Fluorescent target and preparation and calibration method of fluorescent target

Abstract

The invention provides a fluorescent target and a preparation and calibration method of the fluorescent target, and relates to the field of fluorescent imaging. A plurality of grooves for accommodating fluorescent materials are formed in the surface of the fluorescent target. Wherein the groove can be set based on a preset calibration image. Therefore, when the fluorescent target is assembled on the imaging equipment, the fluorescent material can release the fluorescent signal corresponding to the preset calibration image through the groove so as to calibrate the imaging view of the imaging equipment. The fluorescent target provided by the invention is simple to prepare and relatively low in cost, and the calibration cost is reduced. When calibration is carried out based on the fluorescent target provided by the invention, the fluorescent material corresponding to the imaging equipment can be selected, so that calibration of different types of fluorescent imaging equipment is realized. Besides, during calibration, the fluorescent material can be directly presented on the contact interface of the imaging equipment and the fluorescent target, so that the requirement on the imaging range is reduced, and other parameters can be measured in the test, thereby improving the universality and reusability of the calibration method.

Description

Technical Field

[0001] The present application relates to the field of fluorescence imaging, and in particular to a fluorescent target and a method for preparing and calibrating the fluorescent target. Background Art

[0002] Fluorescence imaging is a highly sensitive imaging technology based on the phenomenon of fluorescence, which refers to the phenomenon that fluorescent substances release light energy and produce fluorescence after being excited. In fluorescence imaging technology, fluorescence refers to the fluorescent signals emitted by fluorescent markers in the sample after being excited. These signals are captured by the detector and converted into images.

[0003] With the development of technology, the internal optical path of fluorescent imaging equipment has become more and more complex, and its application scenarios and imaging objects have become more and more diverse. Therefore, errors (such as lens distortion) may occur during actual imaging. In order to ensure the imaging accuracy of the fluorescent imaging equipment, some parameters of the fluorescent imaging equipment can be calibrated before use. Therefore, how to achieve the calibration of the fluorescent imaging equipment is a technical problem that needs to be solved urgently by those skilled in the art. Summary of the invention

[0004] In view of this, an embodiment of the present application provides a fluorescent target and a method for preparing and calibrating the fluorescent target to solve the calibration problem of the fluorescent imaging device.

[0005] In a first aspect, the present application provides a fluorescent target applied to a contact imaging device based on weak light fluorescence imaging technology, wherein the fluorescent target comprises a substrate, wherein the substrate comprises: a first surface for contacting with an imaging window of the imaging device; a second surface opposite to the first surface; and a plurality of grooves, wherein the plurality of grooves are arranged on the first surface and extend toward the second surface. The arrangement of the plurality of grooves matches a preset calibration image, and is used to accommodate fluorescent materials to release fluorescent signals corresponding to the preset calibration image, and the preset calibration image is used to calibrate the imaging field of view of the imaging device.

[0006] In a second aspect, the present application provides a method for preparing a fluorescent target, the method comprising: determining a light-transmitting substrate. Based on a preset calibration image, an image transfer technology is used to generate a light-shielding mask on the surface of the light-transmitting substrate, wherein the light-shielding mask matches the preset calibration image. Based on the light-shielding mask, an exposure process, a development process, and an etching process are sequentially performed on the light-transmitting substrate to form a plurality of grooves corresponding to the preset calibration image on the first surface of the light-transmitting substrate. The light-shielding mask is removed.

[0007] In a third aspect, the present application provides an imaging device calibration method based on a fluorescent target, which is applied to a contact imaging device based on weak light fluorescent imaging technology, and the method includes: determining a fluorescent material and a fluorescent target, wherein the fluorescent target is the fluorescent target of the first aspect, and the fluorescent material is used to fill the fluorescent target. In response to the contact cooperation between the fluorescent target and the imaging window of the imaging device, the focal plane position of the imaging device is adjusted to the target focus depth, fluorescent imaging is performed, and a fluorescent calibration image of the target focus depth is determined, wherein the target focus depth matches the multiple grooves of the fluorescent target. And the imaging field of view of the imaging device is calibrated based on the fluorescent calibration image to determine the imaging field of view and / or imaging distortion of the imaging device.

