Light sheet microscopy and methods for light sheet microscopy
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- CARL ZEISS MICROSCOPY GMBH
- Filing Date
- 2021-10-28
- Publication Date
- 2026-07-21
Smart Images
Figure CN114428392B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to a light-sheet microscope having a sample cavity with a sample stage for a sample carrier containing a sample. The light-sheet microscope also has a detection device for imaging the sample cavity along an optical imaging axis. Additionally, the light-sheet microscope has an illumination device for illuminating the sample using a light sheet, characterized by a displaceable light sheet plane within the sample cavity. The light sheet plane is substantially aligned perpendicular to the optical imaging axis, and the light sheet has an illumination width b perpendicular to the light sheet plane. The light-sheet microscope also has a processing device connected to the detection device, the illumination device, and / or the sample stage. The processing device manipulates the illumination device and / or the sample stage to adjust the position of the light sheet plane within the sample cavity. The processing device is configured to manipulate the detection device to capture multiple images of the sample cavity at different positions of the light sheet plane, thereby generating a z-stack of images of the sample cavity.
[0002] The present invention also relates to a method for light-sheet microscopy, wherein, in a first step, a sample is illuminated using a light sheet. The light sheet is characterized by a light sheet plane arranged substantially perpendicular to the optical imaging axis, and the light sheet having an illumination width b perpendicular to the light sheet plane. In a second step, the position of the light sheet plane is adjusted along the optical imaging axis, wherein multiple images of the sample are captured along the optical imaging axis at different positions of the light sheet plane, thereby generating a z-stack consisting of multiple images of the sample. Background Technology
[0003] DE10257423A1 describes a microscope in which a sample is illuminated by a thin band of light and observed in a plane perpendicular to the light band. The light band is moved for image capture, and a plane detector is used to capture fluorescence and / or scattered light. Other published documents on light-sheet microscopy include DE102007063274A1, DE102013107298A1, DE102013112596A1, US2013 / 0286181A1, DE102016103182A1, and WO2010 / 012980A1. Summary of the Invention
[0004] Therefore, a light sheet microscope and a method for light sheet microscopy are proposed, which reduces the photon sample load and improves the detection speed.
[0005] The invention is defined in the independent claims. Advantageous further constructions are given in the dependent claims. Preferred embodiments are applicable in the same manner to the light sheet microscope and the method.
[0006] A light slide microscope and a method for light slide microscopy are proposed.
[0007] The light-sheet microscope includes a sample cavity with a sample stage and an optical detection device. The sample stage is used for a sample carrier with the sample, and the optical detection device is used for imaging the sample cavity along an optical imaging axis. The light-sheet microscope also includes an illumination device for illuminating the sample using a light sheet. The light sheet is characterized by a movable light sheet plane within the sample cavity, with the illumination axis of the illumination unit located within the light sheet plane. The light sheet plane is substantially perpendicular to the optical imaging axis, and the light sheet has an illumination width b perpendicular to the light sheet plane. The light-sheet microscope also includes a processing device connected to the optical detection device, the illumination device, and / or the sample stage. The processing device manipulates the illumination device and / or the sample stage to adjust the position of the light sheet plane within the sample cavity. The processing device is configured to manipulate the optical detection device to capture multiple images of the sample cavity at different positions of the light sheet plane, thereby generating a z-stack of multiple images of the sample cavity (in which the sample is present). This arrangement in the light-sheet microscope allows for the imaging of defined individual sample planes located within the sample in the sample cavity, without the need for mechanically cutting the sample.
[0008] The light sheet is generated, for example, by a cylindrical optics device or a scanning laser beam. By illuminating with the light sheet, the dye of the sample in the light sheet plane, which corresponds to the respective sample plane in the embodiments, is selectively excited only. This excitation produces fluorescence radiation from the light sheet plane. The fluorescence radiation of the light sheet plane is detected by creating a focal plane in the light sheet plane, i.e., the plane in which the optical detection device is focused (the focal point of the optical detection device is located in this plane). The focal plane may also be offset axially from the light sheet plane along the optical imaging axis, but must be inside the light sheet.
