Fluorescent target and preparation and calibration method of fluorescent target

Abstract

The invention discloses a fluorescent target and a preparation and calibration method of the fluorescent target. The fluorescent target comprises a shading area; the light-transmitting area corresponds to the preset calibration image; wherein the first side of the fluorescent target is matched with the fluorescent imaging equipment, and the second side of the fluorescent target is matched with the target fluorescent material; the light-transmitting area is used for forming a light-transmitting light path corresponding to the preset calibration image between the first side and the second side. Based on the fluorescent target provided by the invention, calibration of various fluorescent imaging devices can be realized by selecting corresponding fluorescent materials, and the usability of related devices is improved. In addition, the fluorescent target provided by the invention is simple to prepare and relatively low in cost, and the calibration cost is reduced.

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, an embodiment of the present application provides a fluorescent target, which is applied to an imaging device based on weak light imaging technology, wherein the fluorescent target includes a substrate, and the substrate includes: a light-shielding area and a light-transmitting area corresponding to a preset calibration image. The first side of the fluorescent target cooperates with the imaging device, and the second side cooperates with the target fluorescent material. The light-transmitting area is used to form a light-transmitting optical path corresponding to a preset calibration image between the first side and the second side, and the preset calibration image is used to calibrate the imaging field of view of the imaging device.

[0006] In some embodiments of the present application, the substrate includes: a transparent substrate and a light shielding layer disposed on the transparent substrate, wherein the area of ​​the transparent substrate shielded by the light shielding layer and the light shielding layer form a light shielding area, and the area of ​​the transparent substrate not shielded by the light shielding layer forms a light transparent area.

[0007] In some embodiments of the present application, the light shielding layer is disposed on the second side.

[0008] In some embodiments of the present application, the distribution range of the light-transmitting area is not less than the imaging range of the imaging device.

[0009] In some embodiments of the present application, the fluorescent target further includes a receiving cavity disposed on the second side, and the receiving cavity is used to receive the fluorescent material so that the fluorescent material forms a fluorescent light ring and cooperates with the second side.

[0010] In some embodiments of the present application, the material of the accommodating cavity is a light-shielding material.

[0011] In certain embodiments of the present application, a light-shielding connector is disposed around the substrate, and the light-shielding connector is used to cooperate with an imaging device and form a weak-light environment between the imaging device and the fluorescent target.

[0012] In certain embodiments of the present application, the light-shielding connector includes a side wall and a physical guide portion disposed on an inner wall of the side wall, wherein the physical guide portion is configured to detachably cooperate with the imaging device.

[0013] In a second aspect, the present application provides a method for preparing a fluorescent target, the method comprising: generating a light-shielding mask on the surface of a light-transmitting substrate: etching the light-shielding mask based on a preset calibration image to form a light-shielding layer, wherein an area of ​​the light-transmitting substrate blocked by the light-shielding layer and the light-shielding layer form a light-shielding area, and an area of ​​the light-transmitting substrate not blocked by the light-shielding layer forms a light-transmitting area.

[0014] 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 field of view imaging field of the imaging device is calibrated based on the fluorescent calibration image.

[0015] 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

[0016] 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.

[0017] Figure 1is a schematic diagram of an application scenario of a fluorescent target provided according to an exemplary embodiment of the present application;

[0018] Figure 2 is a top view of a fluorescent target provided according to an exemplary embodiment of the present application;

[0019] Figure 3 According to this manual Figure 2 A cross-sectional view of the fluorescent target in the AA region is shown.

[0020] Figure 4 is a cross-sectional schematic diagram of a fluorescent target provided according to an exemplary embodiment of the present specification.

[0021] Figure 5 is an exemplary flow chart of a method for preparing a fluorescent target according to an exemplary embodiment of this specification.

[0022] Figure 6 is an exemplary flow chart of a fluorescence imaging device calibration method provided according to an exemplary embodiment of the present specification. DETAILED DESCRIPTION

[0023] 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.

[0024] Application Overview:

[0025] 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.

