Orthogonal line illumination optical three-dimensional imaging device

Abstract

The application discloses a kind of orthogonal line illumination optical three-dimensional imaging devices, belong to optical imaging field.Light modulation module provides two orthogonal line light spots, project on the surface of sample, two orthogonal line light spot focusing surface is same, focusing direction is perpendicular to each other;Light detection module includes detector, and objective lens and dichroic mirror are sequentially arranged between sample and detector, for detecting the signal light generated after sample is excited by orthogonal line light spot, light detection module further includes sample stage, for driving sample moves in the diagonal direction of two orthogonal line light spots.By two orthogonal line light spot irradiation to sample, two images corresponding to acquisition only need simple displacement operation to realize registration, overcome the problem that line scanning system is not uniform in transverse spatial resolution distribution after algorithm fusion, optimize the imaging performance of system, combined with cutting can quickly obtain the three-dimensional imaging result of large sample.

Description

An orthogonal line illumination optical three-dimensional imaging device Technical Field

[0001] This application belongs to the field of optical imaging, and more specifically, relates to an orthogonal line illumination optical three-dimensional imaging device. Background Technology

[0002] In the field of optical imaging, background interference makes it difficult to acquire clear focal plane images, significantly affecting the imaging quality of microscopy systems. To remove background interference, laser scanning confocal microscopy (LSCM) has been developed, which uses hardware such as pinholes to directly prevent the background from entering the detector. LSCM offers superior imaging performance, with significantly improved spatial resolution and tomographic capabilities compared to traditional wide-field microscopy techniques. However, LSCM typically uses point scanning to acquire sample images, resulting in very low imaging speed, making it unsuitable for samples on the centimeter scale and larger. To address this, line scanning-based imaging methods have been proposed. Line scanning allows for single-frame acquisition of linear array detection results, greatly reducing imaging time compared to point scanning. However, its lateral spatial resolution distribution is uneven, meaning the spatial resolution in non-focusing directions is worse than in the focusing direction. Furthermore, the tomographic capability of line scanning imaging systems is also reduced compared to point scanning-based LSCM.

[0003] Therefore, developing a simple design and algorithm to improve the uneven distribution of lateral spatial resolution in line scan imaging systems is of great significance for obtaining high-quality 3D imaging results for large-size samples. Summary of the Invention

[0004] In view of the above-mentioned defects or improvement needs of the prior art, this application provides an orthogonal line illumination optical three-dimensional imaging device, which aims to overcome the shortcomings of the line scan imaging system in terms of uneven distribution of lateral spatial resolution, and thus solve the technical problem that it is difficult to achieve both imaging quality and imaging speed for large-size samples in the prior art.

[0005] To achieve the above objectives, according to one aspect of this application, an orthogonal line illumination optical three-dimensional imaging device is provided, comprising a light modulation module and a light detection module:

[0006] The optical modulation module provides two orthogonal line spots that are projected onto the surface of the sample. The two orthogonal line spots have the same focal plane and their focal directions are perpendicular to each other.

[0007] The light detection module includes a detector, and an objective lens and a dichroic mirror are sequentially arranged between the sample and the detector to detect the signal light generated by the sample after being excited by the orthogonal line light spots. The light detection module also includes a sample stage to move the sample in the diagonal direction of the two orthogonal line light spots.

[0008] Through the above technical solution, the optical modulation module provides two orthogonal line spots with the same focal plane and mutually perpendicular focusing directions. Illuminating the sample with these orthogonal line spots and then detecting it with a detector, registration of the imaging results corresponding to the two line spots can be achieved with only simple displacement. This significantly reduces the amount of data processing and improves the problem of drastically reduced imaging speed when imaging samples from different angles. Furthermore, the fusion of the two images acquired under the illumination of the two orthogonal line spots yields better imaging quality than that under single-beam line spot illumination, overcoming the problem of uneven lateral spatial resolution distribution in unidirectional line scanning systems. Therefore, this application can simultaneously achieve high imaging quality and fast imaging speed for large-sized samples. Attached Figure Description

[0009] Figure 1 is a schematic diagram of the imaging device using a single detector;

[0010] Figure 2 is a schematic diagram of the structure that yields two orthogonal line spots when two excitation beams are provided;

[0011] Figure 3 is a schematic diagram of the structure of obtaining two orthogonal line spots using a spatial light modulator when a beam of excitation light is provided;

[0012] Figure 4 is a schematic diagram of the structure for obtaining two orthogonal line spots using a first cross-shaped mask when an excitation beam is provided.

