fluorescence microscopy, Journal of Microscopy 232, 99–105 (2008); Gliko O.Fiolka R. et al.: Fast two-dimensional standing-wave total-internalreflection fluorescence microscopy using acousto-optic deflectors, Optics Letters 34, 836–838 (2009); Fiolka R.: Clearer view for TIRF and obliqueillumination microscopy, Optics Express 24, 29556–29567 (2016); Young LJ et al.: A Guide to Structured Illumination TIRF Microscopy at High Speed ​​with Multiple Colors, Journal of Visualized Experiments 111 (2016) (2016); Kner P. et al., “Super-Resolution Video Microscopy of Live Cells by Structured Illumination”, Nature Methods, Vol. 6, S.339 (2009) and Guo M. et al., “Single-shot super-resolution total internal reflection fluorescence microscopy”, Nature Methods 15, 425–428 (2018).

[0009] The number of raw images limits the shooting rate. More raw images require more time. In addition, the number of raw images reduces the number of high-resolution images of biological samples, thereby reducing the number of repetitions of the microscopy process, as each exposure causes optical damage until the sample is eventually damaged to an undesirable degree.

[0010] Lattice SIM requires 13 raw images for image processing to generate a high-resolution image. US11867894B1 reduces the number of raw images required through a special iterative method. This speeds up image acquisition but is computationally expensive. Kner et al. achieved the same result using a different dedicated evaluation algorithm. Traditional evaluation algorithms cannot achieve this. Summary of the Invention

[0011] The objective of this invention is to accelerate image acquisition in lattice SIM without using special evaluation algorithms. Specifically, this acceleration should be achieved without increasing computational load compared to conventional evaluation algorithms.

[0012] The invention is defined in the independent claims. The dependent claims relate to preferred designs.

[0013] A method for high-resolution SIM microscopy imaging of samples is presented. The sample is illuminated with a three-dimensional periodic illumination pattern. This illumination pattern is designed as a three-dimensional grating structure composed of bright spots, primarily exhibiting a periodicity of bright spots along the sample depth direction. Diffraction orders correspond to this illumination pattern. These diffraction orders are the discrete spatial frequency components of the illumination pattern; in English literature, they are referred to as "spatial frequency components of illumination intensity," as used by Gustafsson in his 2008 work, for example. This can be understood as the bright spots in the three-dimensional grating structure being physically interpreted as diffraction orders of interfering beams arranged in illumination pupils with corresponding illumination optical paths, as described above in the overview of SIM techniques. Here, the terms "diffraction order" and "discrete spatial frequency components of the illumination pattern" are used interchangeably.

[0014] The sample is imaged along the optical axis onto a detector that receives radiation from the sample plane. The sample is repeatedly illuminated and imaged, with a raw image of the sample captured during each illumination and imaging process. A total of n raw images are captured in this manner. These raw images differ in the specific position of the illumination pattern in the sample plane perpendicular to the optical axis. For each raw image, a specific position of the illumination pattern is defined, which is known in SIM technology. A high-resolution wide-field image of the sample is generated from the n raw images captured. To reduce the number of raw images, specifically to n=9 raw images, the illumination pattern is moved relative to the sample along the optical axis during the reception of radiation for the corresponding raw image.

[0015] The relative movement is an integer multiple of the period of the lighting pattern. This axial movement is either the length of one axial period of the three-dimensional periodic lighting pattern, or an integer multiple of that axial period length.

[0016] This achieves the use of fewer original images, n=9, compared to 13 original images required for 3D illumination patterns in existing techniques. This shift reduces the effective number of discrete spatial frequency components (diffraction orders) in the illumination pattern relevant to reconstruction to 9. In this way, conventional reconstruction methods based on n=9 can be used because the effects of zero-order diffraction are averaged in the 3D illumination pattern due to the relative axial shift during each radiation reception of the original image. Therefore, interference terms caused by zero-order diffraction in the 3D illumination pattern do not appear in the original images, thus reducing the number of original images in conventional image processing.

[0017] The optics used to set the illumination optical path for generating the illumination pattern can largely remain unchanged from existing techniques. Illumination for each original image can be achieved by simply supplementing the structure to move the illumination pattern relative to the sample along the optical axis during image capture (typically within the detector's integration time). For this purpose, relatively simple optics can be used in the illumination optical path. For example, existing tube lenses can be designed to be adaptive and thus adjustable. Alternatively, a grating arranged in the optical path for generating the illumination pattern can be moved axially. A sample stage can also be used to move the selected sample plane relative to the three-dimensional illumination pattern. Any defocusing caused by this movement in the detection path typically needs to be corrected.

