Prism type laser excitation fluorescence detection system

Abstract

The invention discloses a prism type laser excitation fluorescence detection system, which relates to the technical field of biological imaging, and comprises a film window, a fluorescent diaphragm, a prism, a laser and a camera, wherein the fluorescent diaphragm is arranged below the thin film window and is attached to the top surface of the prism; the film window is used for containing a biological sample to be detected; laser emitted by the laser device takes the side surface of the prism as an incident surface, is refracted after entering the incident surface, and is incident to the fluorescent diaphragm to generate a fluorescent signal, so that the camera detects that the fluorescent signal irradiates a biological image of the biological sample; wherein the laser device is set to have a first incident angle, so that when the laser is incident to the fluorescent diaphragm, internal total reflection occurs in the fluorescent diaphragm. By applying the system provided by the embodiment of the invention, the biological sample can be prevented from being damaged or influenced by laser.

Description

Technical Field

[0001] The invention relates to the technical field of biological imaging, and in particular to a prism-type laser excitation fluorescence detection system. Background Art

[0002] QDM imaging (Quantum Dot Magnetic) is a biological imaging technology that uses micromagnetic nanoparticles (MNP) to mark and fit NV color centers. The spin quantum state of the luminescent defect of the NV color center in diamond is easily affected by the surrounding microwave field and static magnetic field, and can be read out by laser. It can measure the magnetic field or microwave field distribution around the biological sample being tested, and can achieve quantitative and non-destructive biomagnetic imaging. It has the characteristics of high spatial resolution, large field of view, large dynamic range of detectable magnetic field, and fast imaging speed.

[0003] In actual work, the lens fits the sample hole on the porous plate and pure gas is passed into the lens-sample hole cavity to pressurize it, so that the bottom of the sample porous cavity fits the fluorescent membrane, thereby realizing the detection of batch biological samples. The fluorescent membrane contains a material with NV color centers, or a membrane with other properties can be replaced.

[0004] NV center (Nitrogen-Vacancy Center) refers to a nitrogen-vacancy defect center, which is a common solid-state quantum bit and is widely used in quantum information science and nano-optics. To excite NV centers to produce light, a common method is to use optical excitation, using a laser of appropriate wavelength to irradiate the NV center. The common excitation wavelength is 532 nanometers. After the laser passes through the center, the photon energy in it is transferred to the electronic energy level of the NV center. This causes the transition of the electronic energy level of the NV center, and photons are emitted when the transition occurs between the ground state electronic energy level and the excited state electronic energy level of the NV center. The wavelength of these emitted photons is usually in the infrared range, about 640-800 nanometers.

[0005] The traditional laser optical path uses optical fiber or various reflectors to guide the laser to be incident on the NV color center. In this case, a technical solution is needed to avoid laser damage or influence on biological samples. Summary of the invention

[0006] The present invention aims to solve one of the technical problems in the related art at least to some extent. To this end, one object of the present invention is to provide a prism-type laser excitation fluorescence detection system to avoid laser damage or influence on biological samples.

[0007] According to a first aspect of an embodiment of the present invention, a prism-type laser excitation fluorescence detection system is provided, the system comprising: a film window, a fluorescent film, a prism, a laser, and a camera; wherein:

[0008] The fluorescent film is placed below the film window and adheres to the top surface of the prism;

[0009] The film window is used to hold the biological sample to be tested;

[0010] The laser emitted by the laser uses the side surface of the prism as an incident surface, is refracted after entering the incident surface, and is incident on the fluorescent film to generate a fluorescent signal, so that the camera detects the biological imaging of the biological sample irradiated by the fluorescent signal;

[0011] In which, the laser is configured to have a first incident angle, the first incident angle satisfies a condition of being greater than or equal to a second incident angle, and the second incident angle is determined based on the angle between the incident surface and the top surface, the refractive index of the laser in the air, and the refractive index of the laser in the prism, so that when the laser is incident on the fluorescent film, total internal reflection occurs in the fluorescent film.

[0012] Optionally, the second incident angle is determined in the following manner:

[0013]

[0014] Among them, θ i represents the second incident angle; θ0 represents the angle between the incident surface and the top surface; n1 represents the refractive index of the laser in the air; and n2 represents the refractive index of the laser in the prism.