[0008] The embodiments of the present application provide a fluorescent target and a method for preparing and calibrating the fluorescent target. The fluorescent target provided by the present application is simple to prepare and has low cost, which reduces the cost of calibration. When calibrating based on the fluorescent target provided by the present application, a fluorescent material corresponding to the imaging device can be selected to achieve calibration of different types of fluorescent imaging devices. In addition, when calibrating, the fluorescent material can be directly presented at the contact interface between the imaging device and the fluorescent target, which reduces the requirements for the imaging range, and other parameters can be measured in the test, thereby improving the versatility and reusability of the calibration method. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0010] Figure 1 This is a fluorescence imaging device calibration scenario provided in some embodiments of this specification.

[0011] Figure 2 is a top view of a fluorescent target according to some embodiments of the present specification.

[0012] Figure 3 is a cross-sectional schematic diagram of a fluorescent target provided according to an exemplary embodiment of the present specification.

[0013] Figure 4 is an exemplary flow chart of a method for preparing a fluorescent target according to an exemplary embodiment of this specification.

[0014] Figure 5 is an exemplary flow chart of a fluorescence imaging device calibration method provided according to an exemplary embodiment of the present specification. DETAILED DESCRIPTION

[0015] The following will be combined with the drawings in the embodiments of the present application to clearly and completely describe the technical solutions in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of this application.

[0016] Application Overview:

[0017] As the existing imaging environment and imaging equipment become more complex, the fluorescence image actually generated by the fluorescence imaging equipment, especially the miniaturized equipment, may be distorted during imaging, so that the fluorescence image cannot correspond to the actual object to be measured. To solve this problem, the fluorescence imaging equipment can be calibrated before performing fluorescence imaging. Among them, the calibration of the fluorescence imaging equipment can at least include the calibration of its imaging field of view, and the imaging field of view can reflect the ratio information between the field of view of the fluorescence imaging equipment and the actual object to be measured.

[0018] Existing fluorescence imaging devices generally include open fluorescence imaging devices and closed fluorescence imaging devices.

[0019] An open fluorescence imaging device may refer to a traditional desktop fluorescence / multiphoton microscope, which performs fluorescence imaging based on an open environment, and the fluorescence generated by the object to be measured is mixed with visible light. An open fluorescence imaging device is generally presented in the form of a desktop microscope, and its calibration method can be based on the relevant technology of a conventional microscope. That is, when performing calibration, it can be performed directly based on the relevant samples of visible light, without the need to use fluorescent samples.

[0020] A closed fluorescence imaging device (also called a low-light imaging device) can be understood as a closed or semi-closed structure formed between the objective lens of the imaging device and the object to be measured to block the external light source, and the fluorescence beam and the activation beam are the main light sources during fluorescence imaging. For example, a fluorescence / multiphoton microscope device that is in direct contact with the object to be measured.

[0021] For closed fluorescent imaging equipment, because its imaging is mainly based on fluorescent light beams in a weak light environment, its calibration process must rely on standard fluorescent samples. In some embodiments, a standard fluorescent slide is generally used to calibrate the fluorescent imaging device (especially the closed fluorescent imaging device). Among them, the standard fluorescent slide can refer to a fluorescent sample encapsulated with a standard fluorescent target. For example, the fluorescent standard sheet provided by Argolight in France, wherein the fluorescent standard sheet is a transparent sheet structure, and fluorescent beads are encapsulated inside it.

[0022] During calibration, the fluorescence imaging device can be controlled to align with a standard fluorescence slide, thereby activating the fluorescent material in the standard fluorescence slide and acquiring relevant images. By comparing the actual morphology of the standard fluorescence slide at the physical level with the imaging morphology presented in the fluorescence imaging device, the fluorescence imaging device can be calibrated based on the difference in morphology.

[0023] Based on the above technical solution, the calibration method based on the fluorescent standard film has at least the following technical problems in actual use:

[0024] 1. Fluorescent materials have a service life problem in actual use. That is, fluorescent materials (especially organic materials) may become inactivated after fluorescence occurs. After a period of use, the standard fluorescent slide formed by directly encapsulating the fluorescent material in a transparent substrate will partially fail the internally encapsulated fluorescent material, causing the actual concentration of the fluorescent material to change, resulting in the fluorescent target provided by it not being able to reflect the standard fluorescent target, thus affecting the calibration process.

[0025] Second, in order to achieve high-precision calibration, the fluorescent material in the transparent substrate needs to be packaged with a high-precision special process. For example, vapor deposition. The production cost of the related technology is high and requires relatively special fluorescent materials.