[0026] Existing fluorescence imaging devices generally include open fluorescence imaging devices and closed fluorescence imaging devices.

[0027] 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.

[0028] 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.

[0029] 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 target fluorescent material. 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.

[0030] 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.

[0031] 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:

[0032] 1. Fluorescent materials have a service life problem in actual use. That is, fluorescent materials (especially organic materials) may become inactivated after fluorescence occurs. When the fluorescent material is directly encapsulated in a transparent substrate, the standard fluorescent slide formed will partially fail after a period of use, causing the actual concentration of the fluorescent material to change, resulting in the target fluorescent material provided not being able to reflect the standard target fluorescent material, which in turn affects the calibration process.

[0033] 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.

[0034] 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.

[0035] 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.

[0036] Example application scenarios:

[0037] In some embodiments, the fluorescent target can be used in a fluorescence imaging device calibration scenario. Figure 1 This is a fluorescence imaging device calibration scene 100 provided in some embodiments of this specification.

[0038] like Figure 1 As shown, the calibration scene 100 may include an imaging device 110 , a fluorescent target 120 , and a target fluorescent material 130 .

[0039] 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.

[0040] 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.

[0041] 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.

[0042] The fluorescent target 120 may be an optical device that can realize a masking function. The fluorescent target 120 may allow only part of the light to pass through based on the optical properties. For example, when the imaging device 110 cooperates with the fluorescent target 120, the fluorescent target 120 may block part of the excitation light, so that the excitation light is only in the focal plane formed corresponding to the fluorescent target 120.

[0043] The target fluorescent material 130 may refer to an external environment composed of fluorescent materials. The target fluorescent material 130 may be a fluorescent material of a preset concentration. The target fluorescent material 130 may release a corresponding fluorescent signal after being excited by the excitation light.

[0044] like Figure 1 As shown, the fluorescent target 120 may be disposed between the imaging device 110 and the target fluorescent material 130. The imaging device 110 activates the target fluorescent material 130 based on the fluorescent target 120 and collects the fluorescent signal of the target fluorescent material 130, thereby generating a corresponding image.

[0045] like Figure 1 As 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 that cooperates with the fluorescent target 120 may be recorded as a first side 123, and the other opposite side may be recorded as a second side 124.

[0046] The substrate can be processed by a preset process to form a light shielding area 128 and a light transmitting area 129. The area corresponding to the light transmitting area 129 can form a light transmitting path between the first side 123 and the second side 124. The light can pass through the fluorescent target 120 along the light transmitting path.

[0047] like Figure 1 As shown, when the imaging device 110 releases the excitation light, the light shielding area 128 blocks part of the excitation light, and only the light transmission path formed by the light transmission area 129 can pass the excitation light, so that the actual focal plane of the imaging device 110 corresponds to the distribution of the light transmission area 129. Then, the corresponding target fluorescent material 130 is activated to release the fluorescent signal, which is collected by the imaging device 110. Therefore, the fluorescent signal collected by the imaging device 110 is basically consistent with the distribution of the light transmission area 129.

[0048] In some embodiments, when the light-transmitting area 129 is distributed according to a preset calibration image, the fluorescence signal collected by the imaging device 110 corresponds to the preset calibration image, and the imaging device 110 can be adjusted and calibrated according to the difference between the fluorescence image formed by the fluorescence signal and the preset calibration image. For more information about the preset calibration image, please refer to Figure 2 and its related description.

[0049] In some embodiments, the imaging field of view calibration of the imaging device 110 can be performed in a weak light environment. For example, the imaging device 110 can be placed in a weak light environment. In some embodiments, the fluorescent target 120 can be improved so that when the fluorescent target 120 and the imaging device 110 cooperate, the target fluorescent material 130 and the imaging device 110 form a weak light environment. For information on forming a weak light environment based on the fluorescent target 120, see Figure 4 and its related description.

