A microscope illumination angle calibration device and method

Abstract

The application discloses a microscope illumination angle calibration device and method, comprising a phantom, a first projection auxiliary imaging element, a second projection auxiliary imaging element and a correction calculation unit, the phantom is provided with a light transmission pinhole, the first projection auxiliary imaging element adjusts the position of a microscope objective of an imaging system, so that a camera of the microscope imaging system can acquire phantom images under different illumination angles of a to-be-tested illumination system and a projection point of the light transmission pinhole in the phantom images, the second projection auxiliary imaging element adjusts the position of the microscope objective, so that light rays with different illumination angles emitted by the to-be-tested illumination system can be projected to the first projection auxiliary imaging element through the light transmission pinhole, and the correction calculation unit calculates the illumination angle under the illumination of the i-th LED according to the intersection point of straight lines formed by the light rays of all the i-th LEDs passing through the p-th light transmission pinhole.

Description

Technical Field

[0001] This invention relates to the field of imaging technology, and in particular to a device and method for calibrating the illumination angle of a microscope. Background Technology

[0002] Microscopic optical imaging is a primary means of acquiring cross-scale information about biological systems, playing a crucial role in structural and functional studies at all levels, from molecules and cells to tissues and organs. Compared to fluorescently labeled imaging, which has strict requirements on the sample, a limited number of fluorescent labeling channels, and the potential for photobleaching and phototoxicity of fluorescent groups, microscopic optical imaging provides label-free imaging that ensures the sample's own life activities are not affected by the imaging process itself. Therefore, it is widely used in fields such as digital pathology, cancer treatment, regenerative medicine, and personalized medicine.

[0003] With the development of computational imaging technology, various label-free imaging techniques based on light field propagation models have emerged in recent years, such as Fourier Ptychography Microscopy (FPM) and Optical Diffractive Tomographic Microscopy (ODT). The core idea of ​​these imaging methods is that, under plane wave illumination modulation from different directions, the imaging system detects different frequency domain components of the sample. By establishing a propagation model of the interaction between the sample and the light field, the reconstruction algorithm demodulates physical features of the sample that are not detectable by conventional microscopes, such as refractive index distribution and absorption coefficient. However, in practical applications, reconstruction based on plane wave illumination modulation relies on an accurate imaging model. The illumination angle directly corresponds to the spatial position of the sample's frequency domain components, and alignment errors in the illumination angle can lead to severe reconstruction artifacts. Therefore, an effective and accurate illumination angle calibration method is crucial.

[0004] In 2018, Regina Eckert et al. proposed an illumination angle calibration method based on image spectral distribution characteristics. This method involves two steps: first, the illumination angle of the bright-field image is calibrated by analyzing the spectrum of the image under tilted plane wave illumination; then, the illumination angles of the bright-field and dark-field images are finely calibrated using spectral correlation. This method has shown robustness in angle calibration with various microscope illumination devices, such as flat-panel LED illumination, laser scanning illumination, and dome-shaped LED illumination. However, the circular edge detection used in this method requires a sufficiently broad Fourier spectrum to encompass the boundaries of two separate circles within the image intensity spectrum, thus necessitating an image with a smaller number of sampling pixels than required by the sampling theorem.

[0005] In 2016, Jiasong Sun et al. proposed an iterative global position correction method for illumination angles based on simulated annealing and nonlinear regression. This method is efficient and robust to noise. In 2018, Ao Zhou et al. introduced a global position misalignment model and updated parameters in addition to the reconstruction algorithm, making their method significantly faster than previous methods. Both the calibration methods proposed by Jiasong Sun et al. and Ao Zhou et al. require LEDs to be arranged according to rules and pre-aligned. Otherwise, the method may become time-consuming if there is a significant misalignment in the illumination angle.

[0006] Therefore, it is desirable to have a technical solution to overcome or at least mitigate at least one of the aforementioned defects of the prior art. Summary of the Invention

[0007] The purpose of this invention is to provide a device and method for calibrating the illumination angle of a microscope, which can achieve position calibration of the illumination angle without relying on prior information about the illumination angle position and the sample.