[0015] Optionally, when the biological sample is placed on a multi-well plate and the fluorescent film sheet is attached to a transparent film of the multi-well plate, the second incident angle is determined in the following manner:

[0016]

[0017] Among them, θ i represents the second incident angle; θ0 represents the angle between the incident surface and the top surface; n1 represents the refractive index of the laser in the air; n2 represents the refractive index of the laser in the prism; n3 represents the refractive index of the laser in the transparent film, and n3<n2.

[0018] Optionally, the system further comprises an objective lens, a lens and a filter;

[0019] The objective lens is located above the film window and is used to collect the fluorescent signal and transmit it to the camera after being focused by the lens.

[0020] The filter is arranged between the lens and the camera.

[0021] Optionally, the angle between the incident surface and the top surface is an acute angle.

[0022] Optionally, the first incident angle is the sum of the angle between the incident surface and the top surface, and the refraction angle of the laser entering the prism.

[0023] Optionally, the target angle of the first incident angle is determined in the following manner:

[0024] Taking the second incident angle as the initial angle of the first incident angle, and starting from the initial angle, adjusting the first incident angle according to a preset step;

[0025] Determine the monitoring power of the laser at the exit surface of the prism after each adjustment, and determine the laser power incident on the fluorescent film based on the determined monitoring power and the corresponding incident power;

[0026] A target angle of the first incident angle is determined based on a maximum power among the laser powers.

[0027] Optionally, when the incident surface and the exit surface are symmetrically arranged relative to the vertical plane where the center of the fluorescent film is located, the power of the laser incident on the fluorescent film is determined in the following manner:

[0028] The first incident angle, the refractive index of the laser in the air, the refractive index of the laser in the prism, and the polarization component of the incident laser on the incident surface are used to calculate the first transmittance of the laser on the incident surface according to the Fresnel formula;

[0029] The second transmittance of the laser incident on the fluorescent film is calculated according to the Fresnel formula using the first incident angle, the angle between the top surface of the prism and the incident surface, the refractive index of the laser in the fluorescent film and the refractive index of the laser in the prism, and the polarization component of the incident laser on the surface of the fluorescent film;

[0030] The third transmittance of the laser on the exit surface of the prism is calculated according to the Fresnel formula using the exit angle of the laser on the exit surface of the prism, the refractive index of the laser in the air and the refractive index of the laser in the prism, and the polarization component of the incident laser on the exit surface;

[0031] The laser power incident on the fluorescent film is determined as follows:

[0032]

[0033] Among them, P represents laser power; a represents the first transmittance; b represents the second transmittance; c represents the third transmittance; P1 represents the determined monitoring power; and P0 represents the incident power corresponding to the determined monitoring power.

[0034] Optionally, the system further comprises a protective frame and a radiation structure;

[0035] The fluorescent film is installed in the middle of the protective frame, and the height of the protective frame is higher than the height of the fluorescent film;

[0036] The radiation structure is installed outside the protection frame.

[0037] Optionally, the system further comprises a prism support and a coating;

[0038] The prism is mounted on the prism bracket, and the coating is connected to the prism bracket and the system grounding electrode.

[0039] In the solution provided by the embodiment of the present invention, a prism is arranged at the bottom of the fluorescent film so that the laser can enter the fluorescent film through the refraction of the prism, and the first incident angle is set to meet the angle condition determined by the second incident angle, so that the laser undergoes total internal reflection in the fluorescent film. On the one hand, the fluorescence excitation effect can be improved, and on the other hand, the laser can be prevented from irradiating the biological sample, thereby damaging or affecting the biological sample.

[0040] Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 It is a structural schematic diagram of a first prism-type laser excitation fluorescence detection system provided by an embodiment of the present invention;

[0042] Figure 2a is a structural schematic diagram of a second prism-type laser excitation fluorescence detection system provided by an embodiment of the present invention;

[0043] Figure 2b is a structural schematic diagram of a third prism-type laser excitation fluorescence detection system provided by an embodiment of the present invention;

[0044] Figure 3a is a schematic structural diagram of a first prism provided by an embodiment of the present invention;

[0045] Figure 3b is a schematic structural diagram of a second prism provided by an embodiment of the present invention;

[0046] Figure 3c is a schematic structural diagram of a third prism provided by an embodiment of the present invention;

[0047] Figure 4a is a scene schematic diagram of a first laser power detection method provided by an embodiment of the present invention;

[0048] Figure 4b 4 is a schematic diagram of a scenario of a second laser power detection method provided by an embodiment of the present invention. DETAILED DESCRIPTION

[0049] Embodiments of the present invention are described in detail below, examples of which are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are intended to be used to explain the present invention, and should not be construed as limiting the present invention.