[0026] 3. The existing standard fluorescent slides only encapsulate a few specific fluorescent materials and cannot meet the different needs of fluorescent imaging equipment. At the same time, the concentration of the encapsulated fluorescent materials is fixed, which affects the fluorescence imaging effect. For example, the fluorescence imaging bands of the corresponding fluorescence microscope and multiphoton microscope are different. The current standard fluorescent slides only select fluorescent materials based on the fluorescence microscope, and the imaging effect for the multiphoton microscope is poor.

[0027] In view of the above problems, the embodiments of this specification provide a fluorescent target, which can achieve calibration of fluorescent imaging equipment by accommodating different fluorescent materials. Various non-limiting embodiments of this specification will be specifically introduced with reference to the accompanying drawings.

[0028] Example calibration scenario:

[0029] Fluorescent targets can be used for calibration of fluorescent imaging devices. To illustrate the calibration process based on fluorescent targets, this application provides an application scenario of fluorescent targets ( Figure 1 ) and top view of the fluorescent target ( Figure 2 ). The following will combine Figure 1 , 2 The calibration scenario of imaging equipment based on fluorescent targets is described.

[0030] Figure 1 This is a fluorescence imaging device calibration scene 100 provided in some embodiments of this specification.

[0031] like Figure 1 As shown, the calibration scene 100 may include an imaging device 110 , a fluorescent target 120 , and a fluorescent object 130 .

[0032] The imaging device 110 may refer to an imaging device that collects light signals (for the convenience of description, uniformly recorded as fluorescence signals) of the object under test based on the photoluminescence effect (such as fluorescence phenomenon, multiphoton phenomenon). In actual use, the imaging device 110 may emit excitation light to the object under test so that the object under test releases fluorescence signals outward in response to the excitation light based on the photoluminescence effect, and the imaging device 110 may collect the fluorescence signals and convert them into fluorescence images. For example, the imaging device 110 may include a fluorescence microscope, a multiphoton microscope (such as a two-photon microscope), etc.

[0033] In some embodiments, the imaging device 110 of the present application may be a low-light imaging device based on low-light imaging technology. That is, in the present application, the imaging device 110 shields or blocks external ambient light during imaging, so that the signals collected by the imaging device 110 are mainly excitation light and fluorescence signals.

[0034] In some embodiments, when the imaging device 110 performs imaging based on the multiphoton effect, the excitation light released by the imaging device 110 can form a focal plane at a preset position, and when the fluorescent material is located at the focal plane, it can be excited by the excitation light to release a fluorescent signal.

[0035] The fluorescent target 120 may be a glass slide that can realize the containing function. The fluorescent target 120 may contain fluorescent materials in the groove through a physical structure, so that when the imaging device 110 releases excitation light to the fluorescent target 120, the fluorescent materials contained in the groove are excited to release fluorescent signals.

[0036] The fluorescent target 130 may refer to an environment formed by a fluorescent material. That is, the fluorescent target 130 may refer to a fluorescent material disposed in a groove. The fluorescent target 130 may be a fluorescent material of a preset concentration. The fluorescent target 130 may release a corresponding fluorescent signal after being excited by the excitation light.

[0037] like Figure 1 As shown, the fluorescent object 130 can be disposed at the cooperation place of the fluorescent target 120 and the imaging device 110. The imaging device 110 activates the fluorescent object 130 based on the fluorescent target 120 and collects the fluorescent signal of the fluorescent object 130, thereby generating a corresponding image.

[0038] like Figure 1As shown in the partially enlarged area A in FIG. 1 , the fluorescent target 120 may be presented as a substrate. The fluorescent target 120 may include two oppositely disposed sides. The side cooperating with the fluorescent target 120 may be recorded as a first surface 121, and the other opposite side may be recorded as a second surface 122.

[0039] The substrate may be formed with a plurality of grooves 123 after a preset processing process. The plurality of grooves 123 are arranged on the first surface 121 and extend toward the second surface 122. That is, when the first surface 121 is in contact with the imaging device 110, a closed accommodation space is formed (within the tolerance range) between the plurality of grooves 123 and the imaging device 110 (such as an imaging window of the imaging device). The accommodation space may be used to accommodate fluorescent materials for calibration of the imaging device.

[0040] like Figure 1 As shown, when the imaging device 110 releases the excitation light, the fluorescent targets 130 disposed in the plurality of grooves 123 are excited and release fluorescent signals. When the arrangement of the plurality of grooves 123 matches the preset calibration image, the fluorescent targets 130 release the fluorescent signals corresponding to the preset calibration image, so that the imaging device can calibrate the imaging field of view of the imaging device 110 based on the preset calibration image.