[0008] To achieve the above objectives, the present invention provides a microscope illumination angle calibration device, comprising a phantom, a first projection-aided imaging element, a second projection-aided imaging element, and a correction calculation unit, wherein:

[0009] The mold body is provided with light-transmitting holes that penetrate both sides of the mold body;

[0010] The first projection-assisted imaging element is used to assist in adjusting the position of the microscope objective of the imaging system, enabling the camera of the microscope imaging system to acquire images of the phantom under different illumination angles of the illumination system under test, as well as the projection point of the light-transmitting aperture in the phantom image. The second projection-assisted imaging element is disposed between the phantom and the first projection-assisted imaging element, and is used to assist in adjusting the position of the microscope objective, enabling light emitted by the illumination system under test at different illumination angles to be projected onto the first projection-assisted imaging element through the light-transmitting aperture. The correction calculation unit is used to calculate the corrected image based on all straight lines. The intersection point x i =(x i ,y i ,z i ), calculate the illumination angle k under the illumination of the i-th LED in the lighting system under test. i , Let be the straight line formed by the light from the i-th LED passing through the p-th aperture.

[0011] Furthermore, the first projection-assisted imaging element uses a single-sided frosted glass plate, with its non-frosted side facing the microscope objective.

[0012] Furthermore, the second projection-assisted imaging element adopts a ring-shaped structure with a central through hole, allowing light emitted from the illumination system under test at different illumination angles to pass through the light-transmitting holes of the model and reach the surface of the first projection-assisted imaging element.

[0013] Furthermore, the method for obtaining the position coordinates of the p-th light-transmitting aperture includes:

[0014] Using a second projection-assisted imaging element, the microscope objective is focused on the surface of the phantom facing the second projection-assisted imaging element. The position of the microscope objective at this point is denoted as z2. All LEDs of the illumination system under test are simultaneously illuminated. The coordinates of the p-th projection point in the obtained phantom image are then determined. Let h be the position coordinates of the p-th light-transmitting aperture, where h = z2 - z1. Let p = 1, 2, ..., N, where N is the total number of light-transmitting holes, and z1 is the reference position of the microscope objective.

[0015] Furthermore, the method for obtaining the position coordinates of the p-th projection point of the light-transmitting aperture includes:

[0016] The first projection-assisted imaging element is used to assist the microscope objective in focusing on the frosted surface. The position of the microscope objective at this time is recorded as z1. The LEDs of the illumination system under test are illuminated sequentially. The coordinates of the p-th projection point in the phantom image obtained when the i-th LED is illuminated are recorded. Let be the coordinates of the projection point of the p-th light-transmitting aperture.

[0017] Furthermore, all lines are solved using the least squares method shown in the following equation. The intersection point x i =(x i ,y i ,z i ):

[0018]

[0019] In the formula, Let x represent the position of the i-th LED to be determined. i =(x i ,y i ,z i ) and straight line The distance.

[0020] The present invention also provides a method for calibrating the illumination angle of a microscope, comprising:

[0021] Step 1: Place the first projection-assisted imaging element on the microscope sample stage, and focus the microscope objective of the imaging system on the surface of the first projection-assisted imaging element facing the illumination system to be tested. The position of the microscope objective at this time is recorded as z1.

[0022] Step 2: Place the phantom with the light-transmitting aperture on the surface of the first projection-assisted imaging element facing the illumination system under test, so that the camera of the microscopic imaging system can acquire images of the phantom under different illumination angles of the illumination system under test and the projection point of the light-transmitting aperture in the phantom image.

[0023] Step 3: First, place the second projection auxiliary imaging element between the phantom and the first projection auxiliary imaging element so that the light emitted by the illumination system under test at different illumination angles can be projected onto the first projection auxiliary imaging element through the light-transmitting aperture. Then, focus the microscope objective on the surface of the phantom facing the second projection auxiliary imaging element. The position of the microscope objective at this time is recorded as z2.

[0024] Step 4: Control the LED illumination mode of the illumination system under test, and obtain the position coordinates of the p-th aperture of the microscope objective in the phantom image of z2 and the position coordinates of the projection point of the p-th aperture of the microscope objective in the phantom image of z1.

[0025] Step 5, based on all straight lines The intersection point x i =(x i ,y i ,z i ), calculate the illumination angle k under the illumination of the i-th LED in the lighting system under test. i , Let be the straight line formed by the light from the i-th LED passing through the p-th aperture.

[0026] Furthermore, in step 4, the method for obtaining the position coordinates of the p-th aperture of the microscope objective in the phantom image of z2 includes:

[0027] Simultaneously illuminate all LEDs of the lighting system under test, and obtain the position coordinates of the p-th light-transmitting hole in the phantom image. h = z2 - z1, Let p = 1, 2, ..., N, where N is the total number of light-transmitting holes in the phantom image.

