A fluorescence detection optical system and a scanning method and a calibration method thereof

Abstract

The application belongs to the technical field of fluorescence signal detection, and particularly relates to a fluorescence detection optical system, a scanning method thereof and a calibration method thereof. The system comprises an excitation light coupling-in optical system and an emission light coupling-out optical system. The excitation light coupling-in optical system comprises an excitation light source, an excitation light coupling lens group, a coupling-in optical fiber and a reaction hole. The excitation light coupling lens group comprises an excitation light collimating lens group, a color filter wheel and an excitation light coupling-in lens. The emission light coupling-out optical system comprises an emission light coupling lens group, a coupling-out optical fiber, a detector and a reaction hole. The emission light coupling lens group comprises an emission light collimating lens group, a color filter wheel and an emission light coupling-out lens. The fluorescence detection optical system, the scanning method thereof and the calibration method thereof can increase the light detection efficiency, improve the stability of light signal output, reduce the system volume, realize the scanning output of multi-waveband excitation light and the scanning detection of multiple reaction holes, improve the consistency between holes, and reduce the inter-bench difference between instruments.

Description

Technical Field

[0001] This invention belongs to the field of fluorescence signal detection technology, specifically relating to a fluorescence detection optical system and its scanning and calibration methods. Background Technology

[0002] Fluorescence is a photoluminescence phenomenon. When certain substances absorb light of a specific wavelength, electrons are excited from the ground state to a higher energy state, and then release energy as longer wavelength light upon returning to the ground state. In quantitative real-time polymerase chain reaction (PCR), the hydrolysis probe contains a fluorescent reporter group. During PCR extension, the Taq enzyme hydrolyzes the probe, emitting light of the corresponding wavelength under excitation light. The intensity of this emitted light is used to monitor the DNA amplification process, enabling highly sensitive and quantitative analysis.

[0003] To detect the fluorescence signal generated in polymerase chain reaction (PCR) and achieve high sensitivity and quantitative analysis, the optical system must maximize the power of the coupled light at the illumination end and the coupled light at the detection end, thereby increasing the coupling-out optical efficiency to enable the detection of even extremely weak light. During optical scanning, the excitation light enters the reaction well as a stable coupled light through the coupling optical system and exits from the detection well as a stable coupled light through the coupling optical system. During scanning, the optical system must ensure the consistency of the coupled light and coupled light in each reaction well. Furthermore, to meet the current demand for miniaturized medical devices, the optical system, as a crucial component of PCR fluorescence equipment, must also meet miniaturization requirements.

[0004] There are currently three main types of optical systems and implementation methods for polymerase chain reaction (PCR): The first type is a top or side scanning system based on a photodiode (PD), silicon photodiode (SIPM), or photomultiplier tube (PMT) with a dichroic mirror, excitation filter, emission filter, and coupling lens. This type of system has the problem of stability between the excitation light coupled into the detector aperture and the emission light coupled out of the detector aperture during the scanning process. In addition, it is difficult for the excitation light and emission light of this type of system to achieve a relatively high coupling efficiency, which affects the sensitivity of the system. The second type is a top-scanning system based on a complementary metal-oxide (CMOS) camera or charge-coupled device (CCD) consisting of an illumination optical system and an imaging optical system composed of a dichroic mirror, an excitation filter, and an emission filter. This type of system has an edge effect, with a high degree of inconsistency between the excitation and emission light at the edges and the excitation and emission light at the center, leading to overall consistency problems. In addition, this type of system is large in size and cannot meet the requirements of current miniaturized medical devices. The third type is a fiber optic scanning system based on a photodiode (PD), silicon photodiode (SIPM), or photomultiplier tube (PMT). This system uses an excitation filter to couple the excitation light into the optical system, and an emission filter to couple the emission light out of the optical system, all connected to an optical fiber. The fiber optic port can be located on the side or top of the reaction aperture. This type of system suffers from alignment issues between the optical system's input / output light spots and the optical fiber during scanning, leading to decreased stability. Currently, all polymerase chain reaction (PCR) fluorescence detection systems do not consider the calibration of absolute light intensity between instruments, which also increases the inter-instrument variability. Summary of the Invention