[0018] During the acquisition of the corresponding original images, relative movement along the movement path can be performed continuously or in steps. For step-by-step adjustments, the period L (or an integer multiple thereof) is preferably evenly distributed, i.e., in the case of n steps, the step size is L / n. The permissible deviation is 10%, preferably 5%, and particularly preferably 1%. For scheme 1, the relative movement must be performed while acquiring the radiation signal of the corresponding original image. Suitablely, a detector is used that receives radiation for each original image within the integration time and moves during the integration time of the corresponding original image.

[0019] In the corresponding microscope, a control device is designed to enable relative movement. The control device manipulates optics (e.g., tube lenses) arranged in the illumination path to move the illumination pattern within the sample.

[0020] Since this discussion pertains to the SIM microscopy method, these designs are naturally applicable to SIM microscopes, and their control devices are constructed accordingly. Conversely, the operating principles performed by the control device are also applicable to the SIM method. The control device preferably includes a processor.

[0021] Of course, the features described above, as well as those explained below, can be used not only in the specified combinations, but also in other combinations or individually, without exceeding the scope of the invention. Attached Figure Description

[0022] The invention will now be described in more detail with reference to the embodiments and accompanying drawings, which also disclose the essential features of the invention. These embodiments are for illustrative purposes only and should not be construed as limiting the invention. For example, the description of embodiments comprising multiple elements or components should not be construed as implying that all such elements or components are necessary for implementation. Rather, other embodiments may also include alternative elements and components, fewer elements or components, or additional elements or components. Unless otherwise stated, elements or components of different embodiments may be combined with each other. Modifications and variations described for one embodiment may also apply to other embodiments. To avoid repetition, the same or corresponding elements in different figures are denoted by the same reference numerals and are explained only once. In the drawings:

[0023] Figure 1 A block diagram illustrating an implementation of a method for lattice SIM microscopy is shown.

[0024] Figure 2 The microscope shown is used to generate raw images, which are then used to... Figure 1 The method shown is used for evaluation.

[0025] Figure 3 Showing according to Figure 2 A schematic diagram of an embodiment of a microscope illumination device, showing a schematic diagram of light spots in an illumination pupil, for generating a SIM illumination pattern composed of multiple points suitable for lattice SIM technology.

[0026] Figure 4 A schematic diagram of the diffraction order (discrete spatial frequency components of the illumination pattern) used to generate the original image is shown.

[0027] Figure 5 Showing according to Figure 2 A schematic diagram of an embodiment of the illumination device for a microscope. Detailed Implementation

[0028] Figure 1 A block diagram is shown for a lattice-SIM microscopy imaging method. The method mainly includes steps S0 to S5, wherein sub-step S2.2 is a step of performing motion during image acquisition. Figure 2A microscope 2 suitable for method execution is schematically shown, operating according to lattice SIM technology. Microscope 2 images a sample 4 located on a sample stage 6. Imaging is achieved via an objective lens 8, which projects the object field in the sample 4 onto a detector 10 through a tube lens (not shown in detail). An illumination device 14 illuminates the sample 4 through the objective lens 8 via a beam splitter 12, thereby achieving structured illumination adjustable with respect to the position within the sample 4, as is known to lattice SIM technology. Microscope 2 is controlled by a control device 16, primarily comprising a processor 18. The sample 4 is located on a stage 6 that is adjustable axially, i.e., perpendicular to the light incident and imaging directions, and is driven by a driver 7 operated by the control device 16.

[0029] The process begins with initial step S0. In step S1, sample 4 is imaged multiple times and illuminated using a SIM illumination pattern known in lattice SIM technology. In this case, sub-steps S2.1 and S2.2 are repeated, each time for the original image to be generated. In sub-step S2.1, the illumination pattern is positioned at a specific lateral position, which is known in SIM technology. In sub-step S2.2, sample 4, illuminated in this manner, is imaged. Here, according to option 1 described above, the relative position of the illumination pattern with respect to sample 4 is adjusted axially, i.e., along the incident direction of the illumination pattern radiation. This adjustment is performed when detector 10 extracts the original image signal. In the embodiment, detector 10 is a detector that receives radiation for image generation during the integration time. Relative movement occurs during this integration time. For example, control device 16 will accordingly manipulate the optics in the illumination optical path, since this movement is a relative movement between sample 4 and the illumination pattern. Here, this relative movement is achieved such that the relative movement takes the (axial) periodic length of the illumination pattern as the travel distance.