[0013] Figure 5 is a schematic diagram of the structure of obtaining two orthogonal line spots using a digital micromirror device when a beam of excitation light is provided;

[0014] Figure 6 is a schematic diagram of the structure of obtaining two orthogonal line spots using two laser emitters when two excitation beams are provided;

[0015] Figure 7 is a schematic diagram of the structure of obtaining two orthogonal line spots using a single laser emitter when two excitation beams are provided;

[0016] Figure 8 is a schematic diagram of the structure of obtaining two orthogonal line spots using a single laser emitter when two excitation beams are provided;

[0017] Figure 9 is a schematic diagram of the imaging device using two detectors;

[0018] Figure 10 is a schematic diagram illustrating the principle of improving the spatial sampling rate in this application (using I as an example). +45° (For example);

[0019] Figure 11 is the strip image I +45° A diagram illustrating pixel repositioning;

[0020] Figure 12 is a schematic diagram of image processing of mouse brain slices in this application;

[0021] Figure 13 is a comparison of simulation results of point confocal imaging, single-direction line scan imaging, and orthogonal line scan imaging of this application in terms of three-dimensional spatial resolution and tomography capability.

[0022] In the diagram, 1. Laser emitter; 2. Half-wave plate; 3. First lens; 4. Second lens; 5. Beam splitter; 6. Second optical element; 7. Beam combiner; 8. First reflector; 9. First optical element; 10. Second mirror; 11. Third lens; 12. Dichroic mirror; 13. Objective lens; 14. Sample stage; 15. Orthogonal line spot; 16. Tube lens; 17. Beam splitter; 18. Emission filter; 19. Detector; 20. Second cross-shaped mask; 21. Spatial light modulator; 22. First cross-shaped mask; 23. Digital micromirror device; 24. Polarizer; 25. Analyzer. Detailed Implementation

[0023] To make the objectives, technical solutions, and advantages of this application clearer, the following detailed description is provided in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application. Furthermore, the technical features involved in the various embodiments described below can be combined with each other as long as they do not conflict with each other.

[0024] This application proposes an orthogonal line illumination optical three-dimensional imaging device, including a light modulation module and a light detection module:

[0025] As shown in Figure 1, the optical modulation module provides two orthogonal line spots 15, which are projected onto the surface of the sample. The two orthogonal line spots 15 have the same focal plane and their focal directions are perpendicular to each other.

[0026] The light detection module includes a detector, and an objective lens 13 and a dichroic mirror 12 are arranged sequentially between the sample and the detector 19 to detect the signal light generated after the sample is excited by the excitation light. The light detection module also includes a sample stage 14 to move the sample along the diagonal direction of two orthogonal linear light spots 15.

[0027] The sample is placed on the sample stage 14. In this device, two orthogonal line beams 15, in conjunction with the sample stage 14, scan the surface of the sample diagonally (i.e., along the X-axis). The orthogonal line beams 15 have the same focal plane, but their focal directions are perpendicular to each other, and they have a nearly uniform spatial distribution in three-dimensional space. The signal light generated after the orthogonal line beams 15 illuminate the sample is collected by the objective lens 13, then passes through the dichroic mirror 12 again, and is finally acquired by the detector 19, obtaining two line scan images. Subsequent image processing only requires translating and repositioning the pixels in the two line scan images, without the need for scaling, rotation, or other registration steps between the two images. Therefore, optical three-dimensional imaging using this device requires less data processing and produces high-quality images, balancing imaging speed and quality for large-size samples.

[0028] Further, as shown in Figure 2, in some embodiments, the optical modulation module provides two excitation beams, including a beam combiner 7 that combines the two excitation beams. The beam combiner also includes a first optical element 9 and a second optical element 6 that convert the excitation beams into line spots on the two incident paths of the beam combiner. The focusing directions of the first optical element 9 and the second optical element 6 are perpendicular, so that the two excitation beams are combined to form two orthogonal line spots 15.