[0050] The prism-type laser-excited fluorescence detection system according to an embodiment of the present invention will be described below with reference to the accompanying drawings.

[0051] In one embodiment of the present invention, see Figure 1 , provides a prism-type laser excitation fluorescence detection system, including: a film window, a fluorescent film, a prism, a laser, and a camera; wherein,

[0052] The fluorescent film is placed below the film window and fits with the top surface of the prism;

[0053] A film window for holding a biological sample to be tested;

[0054] The laser emitted by the laser uses the side of the prism as the incident surface, is refracted after entering the incident surface, and is incident on the fluorescent film to generate a fluorescent signal, so that the camera detects the fluorescent signal to illuminate the biological sample for biological imaging;

[0055] In which, the laser is set to have a first incident angle, the first incident angle satisfies the condition of being greater than or equal to a second incident angle, and the second incident angle is determined based on the angle between the incident surface and the top surface, the refractive index of the laser in the air and the refractive index of the laser in the prism, so that when the laser is incident on the fluorescent film, total internal reflection occurs in the fluorescent film.

[0056] exist Figure 1 In the illustrated embodiment, the camera may specifically be a CMOS (Complementary Metal Oxide Semiconductor) camera.

[0057] In one embodiment of the present invention, a diamond thin film containing NV color centers may be used as a fluorescent film.

[0058] like Figure 1As shown, the film window can be a film window of a porous plate, and several biological samples to be detected are contained in several film windows. By moving the porous plate, the film windows are moved to the top of the fluorescent film in turn, and the measurement and analysis of the biological samples are achieved based on the fluorescent signal generated by the fluorescent film. A prism is set at the bottom of the fluorescent film so that the fluorescent film fits the top surface of the prism, and the laser enters the fluorescent film through the prism, that is, Figure 1 The incident laser in the prism reaches the top surface of the prism after refraction and illuminates the fluorescent film; the side of the prism on the side of the incident laser is the incident surface, and conversely, after being reflected by the fluorescent film, the side of the prism on the side of the reflected laser is the exit surface; the detector receives the exiting laser and obtains the power of the exiting laser, that is, the monitoring power.

[0059] Another example Figure 1 As shown, the system also includes an objective lens, a lens, and a filter; the objective lens is located above the film window and is used to collect the fluorescent signal, which is focused by the lens and then transmitted to the camera. In this way, the biological imaging generated by the fluorescent signal can be observed in the camera.

[0060] The filter is set between the lens and the camera. On the one hand, it filters out light other than the fluorescent signal, and on the other hand, it blocks the camera lens to prevent the noise light signal around the camera lens from entering the camera lens and interfering with the detection.

[0061] Biological imaging can include microwave field imaging and static magnetic field imaging. For example, microwave field imaging can be performed using a radiation structure composed of microstrip lines. Figure 2a As shown, the system also includes a protective frame and a radiation structure; the fluorescent diaphragm is installed in the middle of the protective frame, the NV color center is inside the fluorescent diaphragm, and the height of the protective frame is higher than the height of the fluorescent diaphragm; the radiation structure is installed on the outside of the protective frame, so as to provide a microwave field for the measurement of the NV color center. By applying an excitation laser to the NV color center, the magnetic field distribution in the biological sample in the porous plate film window above the diamond slice can be detected according to the fluorescence signal of the NV color center, or whether the biological sample in the film window contains magnetic beads can be detected. By providing a microwave field, the size of the magnetic field at each position can be known more specifically, or the noise light information can be eliminated by modulating the microwave field, thereby improving the detection sensitivity of the fluorescence signal.

[0062] In the presence of a protective frame, on the one hand, it is easier for the silicon nitride film in the film window to fit better with the diamond slice, and when the film window is inflated, it can limit and guide the deformation direction of the silicon nitride film; on the other hand, it can protect the diamond slice from damage.