[0030] If, in sub-step S2.2, the image signal of the original image is received while the movement is occurring simultaneously, the process returns to sub-step S2.1, moves the illumination pattern to different lateral positions, and then repeats sub-step S2.2. Therefore, the number of times sub-steps S2.1 and S2.2 are executed is the same as the number of original images that need to be received. Preferably, nine original images need to be received, meaning sub-step S2.1 needs to be executed nine times. Sub-step S2.2 is then performed. The resulting nine original images differ in the lateral position of the illumination pattern, as described at the beginning for lattice SIM.

[0031] For each lateral position of the illumination pattern, a raw image is obtained by imaging the sample 4 onto the detector 10. Therefore, at the end of step S1, nine raw images are obtained. These nine raw images are all imaged from the same sample under the illumination pattern. The difference between these raw images lies in the lateral position of the illumination pattern. (During image acquisition, the illumination pattern moves only axially and remains laterally stationary in each raw image.) The specific lateral position is defined by an illumination pattern function, which defines or represents the position of the illumination pattern. The value n refers to the number of lateral positions the illumination pattern moves in the sample 4 due to the lattice SIM technique.

[0032] The illumination pattern has an illumination pattern frequency, which is generated, for example, by interference. As described above and further elaborated below, the illumination pattern frequency represents m diffraction orders, i.e., the discrete spatial frequency components of the illumination pattern. This interpretation is generally applicable and reasonable, even if the generation methods differ. Considering that the illumination pattern frequency is a complex conjugate, the frequencies of these m diffraction orders correspond to the discrete spatial illumination pattern frequencies. Therefore, above the zeroth order diffraction (i.e., the fundamental frequency, and thus the common-mode component), for each illumination pattern frequency defined in space, there is always a pair of diffraction orders with equal magnitudes in the frequency domain. This is known in the prior art, for example, in the publications of Gustafsson (2008). Due to the use of lattice SIM technology, the value of m for the 3D periodic illumination pattern used in Option 1 is typically 13.

[0033] In step S3, the nine original images generated in step S1 are processed, for example, using a Wiener filter. As mentioned earlier, since a signal is generated each time the original image is received, only nine original images are needed, and only these nine original images are considered in step S3. This simplifies the deconstruction process and reduces computational requirements.

[0034] After step S3 is completed, a high-resolution image of sample 4 is output in step S4. This image is based on the original images. However, unlike existing technologies, this method uses only a smaller number of original images, namely 9.

[0035] Figure 3An example of the implementation of the illumination device 14 is shown schematically. A light source 20 provides an initial beam 22, such as from a laser (not shown in detail). Using a two-dimensional crosshair grating 24, the initial beam 22 is split into five sub-beams 28a to 28e. These sub-beams are optically shaped by suitable lenses or other optics (not shown in detail) to form five light spots 30a to 30e in the entrance pupil 26 (also referred to as the "back focal plane" in English literature) of the objective lens 8. The center point 30a corresponds to the central beam, and four adjacent points 30b to 30e are arranged in a square around the center point, corresponding to the lateral beams. These five light spots in the objective lens pupil 26 interfere with each other, causing the objective lens 8 to form a three-dimensional SIM illumination pattern 32 in the sample 4. Here, according to... Figure 3 The illustration shows a SIM illumination pattern 32 presented as dots, in the form of its Fourier transform image. The four Fourier components 36 are marked with hollow dots, which will be discussed later. Spatially, this SIM illumination pattern is a three-dimensional pattern composed of luminous dots arranged in a square grid. However, Figure 3 The Fourier image of the SIM illumination pattern 32 shows that the radiation interference from the emission points 30a to 30e produces diffraction orders; each of these 13 diffraction orders (discrete spatial frequency components of the illumination pattern) is represented by a point.