[0005] To address at least one of the technical problems of "how to increase optical detection efficiency, how to improve the stability of optical signal output, how to reduce the overall size of the fluorescence detection optical system, and how to reduce the inter-instrument difference," this invention provides the following technical solution: In a first aspect, the present invention provides a fluorescence detection optical system, comprising an excitation light coupling optical system and an emission light coupling optical system; The excitation light coupling optical system includes an excitation source, an excitation light coupling lens group, a coupling fiber, and a reaction aperture; the excitation light coupling lens group includes an excitation light collimating lens group, a color filter wheel, and an excitation light coupling lens. The emitted light coupling optical system includes an emitted light coupling lens group, a coupling fiber, a detector, and a reaction aperture; the emitted light coupling lens group includes an emitted light collimating lens group, another color filter wheel, and an emitted light coupling lens. The excitation light collimating lens group and the emission light coupling lens are fixed on a fixed plate, which connects the detector and the excitation light source; the excitation light coupling lens and the emission light collimating lens group are fixed on another fixed plate, which connects the coupling fiber and the coupling fiber; the other end of the coupling fiber and the coupling fiber is fixed on the reaction hole.

[0006] Furthermore, the excitation collimating lens group includes a first lens having positive refractive power and both surfaces being spherical or aspherical, and a second lens having positive refractive power and both surfaces being spherical or aspherical.

[0007] Furthermore, the light-emitting collimating lens group includes a fourth lens with positive refractive power and both surfaces being spherical or aspherical, and a fifth lens with positive refractive power and both surfaces being spherical or aspherical.

[0008] Furthermore, the excitation light collimating lens group satisfies the following condition: D1≤5mm, D2≤7.5mm, where D1 is the aperture of the first lens and D2 is the aperture of the second lens; 0.43≤f_excitation_collimation / f1≤0.63, 0.56≤f_excitation_collimation / f2≤0.76, where f_excitation_collimation is the total focal length of the excitation_collimation lens group, and f1 and f2 are the focal lengths of the first lens and the second lens, respectively; 1.47≤n1≤1.56, 1.47≤n2≤1.56, where n1 and n2 are the refractive indices of the first lens and the second lens, respectively; 55≤v1≤70, 55≤v2≤70, where v1 and v2 are the Abbe numbers of the first lens and the second lens, respectively.

[0009] Furthermore, the excitation light-coupled lens satisfies the following condition: D3≤7.5mm, 0.75≤fexcitationcouple / f3≤1, where D3 is the aperture of the excitationcouple lens, f3 is the focal length of the excitationcouple lens, and fexcitationcouple is the total focal length of the excitationcouple lens group. 1.47≤n3≤1.56, 55≤v3≤70, where n3 is the refractive index of the excitation light coupler lens and v3 is the Abbe number of the excitation light coupler lens.

[0010] Furthermore, the light-emitting collimating lens group satisfies the following condition: D4≤7.5mm, D5≤12mm, where D4 is the fourth lens diameter and D5 is the fifth lens diameter; 0.68≤f_emitted_beam_collimation / f4≤0.88, 0.29≤f_emitted_beam_collimation / f5≤0.49, where f_emitted_beam_collimation is the total focal length of the emitted_beam_collimation lens group, and f4 and f5 are the focal lengths of the fourth and fifth lenses, respectively; 1.47≤n4≤1.56, 1.47≤n5≤1.56, where n4 and n5 are the refractive indices of the fourth and fifth lenses, respectively; 55≤v4≤70, 55≤v5≤70, where v4 and v5 are the Abbe numbers of the fourth and fifth lenses, respectively.