[0063] Based on the above principle, there should be no magnetic field or electric field that interferes with detection around the NV color center. Since the porous plate needs to move to detect samples in the film windows at different positions, the movement of the porous plate is prone to generate static electricity. In order to overcome the interference of static electricity, the system is also equipped with a prism bracket and a coating. Figure 2b As shown, the prism is mounted on the prism bracket so that the incident surface and the exit surface are exposed on both sides of the prism bracket, and the substrate of the base prism bracket is coated with a coating on the upper and lower sides, and the coating is connected to the prism bracket and the system grounding electrode to conduct static electricity. In this installation method, the microstrip line, NV color center, radiation structure and etching area are all facing the top of the prism bracket, which is convenient for camera observation.

[0064] Specifically, for the etching area, the middle of one side of the substrate with coatings on both sides is etched to remove the coating, leaving the microstrip line and preventing the microstrip line from being connected to other ground coatings. In addition, the microstrip line can also be processed and pasted on the position where the coating is removed by etching the substrate.

[0065] From the above, in the solution provided by the embodiment of the present invention, a prism is set at the bottom of the fluorescent film so that the laser can enter the fluorescent film through the refraction of the prism, and the first incident angle is set to meet the angle condition determined by the second incident angle, so that the laser undergoes total internal reflection in the fluorescent film. On the one hand, the fluorescence excitation effect can be improved, and on the other hand, the laser can be prevented from irradiating the biological sample, damaging or affecting the biological sample.

[0066] In one embodiment, the second incident angle is determined as follows:

[0067]

[0068] Among them, θ i represents the second incident angle; θ0 represents the angle between the incident surface and the top surface; n1 represents the refractive index of the laser in the air; and n2 represents the refractive index of the laser in the prism.

[0069] In one embodiment, when the biological sample is placed on a multi-well plate and the fluorescent film sheet is attached to the transparent film of the multi-well plate, the second incident angle is determined as follows:

[0070]

[0071] Among them, θ i represents the second incident angle; θ0 represents the angle between the incident surface and the top surface; n1 represents the refractive index of the laser in the air; n2 represents the refractive index of the laser in the prism; n3 represents the refractive index of the laser in the transparent film, and n3<n2.

[0072] In one embodiment, the angle between the incident surface and the top surface is an acute angle. Figure 3aThe prism may have a trapezoidal cross section, and the upper base length of the trapezoidal cross section is greater than the lower base length. In this case, the angle θ0 between the incident surface and the top surface is an acute angle. In other embodiments, the prism may also have an inverted triangular cross section.

[0073] like Figure 3a , 3b , 3c, the first incident angle is θ i ,θ t is the refraction angle, and θ0 is the angle between the incident surface and the top surface. Figure 3a In the case where the length of the upper base is greater than the length of the lower base, Figure 3b In the case where the length of the upper base is equal to the length of the lower base, Figure 3c In the case where the length of the upper base is less than the length of the lower base. Figure 3a In the structure, the incident angle can be adjusted more freely. While satisfying the condition that the second incident angle is greater than or equal to the second incident angle, the incident angle can be made large or small. Figure 3b And Figure 3c As shown, the incident angle of the laser on the incident surface has little room for adjustment, and the incident angle is basically too large, which will lead to excessive reflectivity of the laser power and insufficient laser power entering the fluorescent film, so that the power of the laser needs to be increased, resulting in energy waste and also affecting the service life of each optical device in the optical path.

[0074] by Figure 3a Taking the most preferred prism structure as an example, since the refractive index of air is small and close to vacuum, when the laser enters the fluorescent film from the prism, no matter which of the two has a larger refractive index, it is only necessary to ensure that the laser incident angle from the prism entering the fluorescent film is greater than the critical incident angle of total reflection from the prism entering the air, so that the laser entering the fluorescent film can be totally reflected.

[0075] And, in Figure 3a In the prism structure, the first incident angle is the sum of the angle between the incident surface and the top surface and the refraction angle of the laser entering the prism. i Two calculation formulas for .

[0076] In order to reduce the reflectivity of the laser on the incident surface of the prism, in actual operation, the incident angle of the laser on the incident surface of the prism can be set slightly larger than θ i to ensure that the laser entering the fluorescent film can be totally reflected.

[0077] In one embodiment, the target angle of the first incident angle is determined as follows:

[0078] Taking the second incident angle as the initial angle of the first incident angle, and starting from the initial angle, adjusting the first incident angle according to a preset step;

[0079] Determine the monitoring power of the laser at the exit surface of the prism after each adjustment, and determine the laser power incident on the fluorescent film based on the determined monitoring power and the corresponding incident power;

[0080] The target angle of the first incident angle is determined based on the maximum power among the laser powers.