[0029] Specifically, the beam combiner 7 can be a polarized beam splitter (PBS) or a beam splitter (BS). The first optical element 9 and the second optical element 6 can be cylindrical lenses or Powell prisms.

[0030] The beam combiner 7 combines the two excitation beams, which are then converged onto the sample surface by the dichroic mirror 12 and the objective lens 13. Since the focusing directions of the first optical element 9 and the second optical element 6 are perpendicular, an orthogonal line spot 15, as shown in Figure 1, is ultimately formed on the focal plane of the objective lens 13. Furthermore, because the beam splitter 5 splits the original laser beam into two beams, which then enter the first optical element 9 and the second optical element 6 respectively, the resulting orthogonal line spots 15 have the same focusing plane, only their focusing directions are perpendicular to each other.

[0031] Furthermore, in some other embodiments, the optical modulation module provides an excitation beam, including a laser emitter 1. A spatial light modulator 21, a first cross-shaped mask 22, or a digital micromirror device 23 is disposed on the emission path of the laser emitter 1. As shown in Figures 3, 4, and 5, the spatial light modulator 21, the first cross-shaped mask 22, or the digital micromirror device 23 can directly form two orthogonal line spots 15 from the excitation beam, avoiding abrupt brightness changes at the intersection. When using the spatial light modulator 21 for modulation, a polarizer 24 needs to be disposed on the incident path of the spatial light modulator 21, and an analyzer 25 needs to be disposed on the emission path for optimal use.

[0032] Regardless of whether the optical modulation module provides one or two excitation beams, a third lens 11 can be added to the incident light path of the dichroic mirror 12 to expand the excitation beam, and a tube mirror 16 can be added between the dichroic mirror 12 and the detector 19 to facilitate the focusing of the signal light onto the detector 19.

[0033] Furthermore, when the optical modulation module provides two excitation beams, to avoid interference between them, the two beams are P-polarized and S-polarized, respectively, with their intensities distributed in a 1:1 ratio. Each beam corresponds to one of the two orthogonal line spots. Since the excitation beam intensity is proportional to the signal beam intensity, to ensure that the beam intensities of the two excitation beams are not significantly different in the line scan detection results of the two directions, it is necessary to ensure that the beam intensities are equal.

[0034] As shown in Figure 6, in some embodiments, the optical modulation module may include two laser emitters 1 with the same center wavelength. Each laser emitter 1 provides an excitation beam. The light intensity and polarization state of the two laser emitters 1 can be finely adjusted by filters and half-wave plates so that the light intensity of the two line spots finally generated on the focal plane of the objective lens 13 is consistent.

[0035] As shown in Figure 7, in some embodiments, the optical modulation module may also include only one laser emitter 1. A beam splitter 5 is provided on the emission path of the laser emitter 1 to split the excitation light provided by the laser emitter 1 into two excitation light beams. The beam splitter 5 can be a polarized beam splitter (PBS) or a beam splitter (BS). Since the PBS splits the light intensity according to the polarization state, when the PBS is used as the beam splitter 5, a half-wave plate 2 can be placed at the emission port of the laser emitter 1 as shown in Figure 8 to adjust the laser polarization state to meet the above conditions. Furthermore, a beam expander group can also be included between the half-wave plate 2 and the beam splitter 5. The beam expander group includes a first lens 3 and a second lens 4. The excitation light with the adjusted polarization state can be incident parallel to the beam splitter 5 after passing through the beam expander group. Furthermore, a first reflector 8 is provided between the first optical element 9 and the beam splitter 5, and a second reflector 10 is provided between the first optical element 9 and the beam combiner 7. After the beam splitter 5 splits a beam of excitation light into two beams, one of the beams first passes through the first reflector and turns 90° before entering the first optical element 9. After exiting the first optical element 9, it passes through the second reflector 10 and turns 90° again before entering the beam combiner 7.

[0036] As shown in Figure 1, in some embodiments, the detector 19 can be an area array detector, and a second cross-shaped mask 20 is also provided on the incident path of the area array detector, with only the middle cross slit allowing light to pass through and blocking the background light, so as to achieve the imaging effect of line scanning.