[0050] Based on the fluorescent target provided by the above embodiment, the partial display of the fluorescent material based on the preset calibration pattern can be realized through the optical structure, so that the fluorescent material is separated from the carrier of the preset calibration image. When calibrating based on the fluorescent target, the fluorescent material corresponding to the fluorescent imaging device can be selected for calibration, which improves the applicability of the relevant equipment. The fluorescent material itself can be replaced at any time, ensuring that the concentration of the fluorescent material is constant and will not fail. In addition, the above fluorescent target has a simple structure and a low preparation cost, which reduces the cost of calibration.

[0051] Exemplary fluorescent targets:

[0052] Figure 2 It 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.

[0053] It should be noted that the presentation form of the fluorescent target 120 is related to the requirements of the imaging device 110. The fluorescent target 120 is generally a sheet-like structure with a relatively thin thickness. It is similar to the window / cover glass form used in conventional fluorescent imaging devices. The fluorescent target 120 can be adjusted accordingly to meet the special requirements of the imaging device 110.

[0054] like Figure 2 As shown, in the top view of the fluorescent target 120, the fluorescent target 120 may include a light shielding area 128 and a light transmitting area 129. The light shielding area 128 may be Figure 2 In the fluorescent target 120, there is a partial area of ​​the oblique stripe shading, and the light-transmitting area 129 can be Figure 2 Some areas without shading.

[0055] It should be noted that it is only necessary to ensure that the light shielding area 128 and the light transmitting area 129 exist in the distribution area 125 of the light transmitting area 129 for presenting the preset fluorescent image in the fluorescent target 120, and this specification does not limit other areas on the fluorescent target 120. For example, in some embodiments, outside the distribution area 125 of the fluorescent target 120, the fluorescent target 120 may be provided with a visual guide (not shown in the figure), and the visual guide may be used to indicate the location of the distribution area 125, so as to facilitate the matching of the fluorescent target 120 with the imaging device 110.

[0056] The light shielding area 128 may refer to an area in the fluorescent target 120 that can block light in the thickness direction. In some embodiments, the light shielding area 128 shields light, and the fluorescent light beam on the second side cannot pass through the light shielding area 128, or the light intensity of the fluorescent light beam is attenuated to the point that it cannot be detected by the imaging device 110 when the fluorescent light beam passes through the light shielding area 128.

[0057] In some embodiments, the light shielding area 128 can be implemented based on the reflective property, shielding property, absorption property, suppression property or other possible optical property of the optical material. For example, the light shielding area 128 can be implemented based on the shielding property of the light shielding material, thereby shielding the fluorescent light beam emitted from the second side, so that the light beam cannot pass through the light shielding area 128. For another example, the light shielding area 128 can be implemented based on the reflective property of a mirror or metal surface, thereby reflecting the fluorescent light beam emitted from the second side, so that the light beam cannot pass through the light shielding area 128. For another example, the light shielding area 128 can be implemented based on the suppression effect of an optical filter, thereby attenuating the fluorescent light beam emitted from the second side, so that its light intensity is attenuated to the point where it cannot be detected by the imaging device 110.

[0058] The light-transmitting area 129 may refer to an area in the fluorescent target 120 that allows light to pass through in the thickness direction. In some embodiments, the light-transmitting area 129 allows light to pass through at least including that the light beam can be transmitted to the first side in the vertical direction, and the light intensity attenuation is small and can be detected by the imaging device 110.

[0059] In some embodiments, the light-transmitting area 129 can be realized based on the light-transmitting property of an optical material. For example, the substrate of the fluorescent target 120 itself can be a light-transmitting substrate, and the light-transmitting area 129 can refer to an area in the distribution area 125 that is not blocked by the light-shielding material.

[0060] In some embodiments, the light-transmitting area 129 may be set based on a preset calibration image. The preset calibration image may refer to an image with scale significance. Figure 2As shown, the light-transmitting area 129 forms a dot pattern in the distribution area 125. When the dot pattern is used as a preset calibration image, the array distribution of each point can have a scale meaning in an actual physical sense. For example, the distance between each point in the horizontal direction and the vertical direction is always a constant value.