[0081] Stepping refers to a fixed angle. The adjustment process involves turning the laser so that the first incident angle increases by a fixed angle each time, for example, one degree each time.

[0082] A thermoelectric laser power meter is used for monitoring, and the laser power at the exit surface is obtained, which is the monitoring power.

[0083] After each adjustment, the incident power corresponding to the monitored power is the laser power at the incident surface when the laser enters the prism from the laser after this adjustment.

[0084] By optimizing the above adjustment process, the first incident angle corresponding to the best laser power is found to realize detection, thereby reducing power loss and saving energy.

[0085] In addition, when the incident angle of the laser on the incident surface of the prism is fixed, for a prism with a certain structure, the incident position of the laser on the incident surface is also determined; in addition to the incident angle on the incident surface of the prism affecting the laser power entering the fluorescent diaphragm, the incident angle of the incident laser at the interface between the fluorescent diaphragm and the prism and the travel of the laser in the prism will also affect the laser power entering the fluorescent diaphragm.

[0086] like Figure 4a and Figure 4b As shown, the relative positions of the film window, the fluorescent film, and the prism remain unchanged. Figure 4a and Figure 4b The incident surface is the same surface of the prism, and the exit surface is also the same surface on the other side of the prism. The difference is that the angle at which the laser emits the laser changes, causing the stroke of the incident laser and the reflected laser to change, and accordingly, the position at which the detector receives the laser also changes.

[0087] Therefore, by obtaining the laser power entering the fluorescent film, the first incident angle of the laser on the incident surface of the prism can be further optimized.

[0088] In order to obtain the laser power entering the fluorescent film, the incident surface and the exit surface of the prism can be firstly arranged symmetrically with respect to the vertical plane where the center of the fluorescent film is located. Figure 4aThe dotted line shown is set in this way so that the incident laser stroke in the prism is equal to the reflected laser stroke. If the incident surface and the exit surface of the prism are asymmetrical with respect to the vertical plane where the center of the fluorescent film is located, when the angles of the incident laser and the reflected laser change at the same time, the stroke of the reflected laser in the prism and the angle at the exit surface will change differently compared with the incident laser, making it difficult to calculate the laser power entering the fluorescent film based on the laser power monitoring value obtained by the detector.

[0089] When the incident surface and the exit surface are symmetrically arranged relative to the vertical plane where the center of the fluorescent film is located, the laser power incident on the fluorescent film is determined in the following manner:

[0090] The first incident angle, the refractive index of the laser in the air, the refractive index of the laser in the prism, and the polarization component of the incident laser on the incident surface are used to calculate the first transmittance of the laser on the incident surface according to the Fresnel formula;

[0091] The second transmittance of the laser incident on the fluorescent film is calculated according to the Fresnel formula using the first incident angle, the angle between the top surface of the prism and the incident surface, the refractive index of the laser in the fluorescent film and the refractive index of the laser in the prism, and the incident laser polarization component on the surface of the fluorescent film where the laser is incident;

[0092] The third transmittance of the laser on the output surface of the prism is calculated according to the Fresnel formula using the output angle of the laser on the output surface of the prism, the refractive index of the laser in the air and the refractive index of the laser in the prism, and the polarization component of the incident laser on the output surface;

[0093] The laser power incident on the fluorescent film is determined as follows:

[0094]

[0095] Wherein, P represents the laser power; a represents the first transmittance; b represents the second transmittance; c represents the third transmittance; P1 represents the determined monitoring power; and P0 represents the incident power corresponding to the determined monitoring power.

[0096] In the above embodiment, the top surface of the prism is the interface between the prism and the fluorescent film. Due to the preset symmetry relationship about the vertical plane where the center of the fluorescent film is located, the exit angle is equal to the first incident angle, which is convenient for measurement and calculation.

[0097] In the above process, there is no order in which the first, second and third transmittances are calculated. They are listed in steps only for the convenience of explanation.

[0098] In order to make the above measurement and calculation accurate, the prerequisite conditions can be controlled in advance so that the thickness of the fluorescent film is uniform and flatly attached to the top surface of the prism, and the plane where the laser incident, refracted and reflected light paths are located is perpendicular to the side edges of the prism. The prism can be regarded as a right prism, and the edge between the two trapezoidal cross sections of the prism is the side edge.