[0037] As shown in Figure 9, in some other embodiments, the detector 19 can be two linear array detectors. In this case, a beam splitter 17 needs to be set between the dichroic mirror 12 and the detector 19 so that the signal light generated by the sample enters the two linear array detectors respectively for line scanning imaging. The two linear array detectors are conjugate with the two unidirectional line light spots respectively.

[0038] Specifically, as shown in Figure 8, an emission filter 18 can be provided between the two detectors 19 and the beam splitter 17. When excited by light of a specific wavelength, the wavelength of the signal light generated by the sample is also certain. Therefore, by using the wavelength-selective emission filter 18, ambient light that is inconsistent with the wavelength of the signal light can be filtered out, thereby improving the image quality of the imaging result.

[0039] Furthermore, as shown in Figure 10, detector 19 continuously captures multiple frames of images of the sample surface using a strip scanning method. The center row pixels of each frame are taken as the image result for that frame, and the n image results are stitched together in chronological order to form a strip image I. +45° with I -45°Alternatively, the single-frame image obtained by processing a single frame or several adjacent frames using a line illumination modulation algorithm can be used as the image result for each frame, and then stitched together in chronological order to form a strip image I. +45° with I -45° The line illumination modulation algorithm can be applied using existing line illumination modulation optical tomography (LiMo) technology, or other line illumination modulation algorithms. LiMo technology processes multiple adjacent frames to obtain a single-frame result, while other line illumination modulation algorithms process a single frame to obtain a single-frame result. The processing results of all frames can be stitched together in chronological order. In other words, this application can directly take the center row pixels of the n frames in S1 and stitch them together to form a strip image I. +45° I -45° Alternatively, one can choose to combine the n frames of images in S1 with existing line illumination modulation algorithms to stitch the processed single frames into a strip image I. +45° I -45° However, combining it with the line illumination modulation algorithm increases the processing steps, but can further improve spatial resolution and tomography capabilities.

[0040] When two linear array detectors detect two orthogonal line spots, one of them detects the signal light corresponding to a single-direction line spot within the orthogonal line spot, thus obtaining a strip image I. +45° The other probe detects a corresponding beam of signal light, obtaining a strip image I. -45° When a single area array detector, in conjunction with a second cross-shaped mask 20, simultaneously detects two orthogonal line spots, it acquires two signal beams corresponding to the two orthogonal line spots. The two signal beams are then separated, and strip images I are obtained separately. +45° I -45° Regardless of whether one or two detectors are used, because each line spot is tilted, the actual spatial sampling rate in the horizontal direction during scanning is [missing information]. Pixel detector For the size of detector 19 on the focal plane of objective lens 13, Pixel scan This refers to the equivalent pixel size during tilted scanning. For example, if the pixel size of detector 19 is 6.5μm × 6.5μm, and imaging is performed using objective lens 13 with a magnification of 20×, then the size of detector 19 on the focal plane of objective lens 13, which is the lateral spatial sampling rate, is Pixel. detector =0.325μm×0.325μm, and in this tilted scanning mode, the actual lateral spatial sampling rate is

[0041] Furthermore, as shown in Figures 11 and 12, the imaging device also includes an image processing module for processing the strip images I obtained by the detector 19 from multiple consecutive frames. +45° with I -45° Perform pixel repositioning and fusion calculations to obtain the fused image I. recon That is, the focal plane image of the sample surface.

[0042] Furthermore, when detector 19 takes multiple consecutive frames of images of the sample surface, sample stage 14 drives the sample to move at a constant speed along the diagonal direction of the orthogonal linear light spot 15, with a speed v = Pixel scan / t exposure , where t exposure For the detector, 19 single-frame exposure times, Pixel scan This is the equivalent pixel size during tilted scanning.

[0043] Furthermore, the imaging device also includes a cutting module (not shown in the figure). After obtaining the focal plane image of the sample surface, the cutting module removes the imaged sample surface to expose a new sample surface. The detector 19 can then be used repeatedly to capture multiple consecutive frames of the new sample surface to obtain a new focal plane image of the sample surface. By removing the image layer by layer through the cutting module, rapid three-dimensional imaging of the entire large sample can be achieved.