[0061] It should be noted that the preset calibration image formed by the light-transmitting area 129 only needs to meet the scale requirements corresponding to the calibration, and there are no excessive requirements for its specific pattern samples. For example, the preset calibration image can include a dot pattern, an equidistant concentric circle pattern, a grid pattern, a checkerboard pattern, etc.

[0062] In some embodiments, the light-shielding region 128 and the light-transmitting region 129 can be implemented based on a light-transmitting substrate and a light-shielding layer. For details about the hierarchical structure of the light-transmitting substrate and the light-shielding layer, see Figure 3 and its related description.

[0063] In some embodiments, the light-shielding area 128 and the light-transmitting area 129 can be implemented based on an electrically controlled liquid crystal medium. The substrate of the fluorescent target 120 can be constructed based on the electrically controlled liquid crystal medium, and the voltage of each area in the substrate can be controlled according to a preset calibration image, so that each area is in a light-transmitting state and a light-shielding state matching the preset calibration image.

[0064] To further clarify the internal hierarchical structure of the fluorescent target, this manual also provides Figure 3 . Figure 3 According to this manual Figure 2 A cross-sectional view of the fluorescent target in the AA region is shown.

[0065] like Figure 3 As shown, the fluorescent target 120 may include a transparent substrate 310 and a light shielding layer 320. The light shielding layer 320 may be partially disposed on the surface of the transparent substrate 310. The light shielding layer 320 may shield light along the thickness direction between the first side and the second side of the area where the light shielding layer 320 is disposed.

[0066] From the thickness direction of the fluorescent target 120, a part of the transparent substrate 310 (referred to as the first part) is covered by the light shielding layer 320, and a part of the transparent substrate 310 (referred to as the second part) is not covered by the light shielding layer 320. Thus, the first part and the light shielding layer 320 form a light shielding region 128, and the second part forms a light transmitting region 129.

[0067] The light-transmitting substrate 310 may refer to a substrate having a light-transmitting property. In some embodiments, the light-transmitting substrate 310 may refer to a substrate used in a microelectronics manufacturing process. For example, the light-transmitting substrate 310 may be a silicon substrate, a sapphire substrate, a glass substrate, etc.

[0068] The light shielding layer 320 may refer to a coating layer that can achieve a light shielding effect. In some embodiments, the light shielding layer 320 may refer to a mask used in a microelectronics manufacturing process. For example, the light shielding layer 320 may be a photoresist mask, a metal mask, etc. The light shielding layer 320 may be formed based on a microelectronics manufacturing process. For a specific process, see Figure 5 and its related description.

[0069] It should be noted that the present application does not limit the specific location of the light shielding layer 320. When combined with other components, the light shielding layer 320 can be configured to shield the light at the corresponding location in the thickness direction according to actual conditions.

[0070] In some embodiments, when assembling the fluorescent target 120, it may be necessary to assemble the fluorescent target 120 with the imaging device 110. After the assembly is completed, the focal plane of the imaging device 110 corresponding to the light-transmitting area 129 can activate the target fluorescent material 130 and collect the fluorescent signal.

[0071] In some embodiments, the fluorescent target 120 may cooperate with the imaging device 110 with the side including the fluorescent region 220 as the first side, thereby directly presenting a fluorescent light beam corresponding to a preset calibration image on the first side.

[0072] In some embodiments, the fluorescent target 120 can be integrated with the imaging device 110 as a part of the imaging device 110. For example, for a desktop fluorescent imaging device, the fluorescent target can be used as a cover glass in a sample slide. For another example, for an immersion fluorescent imaging device, the fluorescent target can be used as a window for carrying an immersion medium. At this time, in order to further ensure that the preset calibration pattern is presented on the sample surface (i.e., the uppermost layer of the target fluorescent material) and reduce the spacing, the light shielding layer 320 can be set on the second side. That is, when the target fluorescent material 130 is assembled with the fluorescent target 120, the surface where the light shielding layer 320 is located can be assembled as the second side of the fluorescent target 120.