[0028] In further step 4, the method for obtaining the position coordinates of the p-th aperture projection point of the microscope objective in the phantom image of z1 includes:

[0029] The LEDs of the illumination system under test are illuminated sequentially. The coordinates of the p-th projection point in the phantom image obtained when the i-th LED is illuminated are... Let be the coordinates of the projection point of the p-th light-transmitting aperture.

[0030] Furthermore, all lines are solved using the least squares method shown in the following equation. The intersection point x i =(xi ,y i ,z i ):

[0031]

[0032] In the formula, Let x represent the position of the i-th LED to be determined. i =(x i ,y i ,z i ) and straight line The distance.

[0033] Furthermore, the first projection-assisted imaging element uses a single-sided frosted glass plate, with its non-frosted side facing the microscope objective.

[0034] Furthermore, the second projection-assisted imaging element adopts a ring-shaped structure with a central through hole, allowing light emitted from the illumination system under test at different illumination angles to pass through the light-transmitting holes of the model and reach the surface of the first projection-assisted imaging element.

[0035] There are three differences between this method and the two types of calibration methods mentioned earlier:

[0036] 1. This invention is an offline calibration method performed before imaging and image reconstruction of the sample.

[0037] 2. This invention is a geometric calibration method that does not require local searching. Therefore, it has no requirements on the illumination angle position and does not require pre-alignment of the illumination angle.

[0038] 3. This invention does not rely on the image reconstruction process of the samples, therefore there are no special requirements for the image sampling rate. Attached Figure Description

[0039] Figure 1 This is a schematic diagram of a microscope illumination angle calibration device in an embodiment of the present invention;

[0040] Figure 2 yes Figure 1 A schematic diagram of the structure of an embodiment of the second projection-assisted imaging element;

[0041] Figure 3 This is a schematic diagram of a microscope illumination angle calibration method in an embodiment of the present invention;

[0042] Figure 4 yes Figure 1 The projection image of the phantom with light-transmitting holes was obtained by the imaging system in the image;

[0043] Figure 5 This is a schematic diagram illustrating the solution of the intersection point of lines using the least squares method in an embodiment of the present invention;

[0044] Figure 6 This is a schematic diagram comparing the LED coordinate position and illumination angle in an embodiment of the present invention before and after calibration. Detailed Implementation

[0045] In the accompanying drawings, the same or similar reference numerals are used to denote the same or similar elements or elements having the same or similar functions. The embodiments of the present invention will now be described in detail with reference to the accompanying drawings.

[0046] In the description of this invention, the terms "center," "longitudinal," "lateral," "front," "rear," "left," "right," "vertical," "horizontal," "top," "bottom," "inner," and "outer," etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They are used only for the convenience of describing this invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limiting the scope of protection of this invention.

[0047] like Figure 1 As shown, the microscope illumination angle calibration device provided in this embodiment of the invention is used to calibrate the illumination angle of the illumination system 1 under test.

[0048] In one embodiment, the microscope illumination angle calibration device includes a phantom 1, a first projection-aided imaging element 2, a second projection-aided imaging element 3, and a correction calculation unit, wherein:

[0049] Please refer to the reference. Figures 4-6 The mold 1 is provided with a light-transmitting hole that extends through both sides of the mold 1, allowing light emitted from the illumination system 4 located on one side A of the mold 1 to pass through and reach the other side B of the mold 1. For ease of description, from... Figure 1 As shown in the paper view, A indicates the upper side of mold 1 and B indicates the lower side of mold 1. However, in actual use, the direction shown in the figure is not strictly followed. It can be adjusted to the left and right or even other directions according to actual needs.

[0050] Furthermore, the shape, spacing, and number of the apertures can be designed according to the illumination angle of the illumination system in the actual microscope system. The shape of the apertures can be a dot, cross, star, etc., with the selection principle being to facilitate the calculation of position coordinates on the image. The number of apertures determines the number of equations when calculating the illumination angle using the least squares method, and in principle should be greater than or equal to 3. The spacing of the apertures is set such that the projection points of the apertures in the phantom image 8 corresponding to different illumination angles of the illumination system 4 under test exactly fill the entire field of view of the imaging system. The imaging system refers to the objective lens 6, the tube lens, and the camera 7 in the microscope.