[0041] The preset calibration image may refer to an image with scale meaning. The scale meaning means that at least part of the image in the preset calibration image can correspond to a specific size in reality. For example, when the preset calibration image may include a dot pattern, the points in the preset calibration image may be distributed according to a preset size. Exemplarily, the distance between two adjacent points in the horizontal direction or the vertical direction in the dot pattern may be a constant value.

[0042] It should be noted that the preset calibration image only needs to meet the scale requirements corresponding to the calibration, and there are no excessive requirements for its specific pattern sample. For example, the preset calibration image can be presented as a dot pattern, an equidistant concentric circle pattern, a grid pattern, or other calibration images that can be used for calibration, or a combination thereof.

[0043] In some embodiments, before calibration, fluorescent materials need to be placed in a plurality of grooves 123 to form a fluorescent target 130. Specifically, it is necessary to pour the fluorescent material (generally a liquid material) on the first surface 121 so that the fluorescent material fills the plurality of grooves 123, and then remove the excess fluorescent material on the first surface 121. However, the excess fluorescent material on the first surface 121 cannot be completely removed, and a residue will be formed on the first surface 121. Therefore, when the fluorescent target 120 filled with fluorescent material is in contact with the imaging device 110, multi-parameter calibration can be performed based on the residue of the first surface 121. Among them, when the focal plane scans the first surface 121, the change process of the fluorescent signal can be no signal → residue + preset calibration image → preset calibration image. Then, the reference plane of the focal plane (i.e., the displacement scale of the focal plane at the contact interface between the imaging object and the imaging device) can be determined based on the image feature of the residue + preset calibration image, so as to perform subsequent imaging tasks. The imaging field of view of the imaging device 110 can be determined based on the image feature of the preset calibration image.

[0044] In some embodiments, the shape of the substrate (fluorescent target 120) can match the imaging window, so as to facilitate the contact-type matching of the substrate (first surface 121) and the imaging window. The imaging window is generally circular, and the shape of the substrate is also a circle of corresponding size.

[0045] In some embodiments, the area where the multiple grooves 123 are located can be larger than the field of view of the imaging device 110 (such as the field of view during calibration), that is, the area of ​​the multiple grooves 123 is not smaller than the imaging area of ​​the imaging device 110, so that the imaging device 110 can easily find the multiple grooves 123.

[0046] In some embodiments, the fluorescent target 120 can be based on an external structure (such as a physical guide structure) and the imaging device 110. At this time, the imaging device 110 and the fluorescent target 120 can present a weak light environment and present the fluorescent target 120 in the center of the field of view of the imaging device 110. For information about the weak light environment, please refer to Figure 3 and its related description.

[0047] In some embodiments, the height difference of the plurality of grooves 123 relative to the substrate surface (first surface 121) may be a preset depth. The preset depth matches the imaging depth range of the imaging device 110. For example, the preset depth may be the maximum depth of the imaging task / imaging device, and thus, whether the imaging task can be completed or the maximum depth of the imaging device can be calibrated based on whether the focal plane of the imaging device 110 can be lower than the preset depth.

[0048] Figure 2It is a top view of the fluorescent target shown in some embodiments of this specification. The top view refers to a view obtained by taking the thickness direction of the fluorescent target 120 (ie, the direction from the first side to the second side) as the observation direction.

[0049] like Figure 2 As shown, the plurality of grooves 123 can be set based on a preset calibration image. The plurality of grooves 123 can include a plurality of dot grooves, each of which forms a dot matrix pattern. When the dot matrix pattern is used as a preset calibration image, the array distribution of each dot can have a scale meaning in an actual physical sense. For example, the distance between each dot in the horizontal direction and the vertical direction is always a fixed value.

[0050] Example low-light environment:

[0051] Figure 3 is a cross-sectional schematic diagram of a fluorescent target provided according to an exemplary embodiment of the present specification.

[0052] like Figure 3 As shown, when the fluorescent target 120 cooperates with the imaging device 110, the fluorescent target 120 can form a weak light environment, so that the imaging device 110 can perform imaging field calibration in any scene.

[0053] like Figure 3 As shown, the fluorescent target 120 may further include a light shielding layer 124. The light shielding layer 124 may be disposed on the second surface 122 to block external light when the imaging windows of the imaging device 110 are in contact with each other to form a weak light environment.