[0073] In some embodiments, to reduce the difficulty of coordination, the corresponding fluorescent target 120 can be determined / selected according to the field of view of the fluorescent imaging device to be calibrated. Among them, the area of ​​the light-transmitting area 129 can be larger than the field of view of the imaging device 110. This reduces the difficulty of alignment and focusing operations.

[0074] The fluorescent target provided by the above embodiment can be constructed based on the light shielding layer and the light transparent substrate, and the light shielding layer and the light transparent substrate are relatively simple to process and have high processing precision, thereby improving the use effect of the fluorescent target and further reducing the production cost of the fluorescent target.

[0075] Example low-light environment:

[0076] Figure 4 is a cross-sectional schematic diagram of a fluorescent target provided according to an exemplary embodiment of the present specification.

[0077] like Figure 4 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.

[0078] like Figure 4 As shown, the fluorescent target 120 may further include a receiving cavity 126. The receiving cavity 126 may be disposed on the second surface 122 of the fluorescent target 120 and used to receive the fluorescent material to form the target fluorescent material 130. For example, the corresponding fluorescent material may be filled into the receiving cavity 126 as required to form the target fluorescent material 130.

[0079] In some embodiments, the target fluorescent material 130 may include a liquid fluorescent material mixture of a preset concentration. The preset concentration refers to the concentration of the fluorescent material in the medium. The presentation form of the target fluorescent material 130 is related to the mixed medium, and the mixed medium used by the target fluorescent material 130 can also be adjusted according to actual conditions. For example, the target fluorescent material 130 can also use a gaseous, solid or colloidal medium mixed with fluorescent materials to present a corresponding state.

[0080] In some embodiments, the material of the receiving cavity 126 may be a light shielding material. The receiving cavity 126 based on the light shielding material may block external light at the fluorescent material 130 , thereby forming a weak light environment between the fluorescent material 130 and the fluorescent target 120 .

[0081] In some embodiments, to achieve imaging field of view calibration at any position, the entire device can be inverted (ie, the fluorescent target 120 is located above the imaging device 110), so that the target fluorescent material 130 is in contact with the second side of the fluorescent target 120 based on its own gravity.

[0082] In some embodiments, the accommodating cavity 126 may also be provided with a liquid injection port (not shown in the figure) through which liquid fluorescent material corresponding to the imaging device 110 may be injected (or discharged) to form the target fluorescent material 130 .

[0083] In some embodiments, in order to facilitate the assembly of the fluorescent target 120 and the imaging device 110, the fluorescent target 120 may further include a light shielding connector 127 disposed around the substrate, wherein the light shielding connector may be perpendicular to the first surface 121 and extend in a direction away from the second surface 122.

[0084] In some embodiments, the light shielding connector 127 can be mechanically matched with the imaging device 110, so that the first surface 121 of the fluorescent target 120 can be detachably matched with the imaging device 110 (such as the imaging window of the imaging device 110). In some embodiments, the light shielding connector 127 can block external light at the matching point between the fluorescent target 120 and the imaging device 110, thereby forming a weak light environment between the fluorescent target 120 and the imaging device 110.

[0085] In some embodiments, the light shielding connector 127 may be a side wall provided with a physical connection portion, that is, the light shielding connector 127 may include a side wall 1271 and a physical guide portion 1272 provided on the inner wall of the side wall 1271 . The inner wall may be a side of the side wall 1271 facing the fluorescent target 120 .

[0086] The side wall 1271 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 1271 may shield external light from the side where the fluorescent target 120 and the imaging device 110 are assembled.

[0087] The physical guide 1272 may be a physical structure that can achieve mechanical matching. For example, the physical guide 1272 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 1272. For example, the physical guide 1272 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.

[0088] In some embodiments, the accommodating cavity 126, the light shielding connector 127 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 5 After the production process, the body and the shell can be assembled to build Figure 4 A fluorescent target 120 is shown.

[0089] Based on the fluorescent target of the aforementioned embodiment, a weak-light environment is formed between the target fluorescent material 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.

[0090] Exemplary fluorescent target preparation method:

[0091] Figure 5is an exemplary flow chart of a method for preparing a fluorescent target according to an exemplary embodiment of this specification.