[0044] Specifically, as shown in Figure 11, the image processing module processes the strip image I. +45° with I -45° The specific process of pixel realignment can be as follows: I +45° (i, n) → I +45° (i, i+n), I -45° (n, j)→I -45° (m-j+n, j); where n represents the nth frame image result of detector 19, and i represents the strip image I. +45° The i-th pixel in the n-th row, j represents the strip image I. -45° The j-th pixel in the n-th row of the image, where m is the total number of pixels in a single row of the image.

[0045] Figure 11(a) shows the scanning of a standard image (as a sample) commonly used in the field of image processing using the orthogonal line spot 15 of this application. The standard image moves along the x-direction, which is the direction in which the sample stage moves the sample. Figure 11(b) shows the strip image I. +45° In Figure 11(c), it represents the strip image I. +45° The repositioned image after pixel repositioning. It can be observed that the strip image I... +45° I -45°The size of the image is m×n, and the size of the image after the pixels are returned to their true positions is m×(m+n), where m represents the total number of pixels in a row in a single frame image, and n represents the number of image frames detected by the detector.

[0046] The fusion calculation in the image processing module specifically involves combining two strip images I +45° with I -45° After pixel repositioning, the pixels are multiplied and fused using the point spread function. Specifically, the point spread function (PSF) of an optical system characterizes the spatial resolution of the imaging device; the PSF for single-beamline spot imaging is denoted as PSF. LC It can be expressed as the illumination point spread function (PSF). ill and probe point spread function PSF det The product of, i.e.: PSF LC =PSF ill ×PSF det ;

[0047] Among them, the illumination point spread function (PSF) ill It is the point spread function (PSF) of linear array illumination. line Point spread function (PSF) of the objective lens obj The convolution, i.e.:

[0048] Among them, the probe point spread function (PSF) det It is the pixel size of the detector and the point spread function (PSF) of the objective lens. obj The convolution, i.e.:

[0049] therefore,

[0050] The point spread function of the image formed by another beam spot in the orthogonal direction corresponding to the single beam spot is: The superscript T indicates matrix transpose;

[0051] therefore,

[0052] Fusion: Furthermore, under ideal conditions, PSF obj Since it is an impulse function, after simplification,

[0053] In the field of optical imaging, single-beam line scanning imaging systems exhibit good longitudinal resolution but uneven lateral resolution. This application employs two line spots with the same focal plane and perpendicular focusing directions for simultaneous imaging. After fusion, the lateral resolution achieves the same advantage as the longitudinal resolution of the other line spot in the orthogonal direction, thus achieving greater uniformity in lateral resolution compared to single-beam line spot imaging. Furthermore, this method utilizes a point illumination imaging device under ideal conditions. It can be observed that the PSF of the line illumination imaging device of this application is consistent with that of the point illumination imaging device in the prior art, which further illustrates that the imaging device of this application can achieve improved lateral resolution, homogenization and tomography capabilities, and achieve the same imaging quality as the point illumination imaging device. Moreover, the line illumination imaging device has a faster imaging speed than the point illumination imaging device and is suitable for large-size samples. Therefore, this application can achieve fast and high-quality imaging of large-size samples.

[0054] During the aforementioned fusion process, it can be achieved through (Pixel) 2 It was found that the dynamic range of the reconstructed images was improved, for example, in strip image I. +45° with I -45° For a 16-bit image, after the fusion algorithm, I recon Convert to a 32-bit image. A 32-bit image has a higher dynamic range, which is beneficial for displaying samples with rich details and complex structures. Of course, a 32-bit image will also increase the amount of data. Therefore, this 32-bit image can also be processed into a 16-bit image through methods such as square root operations, which can avoid increasing the amount of data.