[0051] like Figure 1 As shown, the first projection auxiliary imaging element 2 is placed on the microscope sample stage 5 and arranged on the other side B of the phantom 1. It is used to adjust the position of the microscope objective 6 of the imaging system so that the camera 7 of the microscope imaging system can acquire the phantom image 8 under different illumination angles of the illumination system 4 under test and the projection point of the light-transmitting aperture in the phantom image 8.

[0052] The first projection-assisted imaging element 2 uses a glass slide made of an optical scattering material. The optical scattering material can be various scattering and light-monopolizing materials such as quartz glass, plain white glass, and ultra-white glass. The shape can be square, rectangular (such as a 25*75mm frosted biological slide), polygonal, etc.

[0053] Furthermore, at least one side of the glass slide is provided with an outer layer to eliminate the effect of light refraction from air into the glass slide. This outer layer is positioned away from the microscope objective 6. Therefore, when the microscope objective 6 focuses on the outer layer used to eliminate the effect of light refraction from air into the glass slide, it can eliminate the effect of light refraction from air into the glass slide, thereby making subsequent calculation results more accurate. Here, "away from" can be understood as the opposite direction to "facing".

[0054] For example, the outer layer of the first projection-assisted imaging element 2, used to eliminate the effect of light refraction from air into the glass slide, is a frosted layer, which is obtained by processing methods such as chemical etching or physical grinding. Preferably, the first projection-assisted imaging element 2 uses a glass slide with a single-sided frosted layer, with its non-frosted side facing the microscope objective 6.

[0055] like Figure 2 As shown, the second projection auxiliary imaging element 3 is disposed between the phantom 1 and the first projection auxiliary imaging element 2, and is used to adjust the position of the microscope objective 6 so that the light emitted by the illumination system 4 under test at different illumination angles can be projected onto the first projection auxiliary imaging element 2 through the light-transmitting aperture.

[0056] In one embodiment, such as Figure 2 As shown, the second projection-assisted imaging element 3 adopts a ring-shaped structure with a central through hole, so that the light emitted by the illumination system 4 under test at different illumination angles can pass through the light-transmitting hole of the model 1 and reach the surface of the first projection-assisted imaging element 2, such as the upper surface.

[0057] Meanwhile, the height of the second projection auxiliary imaging element 3 makes the phantom 1 higher than the first projection auxiliary imaging element 2, so that the light emitted by the illumination system 4 at different illumination angles will be projected onto different positions on the upper surface of the first projection auxiliary imaging element 2 through the light-transmitting hole of the phantom 1.

[0058] Of course, the second projection-assisted imaging element 3 can be in other shapes besides a circular ring structure, such as a square ring or a polygonal ring, as long as it has a central through hole so that light emitted from the illumination system 4 at different illumination angles can pass through the light-transmitting hole of the mold 1 and reach the upper surface of the first projection-assisted imaging element 2. The material and manufacturing process of the second projection-assisted imaging element 3 have no special requirements; it can be 3D-printed resin material, machined metal material, etc.

[0059] The correction calculation unit is used to calculate based on all straight lines. The intersection point x i =(x i ,y i ,z i ), calculate the illumination angle k under the illumination of the i-th LED in the lighting system 4 under test. i , Let k be the straight line formed by the light from the i-th LED passing through the p-th aperture. The illumination angle is k. i The calculation formula is shown in equation (1) below:

[0060]

[0061] Where k0 = 2π / λ is the illumination wave vector in vacuum, and λ is the wavelength of the illumination light.

[0062] In one embodiment, the method for obtaining the position coordinates of the p-th light-transmitting aperture includes:

[0063] Using the second projection-assisted imaging element 3 to assist the microscope objective 6 in focusing on the surface of the phantom 1 facing the second projection-assisted imaging element 3, the position of the microscope objective 6 at this time is recorded as z2. All LEDs of the illumination system 4 under test are illuminated simultaneously, and the coordinates of the p-th projection point in the obtained phantom image (8) are obtained. Let h be the position coordinates of the p-th light-transmitting aperture, where h = z2 - z1. The horizontal coordinates of the light-transmitting apertures in phantom image 8 can be obtained through image processing algorithms, where p = 1, 2, ..., N, N is the total number of light-transmitting apertures, and z1 is the reference position of microscope objective 6.