[0054] In some embodiments, the fluorescent target 120 further includes a physical guiding structure 125. The physical guiding structure 125 can be used to be detachably connected to the imaging device 110 based on a physical structure, so that the first surface 121 is in contact with the imaging window.

[0055] In some embodiments, the physical guide structure 125 can also be used to detachably assemble the plurality of grooves 123 to the center of the field of view of the imaging device 110. That is, the fluorescent target 120 is detachably connected to the imaging device 110 based on the shaft-core fit.

[0056] In some embodiments, in order to ensure a weak light environment between the fluorescent target 120 and the imaging device 110, a physical guide structure 125 can be arranged around the substrate and made of a light shielding material. Therefore, the physical guide structure 125 can also be used to block external light when the imaging window of the imaging device 110 is in contact with each other, so as to form a weak light environment, especially the side external light source where the fluorescent target 120 and the imaging device 110 are in contact.

[0057] As shown in FIG. 3 , the physical guide structure 125 may be a side wall provided with a physical connection portion. That is, the physical guide structure 125 may include a side wall 1251 and a physical guide portion 1252 provided on the inner wall of the side wall 1251. The inner wall may be a side of the side wall 1251 facing the fluorescent target 120.

[0058] The side wall 1251 may be a light shielding structure that is perpendicular to the first surface 121 and extends in a direction away from the second surface 122. The side wall 1251 may shield external light from the side where the fluorescent target 120 and the imaging device 110 are assembled.

[0059] The physical guide 1252 may be a physical structure that can achieve mechanical matching. For example, the physical guide 1252 may be matched with the imaging device 110 based on mechanical structures such as threads and buckles. The imaging device 110 has a structure that matches the physical guide 1252. For example, the physical guide 1252 may be embodied as an elastic convex block, and the imaging device 110 may be provided with a groove at a corresponding position. When in use, the fluorescent target 120 may be assembled on the imaging device 110 through the buckle matching of the elastic convex block and the groove.

[0060] In some embodiments, the light shielding layer 124, the physical guide structure 125 and other structures can form the outer shell of the fluorescent target 120, and the body (i.e., the substrate) of the fluorescent target 120 can be produced in a different manner from the outer shell. Figure 4 After the production process, the body and the shell can be assembled to build Figure 3 A fluorescent target 120 is shown.

[0061] Based on the fluorescent target of the aforementioned embodiment, a weak-light environment is formed between the fluorescent target object and the fluorescent target through the accommodating cavity, and a weak-light environment is formed between the fluorescent target and the imaging device through the shading connecting piece, thereby ensuring that the imaging device is in a weak-light environment when calibrating the imaging field of view, thereby getting rid of the environmental requirements of the imaging device when calibrating the imaging field of view and expanding the use scenarios of the imaging device.

[0062] Exemplary fluorescent target preparation method:

[0063] Figure 4 is an exemplary flow chart of a method for preparing a fluorescent target according to an exemplary embodiment of this specification. Figure 4 The fluorescent target (the main structure) shown can be produced based on microelectronic manufacturing technology.

[0064] like Figure 4 As shown, process 400 may include:

[0065] S410, determining a light-transmitting substrate, and generating a light-shielding mask on the surface of the light-transmitting substrate.

[0066] In some embodiments, the transparent substrate may be the object to be processed in process P400. The transparent substrate may be the main structure used for the aforementioned fluorescent target 120 in this application, that is, the grooves involved in this application may be formed on the first surface (upper surface) of the transparent substrate in S410.

[0067] The light shielding mask (also called photoresist) can block external etching light. In some embodiments, the present application can adopt an image transfer process to etch the light shielding mask to expose a portion of the transparent substrate corresponding to the preset calibration image, and then form a plurality of grooves.

[0068] S420, using image transfer technology to sequentially perform an exposure process, a development process, and an etching process on the light-transmitting substrate to form a plurality of grooves corresponding to a preset calibration image on the first surface of the light-transmitting substrate.

[0069] Image transfer technology may refer to a technology for performing semiconductor etching based on a preset image (i.e., a preset calibration image). In implementation, it may include three steps: an exposure process, a development process, and an etching process. Among them, the exposure process forms a desired pattern by exposing light to the mask using a mask and a photolithography machine. The development process may refer to removing the mask from a specific area by a developer to form a desired pattern. The etching process may refer to removing the area not protected by the photoresist by an etcher to expose the underlying material.