[0092] in, Figure 5 The fluorescent target (the main structure) shown can be produced based on microelectronic manufacturing technology.

[0093] like Figure 5 As shown, process 500 may include:

[0094] S510, generating a light-shielding mask on the surface of the light-transmitting substrate.

[0095] In some embodiments, step S510 may refer to processing the light-transmitting substrate 310 to form a light-shielding mask on the surface of the light-transmitting substrate 310 .

[0096] In some embodiments, the specific execution method of step S510 may be related to the type of the light shielding mask. For example, if the light shielding mask is a photoresist, step S510 may be to spin-coat the photoresist on the transparent substrate 310. For another example, if the light shielding mask is a metal layer, step S510 may be to vapor-deposit the corresponding metal layer on the transparent substrate 310.

[0097] In some embodiments, the execution range of step S510 may be related to the area used to present a preset fluorescent image in the finished fluorescent target 120. For example, the light shielding mask generated in step S510 may completely cover the distribution area 125.

[0098] S520, etching a light shielding mask based on a preset calibration image to form a light shielding layer.

[0099] In some embodiments, step S510 may refer to laser etching the light shielding mask, thereby transferring the preset calibration image to the etched light shielding mask, thereby forming the light shielding layer 320. The laser etching based on the preset pattern may be performed based on an existing mask etching process.

[0100] After etching in step S520, the area in the transparent substrate corresponding to the preset calibration image does not have the etched light shielding mask. Therefore, the area covered by the light shielding layer in the transparent substrate can be used as the light shielding area, and the area not covered by the light shielding layer can be formed as the light transmitting area, so as to achieve the above Figure 1-Figure 4 Fluorescent targets as described in .

[0101] In some embodiments, considering that the excitation light needs to pass through the transparent substrate and form a focal plane below the transparent substrate during calibration, in order to reduce the restrictions of the transparent substrate on the imaging device, the aforementioned fluorescent target (transparent substrate portion) can be made as thin as possible. In some embodiments, based on the aforementioned P500, the total thickness of the aforementioned transparent substrate and its light shielding mask can be within 100nm.

[0102] In some embodiments, after determining the above-processed transparent substrate (fluorescent target body), subsequent assembly steps can be performed to determine the fluorescent target. That is, the above-mentioned P500 can also include the following steps:

[0103] S530, assembling the light-transmitting substrate with the light-shielding layer, the receiving cavity, and the target fluorescent material to obtain a fluorescent target.

[0104] In some embodiments, considering that the accommodating cavity 126 and the light-transmitting substrate form a closed space, in order to allow the target fluorescent material 130 to fill this space, the accommodating cavity 126 can be split for assembly. That is, the accommodating cavity 126 can be processed into two parts during processing, the first part can be used to place the processed light-transmitting substrate (fluorescent target 120 body), and the second part can be used to hold the target fluorescent material 130. After the two parts are assembled, the two parts can be assembled and sealed with materials such as rubber to form a fluorescent target.

[0105] In some embodiments, in order to form a sealed environment, an injection port and an exhaust port can be set on the aforementioned accommodating cavity 126 and the transparent substrate, and the accommodating cavity 126 and the transparent substrate are directly assembled to form a accommodating space for the target fluorescent material 130, and then the target fluorescent material 130 is injected based on the injection port and the exhaust port. After the space is filled, the injection port and the exhaust port can be closed (such as by using ultraviolet glue to cure in an ultraviolet environment) to construct a sealed environment.

[0106] It should be noted that, considering that the light-transmitting substrate is provided with a light-shielding layer only on one side, the assembly can be performed according to the location of the light-shielding layer. To improve the presentation effect, the side where the light-shielding layer is located can be close to the target fluorescent material 130 to construct the fluorescent target 120.

[0107] 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.

[0108] Exemplary imaging field of view calibration method:

[0109] Figure 6 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 6 The illustrated method may be performed by a processor within the imaging device 110 .