[0064] In one embodiment, the method for obtaining the position coordinates of the p-th projection point of the light-transmitting aperture includes:

[0065] The first projection-assisted imaging element 2 is used to assist the microscope objective 6 in focusing on the frosted surface. The position of the microscope objective 6 at this time is recorded as z1. The LEDs of the illumination system 4 under test are illuminated sequentially. The coordinates of the p-th projection point in the p-th projection point of the phantom image 8 obtained when the i-th LED is illuminated are recorded. Let be the coordinates of the projection point of the p-th light-transmitting aperture.

[0066] In one embodiment, such as Figure 5 As shown, all straight lines are solved using the least squares method shown in equation (2). The intersection point x i =(x i ,y i ,z i ):

[0067]

[0068] In the formula, Let x represent the position of the i-th LED to be determined. i =(x i ,y i ,z i ) and straight line The distance.

[0069] It should be noted that the intersection point x i =(x i ,y i ,z i In addition to using the least squares method provided in the above embodiments, other algorithms such as matrix pseudo-inverse, solving linear equations, gradient descent, and iterative optimization can also be used to obtain the result.

[0070] The lighting system under test provides tilted lighting at different angles, and the lighting methods can be flat LED lighting, laser scanning lighting, dome-shaped LED lighting, etc.

[0071] The following example uses a flat LED illumination panel, where each LED is arranged in a 15*15 array with a spacing of approximately 4mm, to illustrate the microscope illumination angle calibration method provided by this invention. Figure 3 As shown. For other lighting methods, such as laser scanning lighting and dome-shaped LED lighting, the calibration methods and procedures for the lighting angle are the same. Figure 3 In the coordinate system, xy refers to the sample plane, and the center of the imaging system's field of view is the origin of xy. In the coordinate system, z refers to the focusing movement direction of the microscope objective, and the focal plane of the imaging system is the origin of the z-axis.

[0072] Step 1: Place the first projection-assisted imaging element 2 on the microscope sample stage 5. Focus the microscope objective 6 of the imaging system on the surface of the first projection-assisted imaging element 2 facing the illumination system 4 under test. Record the position of the microscope objective 6 as z1. Before focusing, a mark can be printed or handwritten on the surface of the first projection-assisted imaging element 2 to assist in clear focusing.

[0073] For example, when the first projection-assisted imaging element 2 is a glass slide with a single-sided frosted layer, and the non-frosted layer of the glass slide faces the microscope objective 6, then when the position of the microscope objective 6 is recorded as z1, the microscope objective 6 is focused on the frosted surface of the first projection-assisted imaging element 2.

[0074] Step 2: Place the phantom 1 with the light-transmitting aperture on the surface of the first projection auxiliary imaging element 2 facing the illumination system 4 under test, so that the camera 7 of the microscopic imaging system can acquire the phantom image 8 under different illumination angles of the illumination system 4 under test and the projection point of the light-transmitting aperture in the phantom image 8.

[0075] Step 3: First, place the second projection auxiliary imaging element 3 between the phantom 1 and the first projection auxiliary imaging element 2, so that the light emitted by the illumination system 4 under test at different illumination angles can be projected onto the first projection auxiliary imaging element 2 through the light-transmitting aperture. Then, focus the microscope objective 6 on the surface of the phantom 1 facing the second projection auxiliary imaging element 3. At this time, the position of the microscope objective 6 is recorded as z2.

[0076] Step 4: Control the LED illumination mode of the illumination system 4 under test, and obtain the position coordinates of the p-th light-transmitting aperture of the microscope objective 6 in the phantom image 8 of z2 and the position coordinates of the projection point of the p-th light-transmitting aperture of the microscope objective 6 in the phantom image 8 of z1.

[0077] Step 5, based on all straight lines The intersection point x i =(x i ,y i ,z i ), calculate the illumination angle k under the illumination of the i-th LED in the lighting system 4 under test. i , Let be the straight line formed by the light from the i-th LED passing through the p-th aperture.

[0078] In one embodiment, in step 4, the method for obtaining the position coordinates of the p-th aperture of the microscope objective 6 in the phantom image 8 of z2 includes:

[0079] Simultaneously illuminate all LEDs in the illumination system 4 under test, acquire the phantom image 8 under bright field illumination through the imaging system, and calculate the position coordinates of the p-th light-transmitting aperture in the phantom image 8. in, Let p = 1, 2, ..., N, where N is the total number of light-transmitting holes in phantom image 8.