[0011] Furthermore, the emitted light coupling lens satisfies the following condition: D6≤12mm, 0.55≤f_emitting_coupling / f6≤0.75, where D6 is the aperture of the emitting_coupling lens, f6 is the focal length of the emitting_coupling lens, and f_emitting_coupling is the total focal length of the emitting_coupling lens group. 1.47≤n6≤1.56, 55≤v6≤70, where n6 is the refractive index of the excitation light coupler lens and v6 is the Abbe number of the excitation light coupler lens.

[0012] Furthermore, the excitation light source wavelength range is [450nm-750nm].

[0013] Furthermore, the type of light source for excitation is not limited to light-emitting diodes, laser diodes, or xenon lamps.

[0014] Furthermore, the detection band of the detector is ∈ [500nm-800nm].

[0015] Furthermore, the detector type is not limited to photodiodes, silicon photodiodes, avalanche photodiodes, and photomultiplier tubes.

[0016] Furthermore, the coupled-in fiber and coupled-out fiber meet the following conditions: 0.40≤numerical aperture≤0.70, 0.2mm≤core or bundled core diameter≤3mm, and the wavelength range of the transmitted light is 400nm-800nm.

[0017] Furthermore, the color filter wheels of the excitation light coupling lens group and the emission light coupling lens group are composed of uniformly spaced filters, which meet the following conditions: the excitation light filtering band is: 455nm-480nm, 510nm-540nm, 575nm-595nm, 620nm-640nm, 672nm-688nm; the emission light filtering band is: 515nm-535nm, 555nm-575nm, 610nm-630nm, 655nm-675nm, 705nm-745nm; the aperture range of the excitation light filter is 8.4mm to 10mm, and the aperture range of the emission light filter is 9.2mm to 12mm.

[0018] By collimating the excitation light and the emission light through the filter, the wavelength shift of the light in the filter can be reduced, thereby reducing crosstalk between different wavelength fluorescent substances.

[0019] Furthermore, the color filter wheel is evenly distributed with N types of excitation filters and N types of emission filters, which are arranged alternately in sequence, where N is an integer and N≥1; M reaction holes are provided, where M is an integer and M≥1; each reaction hole is matched with an excitation light coupling optical system and an emission light coupling optical system, the spacing angle between each type of excitation filter is consistent, the distance from each type of excitation filter to the center of the color filter wheel is consistent, the spacing angle between each type of emission filter is consistent, and the distance from each type of emission filter to the center of the color filter wheel is consistent.

[0020] N types of excitation filters and N types of emission filters represent N types of excitation filters and emission filters with different optical specifications, respectively.

[0021] The excitation light is coupled into the optical system through the corresponding reaction aperture for timed emission. The excitation light coupled into the optical system at the corresponding position of different excitation filters is output sequentially to reduce the interference of excitation stray light between different bands.

[0022] Furthermore, the arrangement of the excitation light coupling optical system and the emission light coupling optical system on the fixed disk is based on the color filter wheel. If N≤M, the angle between the excitation collimating lens group and the emission excitation lens on their fixed disk is 360 / N°, and the distance to the center of their fixed disk is the same as the distance from the excitation filter to the center of the color filter wheel; the angle between the excitation input lens and the emission collimating lens group on their fixed disk is 360 / N°, and the distance to the center of their fixed disk is the same as the distance from the emission filter to the center of the color filter wheel.

[0023] Furthermore, if N > M, u is the quotient of M / N, u is rounded down to the nearest integer, the angle between the excitation collimating lens group and the emission coupling lens on its fixed disk is 360 / (N×(u+1))°, and the distance to the center of its fixed disk is the same as the distance from the excitation filter to the center of the color filter wheel; the angle between the excitation input lens and the emission collimating lens group on its fixed disk is 360 / (N×(u+1))°, and the distance to the center of its fixed disk is the same as the distance from the emission filter to the center of the color filter wheel.