[0070] In some embodiments, the above process can be performed in two ways: intaglio and embossment. When intaglio is used, part of the transparent substrate below the light shielding mask is retained, and the area not covered by the light shielding mask is etched to form a groove. When embossment is used, part of the transparent substrate below the light shielding mask is removed to form a groove.

[0071] Those skilled in the art can adjust the above process according to the adaptability of the specific selected process.

[0072] S430, clear the light-shielding mask to obtain a fluorescent target.

[0073] In some embodiments, the aforementioned S410 may refer to processing a substrate to form a light shielding mask on the surface of the substrate (generally silicon oxide). The specific execution method of S410 may be related to the type of the light shielding mask. For example, if the light shielding mask is a photoresist, then step S410 may be spin coating the photoresist on the substrate. For another example, if the light shielding mask is a metal layer, then step S410 may be vapor deposition of the corresponding metal layer on the substrate.

[0074] The above preparation method has fewer process steps, and only the etching step has errors. The processing accuracy is high and the processing cost is very low. Therefore, the processed fluorescent target itself can have better performance and lower production cost.

[0075] In some embodiments, the fluorescent target obtained based on the aforementioned P400 can be subsequently processed, such as segmentation and assembly, to form the aforementioned Figure 3 Fluorescent targets are shown.

[0076] Exemplary imaging field of view calibration method:

[0077] Figure 5 is an exemplary flow chart of a fluorescence imaging device calibration method provided according to an exemplary embodiment of this specification. In some embodiments, Figure 5 The illustrated method may be performed by a processor within the imaging device 110 .

[0078] like Figure 5 As shown, process 500 may include:

[0079] S510, determining a target fluorescent material and a target fluorescent target.

[0080] The target fluorescent material and the target fluorescent target may refer to the fluorescent material and the fluorescent target required for performing the current imaging field calibration. In some embodiments, the target fluorescent material and the target fluorescent target may be determined according to the imaging device 110. The target fluorescent material may be determined according to the imaging effect of the imaging device 110, and the target fluorescent target may be determined according to the imaging accuracy of the imaging device 110.

[0081] Imaging equipment uses different excitation lights based on different photoluminescence effects. For example, for NADH enzyme, under single-photon excitation (fluorescence effect), the excitation light is generally 350nm, and the fluorescence signal is 450nm. Under two-photon excitation (multi-photon effect), the excitation light can be infrared or near-infrared light (such as 700nm), and the fluorescence signal is 450nm.

[0082] In some embodiments, the target fluorescent material may be a fluorescent material having a better display effect (eg, a higher intensity of fluorescent signal) after being excited by the excitation light of the imaging device. The target fluorescent material may be determined based on the type of the excitation light.

[0083] In some embodiments, the fluorescent target can be produced according to different preset calibration patterns, so as to have calibration capabilities with different imaging fields of view. The target fluorescent target can be a fluorescent target that can meet the calibration requirements of the imaging device. For example, it can be a fluorescent target whose imaging field of view accuracy is greater than the imaging accuracy.

[0084] In some embodiments, the determination process of S510 may also be omitted, and commonly used fluorescent materials and fluorescent targets may be directly used as the target fluorescent materials and target fluorescent targets for this calibration.

[0085] S520, in response to the target fluorescent target cooperating with the imaging device, adjusting the focal plane position of the imaging device to the target focal depth, and determining a fluorescent calibration image at the target focal depth.

[0086] In some embodiments, the user may assemble the target fluorescent target with the imaging device when performing imaging field calibration. For example, the target fluorescent material may be injected into the receiving cavity of the target fluorescent target, and the target fluorescent target may be matched with the imaging device through the light shielding connector of the target fluorescent target.

[0087] In some embodiments, after the target fluorescent target is assembled with the imaging device (ie, the target fluorescent target cooperates with the imaging device), the imaging device can be controlled to perform fluorescent imaging.

[0088] In some embodiments, when the imaging device is based on multiphoton imaging technology, the excitation light can form a focal plane, and the fluorescent material located at the focal plane will be activated and release a fluorescent signal. When performing fluorescence imaging, the focal plane position can be adjusted by adjusting the internal device of the imaging device (such as a translation stage) so that the focal plane position is located at the target focal depth, so as to obtain the fluorescent signal at the target focal depth to generate a fluorescent calibration image.

[0089] The target focus depth may be a focus depth required for performing imaging field of view calibration. In some embodiments, the target focus depth may be a focus depth located within the fluorescent target. Considering that the target fluorescent target is located between the imaging device and the fluorescent target, the target focus depth corresponds to the thickness of the target fluorescent target between the first side and the second side, that is, the focal plane at least needs to cross the target fluorescent target, be located on the second side or enter the interior of the fluorescent target.