[0009] The processing apparatus is configured to manipulate the illumination device and / or the sample stage to move the light plate plane and the imaging focal plane within the sample cavity, such that the light plate plane and the focal plane gradually move through the sample cavity and then through the sample, thereby progressively scanning the sample. This movement of the light plate plane and the focal plane can be achieved by moving the sample itself (e.g., by moving the sample stage with the aid of a driver), or by keeping the sample in its fixed position while the illumination device moves relative to the sample.
[0010] According to the implementation method, the displacement of the light plate plane in the sample cavity and the displacement of the focal plane in the same orientation in the sample cavity are coupled or automatically connected in such a way that the focal plane is always inside the light plate.
[0011] The processing device is also configured to manipulate the detection device according to each shift of the light sheet plane. Therefore, multiple images of each sample plane are sequentially captured, with the light sheet plane and focal plane positioned relative to the sample in each image. These images are generated from a stack at different depths along the imaging axis. In this case, a gap 'a' is created between the positions of the light sheet planes in successive images along the stack's axis. This gap, measured along the optical imaging axis, must always be greater than / equal to half the illumination width 'b', but not greater than the illumination width 'b', also measured along the optical imaging axis. In this case, the light sheets overlap in images captured at adjacent positions of the light sheet planes. This paired overlap ensures that all sample areas are exposed at least once, although for image capture, the light sheet always illuminates only the same sample area being detected within the sample cavity. Preferably, the light sheet thickness is 0.5 to 10 μm, thus a stack of samples, for example, millimeter in size, consists of thousands of images.
[0012] For imaging, it ultimately depends on the relative movement of the sample cavity relative to the detection device. This relative movement can be achieved through various scanning principles. The movement of the detection device (and the illumination device coupled to it and usually perpendicular to it) along the optical axis of imaging is known. In microscopy, this direction is usually referred to as the z-direction and represents the axial direction associated with imaging. In this document, if the term axial is used, it refers to the direction of depth in imaging. In conventional microscopy, this is the z-axis. If the unit consisting of detection and illumination is moved along the optical axis of imaging, an image stack is obtained accordingly, with the images of the image stack placed sequentially in the z-direction. An alternative to this is known in DE102013107297A1. In this, the illumination axis and the detection axis are also substantially perpendicular, and scanning is performed by moving the sample in a plane that does not contain either the illumination axis or the imaging axis. Thus, different depth regions in the sample cavity are also scanned in the same manner, producing an image stack. However, the images are not only separated along the optical imaging axis, i.e., not only in the conventional z-direction, but also laterally offset from each other with respect to the imaging axis. This offset can be compensated for by simply shifting the individual images backward. This backward shift compensates for the lateral (transverse to the imaging axis) offset caused by the angle between the plane of the sample displacement and the illumination axis (or the perpendicular line to the imaging axis). The technique described herein can be implemented in two variations. If these two variations are mentioned below, they are purely exemplary.
[0013] The processing apparatus is configured to compute intermediate images for use in the stack, the intermediate images being equivalent to images captured about virtual positions of the light sheet plane. This reduces the number of images required for sample imaging, where the stack is captured with a number of images insufficient on its own for high-quality imaging of the sample. However, to produce high-quality imaging of the sample, the intermediate images are mathematically reconstructed for use in the stack. Each image in the stack is a projection along the optical imaging axis according to the point spread function (PSF). This means that each image in the stack contains image information from sample planes spaced apart by other axes. Based on the overlap, the intermediate images can be mathematically reconstructed from this image information. This is known, for example, in DE102018009056A1. In this, the stack, consisting of individual images, is also reconstructed, producing additional intermediate images so that fewer individual images are used than previously generally; in particular, this does not exceed the requirements derived from the (Nyquist) theorem. Although this publication does not relate to light sheet microscopy, the mathematical calculations used therein for individual images stacked in the depth direction can also be advantageously and precisely applied to the light sheet microscopy described herein. In particular, refer to paragraphs
[0006] ,
[0014] -
[0017] and
[0073] of the published documents.
[0014] The number of images based on stacking is reduced, and the photon load on the sample is significantly reduced. Furthermore, the image capture speed with stacking is increased, preferably by a factor of 3, compared to captures without intermediate image reconstruction. The image quality of the sample remains high due to the reconstruction of intermediate images.
[0015] The quality of the reconstruction can preferably be improved by constructing illumination in the axial direction, i.e., along the optical imaging axis, during image capture of the stack. Suitable means and structures for this are known in DE102018207821A1, the disclosure of which is fully incorporated herein by reference. This results in a smaller planar contribution to the projection within the point spread function.