[0024] The excitation light source of the excitation light coupling optical system emits excitation light in the 450nm-750nm band or a wider spectrum. This excitation light enters the coupling optical fiber through the excitation light coupling lens group of the system. The excitation light is then collimated by the excitation light collimating lens group of the excitation light coupling lens group, generating collimated light with an aperture of less than 7.5mm and a divergence of less than ±3°. This collimated excitation light is then incident on a color filter wheel. The spot of the collimated excitation light at the filter position on the color filter wheel is smaller than the size of the filter. During the movement of the color filter wheel, the filter has a large range of motion to receive the collimated excitation light, thereby improving the stability during the scanning process. After passing through the color filter wheel, the collimated excitation light enters the excitation light coupling lens of the excitation light coupling lens group. The excitation light coupling lens focuses the collimated excitation light into an optical fiber coupling spot at the optical fiber coupling position. The spot is then transmitted through the coupling optical fiber and enters the reaction aperture. The fluorescent material in the reaction aperture is affected by the excitation light, and electrons are excited from the ground state to a high-energy state. Subsequently, when returning to the ground state, it emits a weak emission light.

[0025] The excitation light coupling optical system has an overall aperture of less than 7.5 mm and an overall working length of less than 22 mm, making it a miniature optical system, thereby reducing the overall volume of the fluorescence detection optical system.

[0026] The fluorescent material in the reaction aperture of the emission light coupling optical system emits emission light distinct from the excitation light wavelength under the influence of excitation light. This emission light is conducted through the coupling fiber and enters the emission light coupling lens group of the emission light coupling optical system. The emitted light is collimated by the emission light collimating lens group of the emission light coupling lens group, generating collimated light with an aperture of less than 8.5 mm and a divergence of less than ±3°. This collimated emitted light enters the color filter wheel. The spot of the collimated emitted light at the filter position on the color filter wheel is smaller than the size of the filter. During the movement of the color filter wheel, the filter has a large range of motion to receive the collimated emitted light, thereby improving the stability during the scanning process. After passing through the color filter wheel, the collimated emitted light enters the emission light coupling lens of the emission light coupling lens group. The emission light coupling lens focuses the collimated emitted light into a detection coupling spot at the detector position, and the detector outputs real-time optical power. The overall aperture of the emission light coupling optical system is less than 12 mm, and the overall working length is less than 30 mm, which is a miniature optical system, thereby reducing the overall size of the fluorescence detection optical system.

[0027] Secondly, the present invention provides a fluorescence detection optical scanning method, employing the aforementioned fluorescence detection optical system, the method comprising: The excitation light is coupled into the optical system through the excitation light and outputs excitation light of different wavelengths into the reaction aperture. Specifically, by rotating the color filter wheel in conjunction with the pulse excitation light output from the excitation source, the color filter wheel rotates to the position of the corresponding excitation filter, which is consistent with the aperture position of the excitation light collimated by the excitation light collimating lens group. Furthermore, the emitted light is output through the emitted light coupling optical system to emit emitted light of different wavelengths into the detector. Specifically, by rotating the color filter wheel, in conjunction with the pulsed emitted light output generated by the fluorescent material in the reaction aperture under the influence of pulsed excitation light, the color filter wheel rotates to the position of the corresponding emission filter, which is consistent with the aperture position of the emitted light collimated by the emission light collimating lens group.

[0028] By rotating the color filter wheel, the positions of the corresponding excitation filter and emission filter are continuously moved to the aperture positions of the collimated excitation light and emission light. The rotation time is different and the positions are different. In conjunction with the excitation light source, pulse light is generated at the corresponding time to realize the scanning output of excitation light and emission light of different wavelengths and detect fluorescence of different wavelengths.

[0029] The fluorescence detection optical scanning method proposed in this invention controls the spot size of the excitation light coupling optical system and the emission light coupling optical system at the filter position during the optical scanning process. This allows the color filter wheel to have a large movement scanning range, ensuring the stability of the excitation light coupling efficiency and the emission light coupling efficiency, and improving the stability of the optical signal output.