[0036] The spot pattern in illumination pupil 9 corresponds to the illumination intensity pattern in the sample plane in Fourier space in its Fourier representation. Each point in the Fourier representation of illumination pattern 32 corresponds to a diffraction order. The farther the point is from the center, the higher its diffraction order. The specific illumination pattern of the original image is generated by changing the phase relationship between the emission points 30a to 30e. This can be achieved, for example, by moving grating 24 located in the image plane at the middle of the illumination optical path, such that the movement is at an angle to the vertical axis of the square grating. This movement in Figure 3 The arrow 34 indicates, for example, an angle of 33.7° relative to the principal axis of the square crosshair grating. This movement is performed in nine steps within a phase range of 0 to 2π, resulting in nine different positions, which in turn cause nine different modulations of the phase relationship between the light-emitting points 30a to 30e in the objective pupil 26. These positions correspond to the lateral positions of the illumination pattern 32, with one position corresponding to each original image.

[0037] The implementation described here uses a square grating as an example only. Hexagonal grating structures are also applicable.

[0038] The method for generating each original image is as follows: illuminating sample 4 with a specially arranged transverse illumination pattern 32 and recording the light reflected by sample 4; or, under fluorescence excitation, recording the fluorescence emitted by sample 4.

[0039] Figure 3 The illumination pattern 32 shown is generated by interference from beams located within the pupil and focused at points 30a to 30e; this is merely an example, but it has become the standard method for historical reasons. In this method, the different discrete frequencies present in the illumination pattern 32 are called diffraction orders. In signal technology terms, these frequencies represent carrier frequencies. Diffraction orders above the fundamental / zero-order diffraction frequency correspond to the frequencies of the spatial illumination pattern, but they are complex conjugates; therefore, above the fundamental frequency, there are always two diffraction orders with the same value. The fundamental frequency corresponds to the zero-order diffraction. In addition, there are 12 additional diffraction orders. Points 30a to 30e can also be generated in other ways, such as by an optical fiber terminating in the pupil or a suitably arranged matrix of tilting mirrors.

[0040] Thirteen diffraction orders (discrete spatial frequency components of the illumination pattern) require thirteen raw images in a lattice SIM to perform image evaluation at the highest resolution (fewer raw images can be used under some potentially acceptable constraints; see US11867894B1). However, this embodiment now generates nine raw images, i.e., using nine shift positions of grating 24, instead of thirteen raw images. Therefore, this embodiment uses fewer raw images compared to the prior art. Nevertheless, the same high resolution can still be obtained using conventional SIM image reconstruction methods because the axial shift of the illumination pattern relative to sample 4 eliminates all diffraction orders based on interference with the zeroth order diffraction. These interference theorems originate from the superposition with diffraction order 28a. They correspond to Figure 3 Hollow points 36 in the Fourier representation of the illumination pattern 32. Therefore, the illumination pattern contains fewer spatial frequencies, allowing us to use nine original images. This naturally increases the speed of the image capture process by 9 / 13 times, or approximately 30%.

[0041] Figure 4 This illustrates the effect of diffraction orders (discrete spatial frequency components of the illumination pattern) when generating images using lattice SIM technology. The movement of grating 24 along the two principal axes affects... Figure 3 The phase of the outer points 30b to 30e in the sample plane is modulated. Through the interference of the in-plane diffraction orders 30b to 30e, 13 orders of the SIM illumination pattern 32 are generated. These orders in... Figure 4 The left side is shown as a Fourier image, with the corresponding amplitudes of the Fourier components generated by interference also indicated. By shifting the image along the axis during the reception of each raw image, the spatial frequency at point 36 (indicated by a dashed circle) can be suppressed, resulting in the situation shown in the right figure. This allows the number of raw images to be set to n=9.