[0016] The method for light-sheet microscopy is performed in three steps. In the first step, the sample is illuminated using a light sheet characterized by a light sheet plane arranged substantially perpendicular to the optical imaging axis and having an illumination width b perpendicular to the light sheet plane. In the second step, the position of the light sheet plane is adjusted within the sample. In this case, multiple images of the sample are taken at different positions within the sample with respect to the light sheet plane and the focal plane, resulting in a stack of multiple sample images from different depth regions of the sample. Two successive positions of the light sheet plane are spaced apart from each other by a distance a, which is greater than or equal to half the illumination width b, but not greater than the illumination width b—measured separately along the optical imaging axis. In the third step, an intermediate image for the stack is calculated, which corresponds to an image of the virtual position of the light sheet plane within the sample cavity. Attached Figure Description
[0017] The invention will now be explained in more detail by way of example with reference to the accompanying drawings. In the drawings:
[0018] Figure 1 Shown as a light slide microscope;
[0019] Figure 2A This shows the illumination of the sample by the light sheet in the first position;
[0020] Figure 2B This shows the illumination of the sample by the light sheet in the second position;
[0021] Figure 2C and Figure 2D The image shows the illumination of the sample by the light sheet in a first position and the illumination of the sample by the light sheet in a second position; and
[0022] Figure 3 A flowchart illustrating a method for light section microscopy is shown. Detailed Implementation
[0023] Figure 1 A light-sheet microscope is shown, which identifies a sample cavity 1 with a sample stage 6 for a sample carrier 2 carrying a sample 4. The sample 4 is illuminated using an illumination device 8 and a light sheet 12. The illumination device 8 is characterized by an illumination axis 10. The light sheet 12 is characterized by a light sheet plane in a first position 22 and has an illumination width b perpendicular to the light sheet plane. An optical imaging axis 16 of an optical detection device 14 is arranged perpendicular to the light sheet plane in the first position 22. The optical detection device 14 is focused in a focal plane 11. In this embodiment, the focal plane 11 is precisely located on the light sheet plane, but it can also be located outside the light sheet plane, as long as it is located inside the light sheet 12. This requirement will also be satisfied in subsequent developments.
[0024] The sample stage 6 is driven by the driver 17, thereby allowing the position of the sample stage 6 and, consequently, the sample carrier 2 bearing the sample 4 thereon to be adjusted relative to the illumination device 8 and synchronously relative to the detection device 14. Consequently, the light plate 12 and the focal plane 11 are synchronously moved within the sample 4 by means of the driver. In this case, the positions of the light plate 12 and the focal plane 11 remain unchanged.
[0025] The lighting device 8, the optical detection device 14, and the driver 17 are connected to the processing device 20 via wire 18. This is an exemplary connection; other data-related connections (such as radio or similar connections) are also possible.
[0026] Alternatively, (not shown) the sample 4 may be left in its position or the driver 17 may be positioned on the illumination device 8 to move the illumination device relative to the sample 4, and consequently the light plate 12, or by pushing the light plate along the optical imaging axis 16, for example, using optical displacement of the scanner. The focal plane 11 is then also shifted together in the same direction and synchronously, for example by refocusing or moving the detection device 14.
[0027] Figure 2A and 2B Showing according to Figure 1 The arrangement described herein, in detail, shows a sample cavity 1 with a sample stage 6, a sample carrier 2, and a sample 4, and is relative to... Figure 2A exist Figure 2B The sample stage 6, which contains the sample carrier 2 and the sample 4, is pushed along the moving axis 13 inside the sample cavity 1.
[0028] The processing device 20 operates the illumination device 8, thereby illuminating the sample 4 using the light plate 12. The light plate plane is thus defined in the sample 4 at a first position 22. The light plate 12 is generated, for example, using cylindrical optics or by scanning laser beams. Illumination with the light plate 12 excites only the dye in the sample 4 located within the light plate plane, thereby generating fluorescence radiation from the light plate plane.