[0055] As shown in Figure 12, two orthogonal linear light spots 15 of this application were used to image a 100 μm thick slice of the Thy1-eGFP-labeled mouse brain. Figure 12 shows the dendritic location of neurons in the brain slice, where multiple dendritic spines are present. Figure 12(a) shows the strip image I acquired by the detector under illumination from one of the two orthogonal linear light spots 15 at a -45° angle. -45° Figure 12(b) shows the strip image I acquired by the detector under illumination from one of the two orthogonal line spots 15 at a +45° angle. +45° In Figure 12(c), the band image I is shown. -45° The repositioned image after pixel repositioning, in Figure 12(d) represents the strip image I. +45° The pixel-aligned image, in Figure 12(e), shows the fused image I obtained by fusing the two aligned images in Figure 12(c) and Figure 12(d). reconThis refers to the focal plane image of the sample. It can be observed that the dendritic spines on the neurons of the mouse brain are on the order of micrometers. When imaging with a line illumination imaging device, due to the non-uniform lateral resolution, shape changes easily occur, as seen in Figure 12(c) and Figure 12(d), where the dendritic spines exhibit tailing in the non-focusing direction. However, this error can be avoided after reconstruction using the orthogonal line illumination imaging device of this application. Furthermore, compared to the single-direction line scan imaging results in Figures 12(c) and 12(d), the final imaging result of orthogonal line illumination in Figure 12(e) also shows an improved signal-to-background ratio, indicating that this application can improve the tomographic capability of the line scan imaging system.

[0056] Specifically, the cutting module performs a flat-push cutting motion on the sample surface. The sample stage 14, used to hold the sample, not only moves the sample along the X-direction but also has a lifting function. After the sample surface is imaged, the sample stage 14 lifts the sample a distance equal to the required cutting thickness. During the cutting process, the sample stage 14 can remain stationary while the cutting tool in the module moves in a flat-push motion, or the tool can remain stationary while the sample stage moves parallel. When a new sample surface is exposed after cutting, it can be brought under the objective lens by the sample stage for re-image formation. Through the coordinated movement of the cutting tool and the sample stage in the cutting module, layer-by-layer cutting and imaging of the sample can be achieved, thus enabling rapid three-dimensional imaging of large-size samples.

[0057] The following section verifies the imaging effect of the two orthogonal line spots in this application.

[0058] Figure 13 shows a comparison of simulation results of point confocal imaging, single-direction line scan imaging, and orthogonal line scan imaging of this application in terms of three-dimensional spatial resolution and tomographic capability. In Figure 13, (a), (b), and (c) represent the point spread function of point confocal imaging, single-direction line scan imaging, and orthogonal line scan imaging on the focal plane of the objective lens, respectively. It can be found that single-direction line scan imaging has the problem of uneven resolution distribution in the focusing and non-focusing directions, while orthogonal line scan imaging can effectively solve this problem.

[0059] In Figure 13, circles (d), (e), (f), and (g) represent simulation results of point confocal imaging (LSCM), triangles (△) represent simulation results of single-direction line scanning imaging (LS), and solid lines (-) represent simulation results of orthogonal line scanning imaging (cLS). The simulation conditions are: numerical aperture of the objective lens is NA = 1, magnification is 20×, and excitation wavelength is λ. ex =488nm, the signal light emission wavelength is λ em =505nm; detector pixel size is Pixel = 6.5μm × 6.5μm.

[0060] Figure 13(d), (e), and (f) show the point spread function curves in the X, Y, and Z directions of three-dimensional space for point confocal imaging, single-direction line scan imaging, and orthogonal line scan imaging, respectively. The full width at half maximum (FWHM) of the curve represents the spatial resolution in that dimension; the narrower the FWHM, the higher the spatial resolution in that dimension. It can be observed that in Figure 13(d), the spatial resolution of the three imaging systems in the X direction is almost identical; in Figure 13(e), the single-direction line scan imaging has poor spatial resolution in the Y direction, while the orthogonal line scan imaging achieves the same level of spatial resolution as point confocal imaging in the Y direction; in Figure 13(f), the orthogonal line scan imaging has better spatial resolution in the Z direction than the single-direction line scan imaging, and the single-direction line scan imaging has better spatial resolution than the point confocal imaging. Comparing Figure 13(d) and Figure 13(e), it can be seen that LS-x = 0.32 μm and LS-y = 0.42 μm in unidirectional line scan imaging, which are not equal, while LSCM-x = 0.32 μm and LSCM-y = 0.32 μm in point confocal imaging, which are equal. This means that point confocal imaging has a uniform resolution distribution in the lateral direction, while unidirectional line scan imaging has an uneven resolution distribution in the lateral direction. In the orthogonal line scan imaging of this application, cLS-x = cLS-y = 0.32 μm, indicating that it can overcome the problem of uneven resolution in the lateral direction of line scan imaging. Furthermore, regarding axial resolution, referring to Figure 13(f), cross-z = 0.54 μm, representing the axial resolution of orthogonal line scan imaging, has the narrowest full width at half maximum (FWHM) and the best axial resolution.