[0099] According to the above method, assuming that the other parameters remain unchanged, the incident angle of the laser on the incident surface of the prism is adjusted according to the preset step, starting from θ0 and gradually increasing. Each time the adjustment is made, the laser power incident on the fluorescent film is calculated based on the monitoring power P1 of the laser on the exit surface, and the maximum laser power entering the fluorescent film is selected accordingly, and the angle of the incident angle that reaches the maximum laser power is determined as the target angle.

[0100] It should be noted that the logic and / or steps represented in the flowchart or otherwise described herein, for example, can be considered as a sequenced list of executable instructions for implementing logical functions, and can be specifically implemented in any computer-readable medium for use by an instruction execution system, device or apparatus (such as a computer-based system, a system including a processor, or other system that can fetch instructions from an instruction execution system, device or apparatus and execute instructions), or in combination with these instruction execution systems, devices or apparatuses. For the purposes of this specification, "computer-readable medium" can be any device that can contain, store, communicate, propagate or transmit a program for use by an instruction execution system, device or apparatus, or in combination with these instruction execution systems, devices or apparatuses. More specific examples of computer-readable media (a non-exhaustive list) include the following: an electrical connection portion with one or more wirings (electronic device), a portable computer disk box (magnetic device), a random access memory (RAM), a read-only memory (ROM), an erasable and programmable read-only memory (EPROM or flash memory), an optical fiber device, and a portable compact disk read-only memory (CDROM). In addition, the computer-readable medium may even be paper or other suitable medium on which the program is printed, since the program may be obtained electronically, for example, by optically scanning the paper or other medium and then editing, interpreting or processing in other suitable ways if necessary, and then stored in a computer memory.

[0101] It should be understood that the various parts of the present invention can be implemented by hardware, software, firmware or a combination thereof. In the above-mentioned embodiments, a plurality of steps or methods can be implemented by software or firmware stored in a memory and executed by a suitable instruction execution system. For example, if implemented by hardware, as in another embodiment, it can be implemented by any one of the following technologies known in the art or their combination: a discrete logic circuit having a logic gate circuit for implementing a logic function for a data signal, a dedicated integrated circuit having a suitable combination of logic gate circuits, a programmable gate array (PGA), a field programmable gate array (FPGA), etc.

[0102] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "examples", "specific examples", or "some examples" means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner.

[0103] In the description of the present invention, it is to be understood that the terms “center”, “longitudinal”, “lateral”, “length”, “width”, “thickness”, “up”, “down”, “front”, “back”, “left”, “right”, “vertical”, “horizontal”, “top”, “bottom”, “inside”, “outside”, “clockwise”, “counterclockwise”, “axial”, “radial”, “circumferential”, etc., indicating orientations or positional relationships based on the orientations or positional relationships shown in the accompanying drawings, are only for the convenience of describing the present invention and simplifying the description, and do not indicate or imply that the referred device or element must have a specific orientation, be constructed and operated in a specific orientation, and therefore should not be understood as limiting the present invention.

[0104] In addition, the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include at least one of the features. In the description of the present invention, the meaning of "plurality" is at least two, such as two, three, etc., unless otherwise clearly and specifically defined.

[0105] In the present invention, unless otherwise clearly specified and limited, the terms "installed", "connected", "connected", "fixed" and the like should be understood in a broad sense, for example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be a direct connection or an indirect connection through an intermediate medium, it can be the internal connection of two elements or the interaction relationship between two elements, unless otherwise clearly defined. For ordinary technicians in this field, the specific meanings of the above terms in the present invention can be understood according to specific circumstances.

[0106] In the present invention, unless otherwise clearly specified and limited, a first feature being "above" or "below" a second feature may mean that the first and second features are in direct contact, or the first and second features are in indirect contact through an intermediate medium. Moreover, a first feature being "above", "above" or "above" a second feature may mean that the first feature is directly above or obliquely above the second feature, or simply means that the first feature is higher in level than the second feature. A first feature being "below", "below" or "below" a second feature may mean that the first feature is directly below or obliquely below the second feature, or simply means that the first feature is lower in level than the second feature.