[0029] An optical detection device 14 is provided for detecting the fluorescence radiation. The processing device 20 manipulates the optical detection device 14, such as a camera of the detection device, to generate an image from a focal plane 11 perpendicular to the optical imaging axis 16. In this embodiment, the focal plane 11 is located in the light sheet plane, but it may also be offset parallel to the light sheet plane along the imaging axis 16; however, in any case, it is located inside the light sheet 12. The optical detection device 14 is focused in the focal plane 11 and detects the fluorescence radiation emitted from the light sheet plane illuminated by the light sheet 12. By according to... Figure 1 With this arrangement, the individual sample planes can be photographed by simply moving the sample 4 laterally using a light sheet microscope with a light sheet 12 and a focal plane 11 in a fixed relative position.
[0030] The light plate 12 and the focal plane 11 are moved synchronously in the sample cavity 1. Figure 2A and Figure 2B The images show two positions, 22 and 26, where the light plate 12 and the focal plane 11 move synchronously within the sample cavity 1.
[0031] At different positions 22, 26 on the light plate plane, the light plate 12 illuminates different sample planes in the sample 4 and images them from the focal plane 11 (not shown). The processing device 20, which performs position adjustment of the optical detection device 14 (e.g., via the driver 17), thus generates a stack of multiple images of the sample 4, each image having a different position 22, 26 on the light plate plane and the focal plane 11.
[0032] Figure 2C Showing with Figure 2A and Figure 2B The same structure is shown, but instead of showing the light plate 12, only the light plate planes in the first position 22 and the second position 26 are shown. Figure 2C Show Figure 2A and Figure 2B The imaging positions 22 and 26 of the light sheet plane in the image are shown; for simplicity, the focal plane is not shown. This illustration shows the interval 'a' between the light sheet planes at the first position 22 and the second position 26 when a first image of the stack is taken while the sample 4 is illuminated by the light sheet 12 at the first position 22, and a second image of the stack is taken while the sample 4 is illuminated by the light sheet at the second position 26.
[0033] remove Figure 2C In addition, Figure 2D The light sheet 12 is shown at two positions 22 and 26. The overlap Δz and the illumination width b of the light sheet 12 can be seen. Both values are measured along the optical imaging axis.
[0034] Processing device 20 manipulates illumination device 8 and / or sample stage 6 in such a way that a distance a is created between the positions of the light sheet planes of the sequentially captured stacked images along the optical imaging axis. This distance a is greater than / equal to half the illumination width b, but not greater than the illumination width b (see [link to documentation]). Figure 2D This creates an overlap Δz between the light sheet 12 corresponding to the light sheet plane in the first position 22 and the sample cavity corresponding to the light sheet plane in the second position 26, thereby ensuring that all areas of the sample 4 are exposed at least once.
[0035] The processing unit 20 is also configured to calculate intermediate images for the stack, which are equivalent to images illuminated using a light sheet 12 with a light sheet plane in a virtual position. By calculating the intermediate images using the processing unit 20, the number of images used to capture the stack (in order to capture sample 4 with high quality) can be selectively reduced. In this way, the sample is protected, and the capturing speed can be improved without significantly reducing the image quality. The mathematical reconstruction of the intermediate images is achieved by displaying a projection of each image of the stack along the optical imaging axis 16 according to the point spread function (PSF). For more information on reconstruction, see DE102018009056A1.
[0036] If illumination is constructed axially (i.e., along the imaging axis) during image capture, the reconstruction quality of the virtual position of the sheet plane can be improved because the contribution of the projected plane within the PSF is smaller. DE102018009056A1 describes a possible means for this, particularly asymmetry in sheet illumination. As already described, this is particularly preferably achieved through different positions of the sheet. Axial construction is also known in DE102018207821A1, which was mentioned at the beginning.
[0037] Figure 3 A flowchart of a method for light-sheet microscopy is shown. In a first step S1, a sample 4 is illuminated using a light sheet 12, characterized by a light sheet plane in a first position 22. The light sheet plane in the first position 22 is located in the illumination axis 8, wherein the light sheet plane is arranged substantially perpendicular to the optical imaging axis 16, and the light sheet 12 has an illumination width b perpendicular to the light sheet plane. In a second step S2, the light sheet plane is adjusted to a second position 26 in the sample cavity. Images of the sample 4 are captured in the first position 22 and the second position 26 of the light sheet plane, wherein the focal plane 11 of the imaging is always located in the light sheet. Therefore, when the sample is illuminated using the light sheet 12 at different positions in the light sheet plane and the focal plane, a stack of multiple images of the sample 4 is captured. In this case, the interval 'a' between successive positions of the light sheet plane in the stack of images measured along the optical imaging axis 16 is determined to be greater than / equal to half the illumination width b, but not greater than the illumination width b. In a third step S3, intermediate images for the stack are calculated.