[0080] Of course, those skilled in the art can also use other existing methods to obtain the position coordinates of the p-th light-transmitting aperture in the phantom image 8 when the microscope objective 6 is at position z2. For example, the position coordinates of the p-th light-transmitting aperture in the phantom image 8 when the microscope objective 6 is at position z2 can be obtained by calculating the origin moment of the image, performing Gaussian fitting on the image to obtain the center position of the spot, calculating the position of the maximum gray value of the image, etc.

[0081] In one embodiment, in step 4, the method for obtaining the position coordinates of the p-th aperture projection point of the microscope objective 6 in the phantom image 8 of z1 includes:

[0082] The LEDs of the controlled illumination system 4 are illuminated sequentially. The coordinates of the p-th projection point in the p-th image 8 obtained when the i-th LED is illuminated are... Let be the coordinates of the projection point of the p-th light-transmitting aperture. Please refer to... Figure 4 Move the microscope objective 6 back to position z1, illuminate each LED sequentially, and capture image 8 of the phantom. Figure 3 The bright spots in each image are projections of the small holes on the mold when a single LED is illuminated. Since there are 225 LEDs illuminated sequentially in this example, therefore... Figure 4 There are 225 projected images.

[0083] It should be noted that those skilled in the art can also use other existing methods to obtain the position coordinates of the p-th light-transmitting aperture projection point in the phantom image 8 when the microscope objective 6 is at position z1, such as: calculating the image origin moment, performing Gaussian fitting on the image to obtain the center position of the spot, calculating the position of the maximum gray value of the image, etc., thereby obtaining the position coordinates of the p-th light-transmitting aperture projection point in the phantom image 8 when the microscope objective 6 is at position z1.

[0084] Figure 6 The comparison results are shown for calibrated and uncalibrated scenarios, where green dots represent the results after illumination angle calibration, and black dots represent the results under the uncalibrated scenario assuming uniform LED distribution. Figure 6 The comparison results show that, in the uncalibrated case, the LED positions are assumed to be uniformly distributed. The calibration method of this invention achieves LED position calibration without relying on an LED presentation matrix or other regular arrangements. Furthermore, the calibration method of this invention is sample-independent and achieves incident angle alignment before reconstruction, thus not affecting the algorithm's reconstruction iteration process.

[0085] The microscope illumination angle calibration method of this invention is compatible with various tilting illumination systems and has no special requirements for the arrangement of illumination angles. Furthermore, this illumination angle calibration method is not mutually exclusive with other online calibration methods and can provide more accurate initial values ​​for existing online calibration schemes. After this illumination angle calibration, the calibrated illumination angle is substituted into the imaging model, such as FPM or ODT, which are highly dependent on the accurate depiction of the actual physical imaging model, to obtain high-quality image reconstruction results.

[0086] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Those skilled in the art should understand that modifications can be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A device for calibrating the illumination angle of a microscope, characterized in that, It includes a phantom (1), a first projection-assisted imaging element (2), a second projection-assisted imaging element (3), and a correction calculation unit, wherein: The mold (1) is provided with light-transmitting holes that penetrate both sides of the mold (1) so that the light emitted by the illumination system (4) located on one side of the mold (1) can pass through and reach the other side of the mold (1). The number of light-transmitting holes is greater than or equal to 3. The first projection auxiliary imaging element (2) is used to adjust the position of the microscope objective (6) of the imaging system so that the camera (7) of the microscope imaging system can acquire the phantom image (8) of the illumination system (4) under different illumination angles and the projection point of the light-transmitting aperture in the phantom image (8). The first projection auxiliary imaging element (2) is made of a single-sided frosted glass plate with its non-frosted surface facing the microscope objective (6). The first projection auxiliary imaging element is arranged on the other side of the phantom (1). The second projection auxiliary imaging element (3) is set between the phantom (1) and the first projection auxiliary imaging element (2). It is used to adjust the position of the microscope objective (6) so that the light emitted by the illumination system (4) under test at different illumination angles can be projected onto the first projection auxiliary imaging element (2) through the light-transmitting hole. The second projection auxiliary imaging element (3) adopts a ring structure and has a central through hole so that the light emitted by the illumination system (4) under test at different illumination angles can reach the surface of the first projection auxiliary imaging element (2) through the light-transmitting hole of the phantom (1). The correction calculation unit is used to calculate based on all straight lines. intersection Calculate the first step of the lighting system under test (4). i Illumination angle under LED lighting , For the first i The light from the LED passes through the first p A straight line formed by a small aperture.