[0090] Considering that there is a weak light environment between the imaging device and the fluorescent target, the collected signals do not contain fluorescent signals before the focal plane reaches the second side of the target fluorescent target. Therefore, the collected fluorescent signal can be used as an indicator of reaching the target focus depth. In some embodiments, the target focus depth can be determined based on a step-by-step detection strategy. That is, the imaging device can gradually adjust the focus depth and perform fluorescent imaging accordingly until a stable fluorescent signal is collected. This position is used as the target focus depth.

[0091] A fluorescence calibration image may refer to a fluorescence image determined by an imaging device based on a target fluorescence target. Among them, the excitation light of the imaging device is blocked by the shading area in the target fluorescence target, and only the partial focal plane corresponding to the light-transmitting area can activate the target fluorescent material and collect the corresponding fluorescence signal. A fluorescence calibration image can be formed based on the fluorescence signal. Considering that the fluorescent target itself is a homogeneous medium, the fluorescence calibration image can reflect the distribution of the light-transmitting area. Furthermore, the light-transmitting area is formed based on a preset calibration image, and the fluorescence calibration image can reflect the imaging result of the preset calibration image.

[0092] S530: Calibrate the imaging device based on the fluorescence calibration image to determine the imaging field of view and / or imaging distortion of the imaging device.

[0093] In some embodiments, based on the aforementioned fluorescent target, the imaging field of view and imaging distortion calibration of the imaging device may be performed.

[0094] The imaging field of view may refer to the actual size of the fluorescent image acquired by the imaging device, that is, the scale between the fluorescent image and the actual image. Imaging distortion may refer to the possible deformation of the fluorescent image acquired by the imaging device. Considering that the imaging device is generally constructed based on micro-multiphoton imaging technology, its imaging distortion is difficult to change by adjusting the internal optical path. Generally, the image is orthogonalized by performing affine transformation or piecewise linear fitting on the fluorescent image. The calibration process of imaging distortion can be understood as the aforementioned image orthogonalization process.

[0095] In some embodiments, the aforementioned calibration process can be implemented based on the actual physical meaning of the pattern in the preset calibration image. Figure 2 In the dot pattern shown, the spacing between each point is a constant value (such as 10 μm). When the fluorescence calibration image contains the dot pattern or part of the dot pattern, the distribution of each point in the fluorescence calibration image (such as the number of pixels between each point) can be used to determine the conversion relationship between the pixels in the fluorescence calibration image and the actual distance, thereby determining the imaging field of view (scale).

[0096] For another example, the acquired fluorescence calibration image can be compared with the preset calibration image to determine the distortion of the fluorescence calibration image and perform orthodontics based on the distortion, so that the fluorescence calibration image is consistent with the preset calibration image after orthodontics. Figure 2 In the dot pattern shown, each point is evenly distributed. When the distribution of each point of the dot pattern in the fluorescence calibration image is different from the preset dot pattern (for example, the spacing of some points is different from that of other points), the fluorescence calibration image can be orthogonalized based on the theoretical distribution of each point (i.e., the aforementioned uniform distribution) to make each point in the fluorescence calibration image evenly distributed.

[0097] All the above optional technical solutions can be arbitrarily combined to form optional embodiments of the present application, and will not be described one by one here.

[0098] Those of ordinary skill in the art will appreciate that the units and algorithm steps of each example described in conjunction with the embodiments disclosed herein can be implemented in electronic hardware, or a combination of computer software and electronic hardware. Whether these functions are performed in hardware or software depends on the specific application and design constraints of the technical solution. Professional and technical personnel can use different methods to implement the described functions for each specific application, but such implementation should not be considered to be beyond the scope of this application.

[0099] Those skilled in the art can clearly understand that, for the convenience and brevity of description, the specific working processes of the systems, devices and units described above can refer to the corresponding processes in the aforementioned method embodiments and will not be repeated here.

[0100] In the several embodiments provided in the present application, it should be understood that the disclosed systems, devices and methods can be implemented in other ways. For example, the device embodiments described above are only schematic. For example, the division of the units is only a logical function division. There may be other division methods in actual implementation, such as multiple units or components can be combined or integrated into another system, or some features can be ignored or not executed. Another point is that the mutual coupling or direct coupling or communication connection shown or discussed can be through some interfaces, indirect coupling or communication connection of devices or units, which can be electrical, mechanical or other forms.