Claims
1. A light-section microscope, comprising: The sample cavity (1) has a sample stage (6) for a sample carrier (2) with a sample (4). A detection device (14) is used to image the sample cavity (1) along the optical imaging axis (16); An illumination device (8) for illuminating a sample (4) using a light plate (12), the light plate being characterized by a light plate plane capable of being displaced within the sample cavity (1), wherein the light plate plane is arranged substantially perpendicular to the optical imaging axis (16), and the light plate (12) has an illumination width b perpendicular to the light plate plane; and A processing device (20) is connected to the detection device (14) and to the illumination device (8) and / or the sample stage (6), and the processing device manipulates the illumination device (8) and / or the sample stage (6) to progressively axially move the light plate plane of the light plate (12) to different axial positions (22; 26) of the light plate plane of the light plate (12) in the sample cavity (1), wherein the processing device (20) is configured to manipulate the detection device (14) to capture multiple images of the sample cavity (1) at different positions (22; 26) of the light plate plane of the light plate (12), thereby generating a stack of multiple images of the sample cavity (1), wherein the images of the stack are from different axial positions (22; 26) of the light plate plane of the light plate (12). Its features are, The processing device (20) is configured to operate the illumination device (8) and / or the sample stage (6) in such a way that, when axially pushing the light plate plane of the light plate (12), the interval a between axially adjacent positions (22; 26) of the light plate plane of the light plate (12) is greater than / equal to half of the illumination width b, but not greater than the illumination width b, which is measured along the optical imaging axis (16) respectively, and The processing device (20) is configured to compute an intermediate image for stacking, the intermediate image being an image of the light sheet plane of the light sheet (12) at an additional axial position in the sample cavity (1).
2. The light-sheet microscope according to claim 1, characterized in that, The processing device (20) is configured to operate the illumination device (8) in such a way that illumination is constructed along the optical imaging axis (16) during the imaging of the stack.
3. The light sheet microscope according to claim 1 or 2, characterized in that, The processing device (20) is configured to operate the lighting device (8) and the detection device (16) in such a way that only the area of the sample cavity (1) to be detected is illuminated.
4. The light sheet microscope according to claim 1 or 2, characterized in that, The detection device (14) is configured to image the sample cavity (1) from the focal plane (11), and the axial displacement of the light plate plane of the light plate (12) in the sample cavity (1) is coupled or connected to the axial displacement of the focal plane (11) in the same direction in the sample cavity (1) in such a way that the focal plane (11) is always located within the light plate (12).
5. A method for light section microscopy, comprising the following steps: a) Illuminate the sample (4) using a light plate (12), wherein the light plate is characterized by being planar, The light plate plane is arranged substantially perpendicular to the optical imaging axis (16), and the light plate (12) has an illumination width b perpendicular to the light plate plane. b) The light plate plane of the light plate (12) is gradually moved axially to different axial positions (22; 26) in the sample cavity (1), and at least one image of the sample (4) is captured at each axial position (22; 26) of the light plate plane of the light plate (12), thereby capturing a stack of multiple images of the sample (4). Its features are, In step b), the interval a between adjacent positions (22; 26) along the optical imaging axis (16) on the axial plane of the light sheet (12) is greater than / equal to half the illumination width b, but not greater than the illumination width b, which is measured along the optical imaging axis (16) respectively, and c) Calculate an intermediate image, which is equivalent to an image for z-stacking at an additional axial position of the light sheet plane of the light sheet (12).
6. The method for light section microscopy according to claim 5, characterized in that, Illumination is constructed along the optical imaging axis (16) during the imaging of the stack.
7. The method for light section microscopy according to claim 5 or 6, characterized in that, Illumination is applied only to the area of the sample cavity (1) that is being tested.
8. The method for light section microscopy according to claim 5 or 6, characterized in that, Each image is acquired from a focal plane (11), and the axial displacement of the light sheet plane of the light sheet (12) in the sample cavity (1) is coupled or connected to the axial displacement of the focal plane (11) in the sample cavity (1) in the same direction such that the focal plane (11) is always located within the light sheet (12).