2. The microscope illumination angle calibration device as described in claim 1, characterized in that, No. p The methods for obtaining the position coordinates of each light-transmitting aperture include: Using the second projection-assisted imaging element (3) to assist the microscope objective (6) in focusing on the surface of the phantom (1) facing the second projection-assisted imaging element (3), the position of the microscope objective (6) at this time is denoted as Simultaneously illuminate all LEDs of the lighting system under test (4), and obtain the phantom image (8) in which the first LED is located. coordinates of the projection points For the first The coordinates of the position of each light-transmitting hole , ( ) represents the horizontal coordinate of the position of the light-transmitting hole in the phantom image (8). , N This represents the total number of light-transmitting apertures. This is the reference position for the microscope objective (6).

3. The microscope illumination angle calibration device as described in claim 2, characterized in that, No. The methods for obtaining the position coordinates of the projection points of each light-transmitting pinhole include: The first projection-assisted imaging element (2) is used to assist the microscope objective (6) in focusing on the frosted surface. The position of the microscope objective (6) at this time is denoted as . The LEDs of the lighting system (4) under test are illuminated sequentially, the first... i The phantom image (8) obtained when illuminated by LEDs coordinates of the projection points For the first The coordinates of the projection points of the light-transmitting apertures.

4. The microscope illumination angle calibration device as described in claim 3, characterized in that, Solving for all lines using the least squares method shown in the following equation intersection : In the formula, The term to be determined is the first one. i LED position With a straight line The distance.

5. A method for calibrating the illumination angle of a microscope, characterized in that, The method employs the microscope illumination angle calibration device as described in any one of claims 1-4, and the method includes: Step 1: Place the first projection-assisted imaging element (2) on the microscope sample stage (5), and focus the microscope objective (6) of the imaging system on the surface of the first projection-assisted imaging element (2) facing the illumination system (4) to be tested. The position of the microscope objective (6) at this time is recorded as follows: ; Step 2: Place the phantom (1) with the light-transmitting aperture on the surface of the first projection-assisted imaging element (2) facing the illumination system (4) under test, so that the camera (7) of the microscopic imaging system can acquire the phantom image (8) of the illumination system (4) under different illumination angles and the projection point of the light-transmitting aperture in the phantom image (8); Step 3: First, place the second projection-assisted imaging element (3) between the phantom (1) and the first projection-assisted imaging element (2), so that the light emitted by the illumination system (4) at different illumination angles can be projected onto the first projection-assisted imaging element (2) through the light-transmitting aperture. Then, focus the microscope objective (6) on the surface of the phantom (1) facing the second projection-assisted imaging element (3). The position of the microscope objective (6) at this time is recorded as follows: ; Step 4: Control the LED illumination mode of the illumination system (4) under test, and obtain the image of the microscope objective (6) in the light. The phantom image (8) of the first The position coordinates of the small apertures and the microscope objective (6) in The phantom image (8) of the first The coordinates of the projection point of each light-transmitting aperture; Step 5, based on all straight lines intersection Calculate the first step of the lighting system under test (4). i Illumination angle under LED lighting , For the first i The light from the LED passes through the first p A straight line formed by a small aperture.

6. The method for calibrating microscope illumination angle as described in claim 5, characterized in that, In step 4, the microscope objective (6) is in The phantom image (8) of the first The methods for obtaining the position coordinates of each light-transmitting aperture include: Simultaneously illuminate all LEDs of the lighting system under test (4) to acquire the phantom image (8). p The coordinates of the position of each light-transmitting hole , , ( ) represents the horizontal coordinate of the position of the light-transmitting hole in the phantom image (8). , N This represents the total number of light-transmitting pinholes.

7. The method for calibrating microscope illumination angle as described in claim 6, characterized in that, In step 4, the microscope objective (6) is in The phantom image (8) of the first The methods for obtaining the position coordinates of the projection points of each light-transmitting pinhole include: The LED lighting of the lighting system under test (4) is controlled to illuminate sequentially, the first... i The phantom image (8) obtained when illuminated by LEDs coordinates of the projection points For the first The coordinates of the projection points of the light-transmitting apertures.

8. The method for calibrating microscope illumination angle as described in claim 7, characterized in that, Solving for all lines using the least squares method shown in the following equation intersection : In the formula, The term to be determined is the first one. i LED position With a straight line The distance.

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