[0107] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. A prism-type laser excitation fluorescence detection system, characterized in that: The system includes: a film window, a fluorescent film, a prism, a laser, and a camera; wherein: The fluorescent film is placed below the film window and adheres to the top surface of the prism; The film window is used to hold the biological sample to be tested; The laser emitted by the laser uses the side surface of the prism as an incident surface, is refracted after entering the incident surface, and is incident on the fluorescent film to generate a fluorescent signal, so that the camera detects the biological imaging of the biological sample irradiated by the fluorescent signal; In which, the laser is configured to have a first incident angle, the first incident angle satisfies a condition of being greater than or equal to a second incident angle, and the second incident angle is determined based on the angle between the incident surface and the top surface, the refractive index of the laser in the air, and the refractive index of the laser in the prism, so that when the laser is incident on the fluorescent film, total internal reflection occurs in the fluorescent film.

2. The prism-type laser excitation fluorescence detection system according to claim 1, characterized in that: The second incident angle is determined as follows: Among them, θ i represents the second incident angle; θ0 represents the angle between the incident surface and the top surface; n1 represents the refractive index of the laser in the air; and n2 represents the refractive index of the laser in the prism.

3. The prism-type laser excitation fluorescence detection system according to claim 1, characterized in that: When the biological sample is placed on a multi-well plate and the fluorescent film sheet is attached to the transparent film of the multi-well plate, the second incident angle is determined as follows: Among them, θ i represents the second incident angle; θ0 represents the angle between the incident surface and the top surface; n1 represents the refractive index of the laser in the air; n2 represents the refractive index of the laser in the prism; n3 represents the refractive index of the laser in the transparent film, and n3<n2.

4. The prism-type laser excitation fluorescence detection system according to claim 1, characterized in that: The system also includes an objective, a lens, and a filter; The objective lens is located above the film window and is used to collect the fluorescence signal and transmit it to the camera after being focused by the lens; The filter is arranged between the lens and the camera.

5. The prism-type laser excitation fluorescence detection system according to claim 1, characterized in that: The angle between the incident surface and the top surface is an acute angle.

6. The prism-type laser excitation fluorescence detection system according to claim 5, characterized in that: The first incident angle is the sum of the angle between the incident surface and the top surface and the refraction angle of the laser entering the prism.

7. The prism-type laser excitation fluorescence detection system according to claim 1, characterized in that: The target angle of the first incident angle is determined as follows: Taking the second incident angle as the initial angle of the first incident angle, and starting from the initial angle, adjusting the first incident angle according to a preset step; Determine the monitoring power of the laser at the exit surface of the prism after each adjustment, and determine the laser power incident on the fluorescent film based on the determined monitoring power and the corresponding incident power; A target angle of the first incident angle is determined based on a maximum power among the laser powers.

8. The prism-type laser excitation fluorescence detection system according to claim 7, characterized in that: When the incident surface and the exit surface are symmetrically arranged relative to the vertical plane where the center of the fluorescent film is located, the laser power incident on the fluorescent film is determined in the following manner: The first incident angle, the refractive index of the laser in the air, the refractive index of the laser in the prism, and the polarization component of the incident laser on the incident surface are used to calculate the first transmittance of the laser on the incident surface according to the Fresnel formula; The second transmittance of the laser incident on the fluorescent film is calculated according to the Fresnel formula using the first incident angle, the angle between the top surface of the prism and the incident surface, the refractive index of the laser in the fluorescent film and the refractive index of the laser in the prism, and the polarization component of the incident laser on the surface of the fluorescent film; The third transmittance of the laser on the exit surface of the prism is calculated according to the Fresnel formula using the exit angle of the laser on the exit surface of the prism, the refractive index of the laser in the air and the refractive index of the laser in the prism, and the polarization component of the incident laser on the exit surface; The laser power incident on the fluorescent film is determined as follows: Among them, P represents laser power; a represents the first transmittance; b represents the second transmittance; c represents the third transmittance; P1 represents the determined monitoring power; and P0 represents the incident power corresponding to the determined monitoring power.

9. The prism-type laser excitation fluorescence detection system according to claim 1, characterized in that: The system also includes a protective frame and a radiating structure; The fluorescent film is installed in the middle of the protective frame, and the height of the protective frame is higher than the height of the fluorescent film; The radiation structure is installed outside the protection frame.

10. The prism-type laser excitation fluorescence detection system according to claim 1, characterized in that: The system also includes a prism holder and a coating; The prism is mounted on the prism bracket, and the coating is connected to the prism bracket and the system grounding electrode.

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