[0110] like Figure 6 As shown, process 600 may include:

[0111] S610, determining a target fluorescent material and a target fluorescent target.

[0112] 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.

[0113] 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.

[0114] 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.

[0115] 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.

[0116] In some embodiments, the determination process of S610 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.

[0117] S620, 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.

[0118] 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.

[0119] 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.

[0120] 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.

[0121] 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 target fluorescent material. Considering that the target fluorescent target is located between the imaging device and the target fluorescent material, 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 target fluorescent material.

[0122] Considering that there is a weak light environment between the imaging device and the target fluorescent material, 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.

[0123] A fluorescence calibration image may refer to a fluorescence image determined by an imaging device based on a target fluorescence target. The excitation light of the imaging device is blocked by the light-shielding 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 may be formed based on the fluorescence signal. Considering that the target fluorescent material itself is a homogeneous medium, the fluorescence calibration image may 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 may reflect the imaging result of the preset calibration image.

[0124] S630: 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.

[0125] 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.

[0126] 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.

[0127] 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).

[0128] 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.

[0129] 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.

[0130] 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.

[0131] 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.

[0132] 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.

[0133] 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.

[0134] 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.

[0135] 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.

[0136] 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.

[0137] 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 an imaging device based on weak light imaging technology, the fluorescent target includes a substrate, and the substrate includes: Shaded areas; and A light-transmitting area corresponding to a preset calibration image; Among them, the first side of the fluorescent target cooperates with the imaging device, and the second side cooperates with the target fluorescent material; the light-transmitting area is used to form a light-transmitting light path corresponding to the preset calibration image between the first side and the second side, 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 substrate comprises: a light-transmitting substrate; and A light shielding layer disposed on the light-transmitting substrate; The area of ​​the light-transmitting substrate blocked by the light-shielding layer and the light-shielding layer form the light-shielding area, and the area of ​​the light-transmitting substrate not blocked by the light-shielding layer forms the light-transmitting area.

3. The fluorescent target according to claim 2, characterized in that: The light shielding layer is disposed on the second side.

4. The fluorescent target according to claim 1, characterized in that: The distribution range of the light-transmitting area is not less than the imaging range of the imaging device. 5 . The fluorescent target according to claim 1 , characterized in that the fluorescent target further comprises a receiving cavity arranged on the second side, the receiving cavity being used to receive a fluorescent material so that the fluorescent material forms the fluorescent light ring and cooperates with the second side.

6. The fluorescent target according to claim 5, characterized in that: The material of the accommodating cavity is a light-shielding material.

7. The fluorescent target according to claim 1, characterized in that: The fluorescent target comprises a light shielding connecting member arranged around the substrate, and the light shielding connecting member is used to cooperate with the imaging device and form a weak light environment between the imaging device and the fluorescent target.

8. The fluorescent target according to claim 1, characterized in that: The light-shielding connecting member comprises a side wall and a physical guide portion arranged on an inner wall of the side wall, wherein the physical guide portion is used for detachably cooperating with the imaging device.

9. A method for preparing a fluorescent target, characterized in that: The method comprises: Generate a light-shielding mask on the surface of a transparent substrate: The light-shielding mask is etched based on a preset calibration image to form a light-shielding layer, wherein the area of ​​the light-transmitting substrate blocked by the light-shielding layer and the light-shielding layer form a light-shielding area, and the area of ​​the light-transmitting substrate not blocked by the light-shielding layer forms a light-transmitting area.

10. A method for calibrating an imaging device based on a fluorescent target, characterized in that: Applied to an imaging device based on low-light imaging technology, the method comprises: Determining a target fluorescent material and a target fluorescent target, wherein the target fluorescent target is the fluorescent target according to any one of claims 1 to 7, and the target fluorescent material forms a target fluorescent material and cooperates with a second side of the fluorescent target; In response to the target fluorescent target cooperating with the imaging device, adjusting the focal plane position of the imaging device to a target focal depth, and determining a fluorescent calibration image at the target focal depth, wherein the target focal depth corresponds to a thickness of a light-transmitting area of ​​the target 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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