[0061] Figure 13(g) compares the tomographic capabilities of point confocal imaging, single-direction line scanning imaging, and orthogonal line scanning imaging. The narrower the full width at half maximum (FWHM) of the curve, the better its tomographic capability. It can be observed that the tomographic capability of orthogonal line scanning imaging is comparable to or even better than that of point confocal imaging.

[0062] Those skilled in the art will readily understand that the above description is merely a preferred embodiment of this application and is not intended to limit this application. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of this application should be included within the scope of protection of this application.

Claims

1. An orthogonal line illumination optical three-dimensional imaging device, characterized in that, The system includes an optical modulation module and an optical detection module: the optical modulation module provides two orthogonal line spots that are projected onto the surface of the sample, the two orthogonal line spots have the same focal plane and their focal directions are perpendicular to each other; the optical detection module includes a detector, and an objective lens and a dichroic mirror are sequentially arranged between the sample and the detector to detect the signal light generated by the sample after being excited by the orthogonal line spots; the optical detection module also includes a sample stage to move the sample along the diagonal direction of the two orthogonal line spots.

2. The orthogonal line illumination optical three-dimensional imaging device according to claim 1, characterized in that, The optical modulation module provides two excitation beams and includes a beam combiner that combines the two excitation beams. The beam combiner also includes a first optical element and a second optical element that convert the excitation beams into line spots on the two incident paths of the beam combiner. The focusing directions of the first optical element and the second optical element are perpendicular so that the two excitation beams are combined to form two orthogonal line spots.

3. The orthogonal line illumination optical three-dimensional imaging device according to claim 2, characterized in that, The optical modulation module includes two laser emitters with the same center wavelength, each laser emitter providing a beam of excitation light; or the optical modulation module includes one laser emitter, and a beam splitter is provided on the output path of the laser emitter to split the beam of excitation light provided by the laser emitter into two beams of excitation light; the two beams of excitation light are P-polarized light and S-polarized light, respectively, and the light intensities are distributed in a 1:1 ratio.

4. The orthogonal line illumination optical three-dimensional imaging device according to claim 1, characterized in that, The optical modulation module provides an excitation beam, including a laser emitter, and a spatial light modulator, a first cross-shaped mask, or a digital micromirror device is disposed in the emission path of the laser emitter.

5. The orthogonal line illumination optical three-dimensional imaging device according to claim 1, characterized in that, The detector is an area array detector, and a second cross-shaped mask is also provided on the incident path of the area array detector.

6. The orthogonal line illumination optical three-dimensional imaging device according to claim 1, characterized in that, The detector consists of two linear array detectors. A beam splitter is also provided between the dichroic mirror and the detector, so that the signal light enters the two linear array detectors respectively, and the two linear array detectors are conjugate with the two line light spots respectively.

7. The orthogonal line illumination optical three-dimensional imaging device according to claim 1, characterized in that, The detector captures multiple consecutive frames of images of the sample surface using a strip scanning method.

8. The orthogonal line illumination optical three-dimensional imaging device according to claim 7, characterized in that, It also includes an image processing module for processing the strip images I obtained by the detector from multiple consecutive frames. +45° with I -45° Perform pixel repositioning and fusion calculations to obtain the fused image I. recon The strip image I +45° with I -45° It is formed by stitching together the center row pixels of multiple frames captured by the detector in chronological order, or by stitching together the single frame processing result obtained after processing a single frame / adjacent multiple frames image with a line illumination modulation algorithm in chronological order.

9. The orthogonal line illumination optical three-dimensional imaging device according to claim 7, characterized in that, It also includes a sample stage, which drives the sample to move at a constant speed along the diagonal direction of the orthogonal linear light spot, with a speed v = Pixel. scan / t exposure , where t exposure For the single-frame exposure time of the detector, Pixel scan This is the equivalent pixel size during tilted scanning.

10. The orthogonal line illumination optical three-dimensional imaging device according to claim 9, characterized in that, It also includes a cutting module for removing the surface of the imaged sample to expose a new sample surface.

Images