[0042] The highest modulation frequency in the illumination intensity determines the maximum frequency of the sample structure that can be visualized using the optical system. By averaging during image acquisition according to Option 1, modulation frequencies 36 in the illumination intensity that cause depth modulation can be eliminated. Therefore, fewer images are required, only nine. This not only improves imaging speed but also simplifies the image reconstruction process, thus accelerating reconstruction. The interference terms avoided by averaging are those generated by zero-order diffraction interference. Figure 4 The interference terms with an intensity of 0.13 are marked with circles on the right, corresponding to... Figure 3 Hollow points in the image. These interference terms additionally modulate the illumination modulation along the axial direction, i.e., the z-direction. This results in improved axial resolution and optical cutting of thick samples. These interference terms can be removed by effective averaging through relative movement during image acquisition. The axial movement amount must be one period length of the three-dimensional illumination pattern, where the period length is related to the depth direction. Of course, it can also be an integer multiple of this movement amount. The relative movement can be performed continuously or in steps. The relative movement removes the axial modulation, thereby removing the aforementioned interference term 36. Since the relative movement changes the phase relationship, term 36 disappears. In this way, only 9 original images are needed to generate the same high-resolution image as 13 original images. For example, axial relative movement can be achieved by using suitable adjustable optics in the illumination optical path, such as using an adaptive lens (e.g., a tube lens) or a tube lens group 38 with an adaptive lens component in the illumination optical path. This is illustrated in Figure 5 As shown in the figure, Figure 5 Enlarged display Figure 2 The illumination device 14 is located within the sample 4. An adaptive tube lens group 38 is arranged between the cross grating 24 and the beam splitter 12, enabling the illumination pattern 23 to be generated axially, i.e., in the depth direction of the sample 4. The control device 16 adjusts the axial position of the illumination pattern 24 in synchronization with the signal integration from the detector 10 by manipulating the adaptive tube lens group 38 and / or the sample stage 6. Alternative implementations using adjustable optics have been described above.

Claims

1. A method for high-resolution SIM microscopy imaging of a sample (4), wherein, The method includes: The sample (4) is illuminated with a periodic illumination pattern (32), which is designed as a grating structure composed of bright spots and corresponds to the diffraction order; The sample (4) is imaged along the optical axis onto the detector that receives the radiation; Among them, n original images of sample (4) are captured by repeated illumination and imaging, wherein for each of the n original images, a specific lateral position of the illumination pattern (32) is used in the sample plane perpendicular to the optical axis; and Generate a high-resolution wide-field image from n original images. Its features are: For each of the n original images (32), During the radiation reception, the illumination pattern (32) moves relative to the sample (4) along the optical axis by an axial period length or a multiple of the axial period length of the illumination pattern.

2. The method according to claim 1, characterized in that, For each original image in the original image (32), during the period of receiving radiation, the illumination pattern (32) moves continuously along the optical axis relative to the sample (4).

3. The method according to claim 1, characterized in that, For each original image in the original image (32), during the receiving radiation, the illumination pattern (32) moves stepwise along the optical axis relative to the sample (4).

4. The method according to any one of claims 1-3, characterized in that, For each original image in the original image (32), the detector receives radiation over an integration time, and the illumination pattern (32) moves relative to the sample (4) over the integration time.

5. The method according to any one of claims 1-4, characterized in that, Choose n=9.

6. A microscope for high-resolution SIM microscopy imaging of a sample (4), the microscope comprising: An illumination device for illuminating a sample (4) with a periodic illumination pattern (32), the periodic illumination pattern (32) being designed as a grating structure composed of bright spots and corresponding to diffraction orders; An imaging device for imaging the sample (4) onto a detector that receives radiation along the optical axis; as well as A control device that manipulates an illumination device and a detector to capture n original images of a sample (4) by repeated illumination and imaging, wherein the control device adjusts a specific lateral position of the illumination pattern (32) in a sample plane perpendicular to the optical axis for each of the n original images; and An evaluation device, optionally designed as part of the control unit, is used to generate a high-resolution wide-field image from n original images. Its features are: For each of the n original images (32), during the radiation reception period, the control device causes the illumination pattern (32) to move relative to the sample (4) along the optical axis by an axial period length or an integer multiple of the axial period length.

7. The microscope according to claim 6, characterized in that, For each original image in the original image (32), during the period of receiving radiation, the control device causes the illumination pattern (32) to move continuously along the optical axis relative to the sample (4).

8. The microscope according to claim 6, characterized in that, For each of the n original images (32), during the radiation reception period, the control device causes the illumination pattern (32) to move in steps relative to the sample (4) along the optical axis.

9. The microscope according to any one of claims 6-8, characterized in that, For each original image in the original images, the detector receives radiation over an integration time, and the control device moves the illumination pattern (32) relative to the sample (4) over the integration time.

10. The microscope according to any one of claims 6-9, characterized in that, The control device uses n=9.

11. The microscope according to any one of claims 6-10, characterized in that, The illumination path includes an adjustable tube lens optics (38), and the control device manipulates the tube lens optics (38) to move the illumination pattern (32).

12. The microscope according to any one of claims 6-11, characterized in that, The illumination path has an axially movable grating (24) in the intermediate image plane, and the control device moves the grating (24) axially to move the illumination pattern (32).

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