[0101] The units described as separate components may or may not be physically separated, and the components shown as units may or may not be physical units, that is, they may be located in one place or distributed on multiple network units. Some or all of the units may be selected according to actual needs to achieve the purpose of the solution of this embodiment.

[0102] In addition, each functional unit in each embodiment of the present application may be integrated into one processing unit, or each unit may exist physically separately, or two or more units may be integrated into one unit.

[0103] If the functions are implemented in the form of software functional units and sold or used as independent products, they can be stored in a computer-readable storage medium. Based on this understanding, the technical solution of the present application can be essentially or partly embodied in the form of a software product that contributes to the prior art. The computer software product is stored in a storage medium, including several instructions to enable a computer device (which can be a personal computer, a server, or a network device, etc.) to perform all or part of the steps of the methods described in each embodiment of the present application. The aforementioned storage medium includes: various media that can store program check codes, such as a USB flash drive, a mobile hard disk, a read-only memory (ROM), a random access memory (RAM), a magnetic disk or an optical disk.

[0104] It should be noted that, in the description of this application, the terms "first", "second", "third", etc. are used for descriptive purposes only and cannot be understood as indicating or implying relative importance. In addition, in the description of this application, unless otherwise specified, the meaning of "plurality" is two or more.

[0105] The above description is only a preferred embodiment of the present application and is not intended to limit the present application. Any modifications, equivalent substitutions, etc. made within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. A fluorescent target, characterized in that: Applied to a contact imaging device based on weak light fluorescence imaging technology, the fluorescent target includes a substrate, and the substrate includes: The first surface is used for contacting with the imaging window of the imaging device; a second surface opposite to the first surface; and A plurality of grooves are provided on the first surface and extend toward the second surface; wherein the arrangement of the plurality of grooves matches a preset calibration image, and is used to accommodate fluorescent material to release a fluorescent signal corresponding to the preset calibration image, and the preset calibration image is used to calibrate the imaging field of view of the imaging device.

2. The fluorescent target according to claim 1, characterized in that The shape of the substrate matches the imaging window.

3. The fluorescent target according to claim 2, characterized in that: The fluorescent target further includes a light shielding layer, which is disposed on the second surface and is used to block external light when the imaging windows of the imaging device are in contact with each other, so as to form a weak light environment.

4. The fluorescent target according to claim 1, characterized in that: The fluorescent target further includes a physical guiding structure, which is used to be detachably connected to the imaging device based on a physical structure, so that the first surface is in contact with the imaging window.

5. The fluorescent target according to claim 4, characterized in that: The physical guide structure is arranged in a ring around the substrate, is made of a light-shielding material, and is also used to block external light when the imaging window of the imaging device is in contact with each other, so as to form a weak-light environment.

6. The fluorescent target according to claim 4, characterized in that: The physical guide structure is also used to removably mount the plurality of grooves to the center of the field of view of the imaging device.

7. The fluorescent target according to claim 1, characterized in that: The height difference between the plurality of grooves and the surface of the substrate is a preset depth, and the preset depth matches the imaging depth range of the imaging device.

8. The fluorescent target according to claim 1, characterized in that: The area range of the plurality of grooves is not smaller than the imaging area of ​​the imaging device.

9. A method for preparing a fluorescent target, characterized in that: The method comprises: Determining a light-transmitting substrate and generating a light-shielding mask on the surface of the light-transmitting substrate; An exposure process, a development process, and an etching process are sequentially performed on the light-transmitting substrate using an image transfer technology to form a plurality of grooves corresponding to a preset calibration image on a first surface of the light-transmitting substrate; The light shielding mask is removed to obtain the fluorescent target.

10. A method for calibrating an imaging device based on a fluorescent target, characterized in that: Applied to a contact imaging device based on weak light fluorescence imaging technology, the method comprises: Determining a fluorescent material and a fluorescent target, wherein the fluorescent target is the fluorescent target according to any one of claims 1 to 8, and the fluorescent material is used to fill the fluorescent target; In response to the fluorescent target being in contact with the imaging window of the imaging device, adjusting the focal plane position of the imaging device to a target focal depth, performing fluorescent imaging, and determining a fluorescent calibration image of the target focal depth, wherein the target focal depth matches the plurality of grooves of the fluorescent target; and The imaging device is calibrated based on the fluorescence calibration image to determine the imaging field of view and / or imaging distortion of the imaging device.

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