[0030] Furthermore, it also includes scanning output of N band excitation light and N band emission light, and scanning detection of M reaction holes, which is achieved by: The excitation and emission filters of the color filter wheel move to the aperture positions of the collimated excitation and emission light, and the excitation light corresponding to the reaction aperture is coupled into the optical system for sequential emission. The excitation light at the corresponding positions of the N excitation filters and N emission filters is coupled into the optical system to output excitation light in sequence. The excitation light is coupled into the optical system to cause the fluorescent material in the reaction aperture to emit light. The intensity of the emitted light is recorded by the detector through the emission light coupled out of the optical system.

[0031] N bands of excitation light and N bands of emission light correspond to N types of excitation filters and N types of emission filters.

[0032] By generating excitation light in different wavelengths, generating specific arrangements of different filters on the color filter wheel, and using specific arrangements of the emission light coupling lens and emission light collimating lens group and the excitation light collimating lens group and emission light coupling lens on the fixed disk, as well as the pulse output of the excitation light and the rotation scanning of the color filter wheel, crosstalk of excitation stray light between different wavelengths is reduced, thereby realizing the scanning output of multi-wavelength excitation light and the scanning detection of multiple reaction apertures.

[0033] Thirdly, the present invention provides a fluorescence detection optical calibration method, characterized in that... The first reaction orifice of the master prototype is used as the orifice for measuring the concentration of the luminescent material, and is called the master reaction orifice. The slanted formula of the emitted light intensity of the master reaction orifice with respect to the concentration of the luminescent material is calibrated. The linear formula relating the emitted light intensity of different instruments and apertures to the concentration of the luminescent material was used to calibrate the intensity of light emitted from different luminescent materials at varying concentrations. The pre-defined formula for the intensity of light emitted from the reaction orifice with respect to the concentration of the substance is applied to the formula for the mother reaction orifice, so that the two formulas are consistent. Under the condition that the concentration of fluorescent material is the same, the emission intensity of the mother reaction well is consistent with the emission intensity of the reaction well after calibration, and the emission intensity of the reaction well after calibration is obtained.

[0034] Compared with the prior art, the present invention has the following beneficial effects: (1) The fluorescence detection optical system proposed in this invention can couple out light from the reaction aperture of the emission light coupling optical system and enter the detector with the full spot diameter and full numerical aperture of the optical fiber, which greatly increases the light detection efficiency.

[0035] (2) The fluorescence detection optical system and its scanning method proposed in this invention, during the optical scanning process, by controlling the collimation and spot size of the coupled light of the excitation light coupling optical system and the emission light coupling optical system on the color filter wheel, the color filter wheel has a large scanning position range, which can ensure the stability of the excitation light coupling efficiency and the emission light coupling efficiency, and improve the stability of the optical signal output.

[0036] (3) The fluorescence detection optical system proposed in this invention, the excitation light coupling optical system and the emission light coupling optical system are both micro optical systems. With a small volume, the excitation light coupling and the emission light coupling are realized, reducing the overall volume of the fluorescence detection optical system, which meets the current demand for small fluorescence detection equipment and medical equipment.

[0037] (4) The fluorescence detection optical scanning method proposed in this invention continuously moves the positions of the corresponding excitation filter and emission filter to the aperture positions of the collimated excitation light and emission light by rotating the color filter wheel. The rotation time is different and the position is different. The excitation light source generates pulse excitation light at the corresponding time to realize the scanning output of excitation light and emission light of different wavelengths and detect fluorescence of different wavelengths.

[0038] (5) The fluorescence detection optical scanning method proposed in this invention reduces crosstalk of excitation stray light between different bands by generating excitation light in different bands, the specific arrangement of different filters on the color filter wheel, the specific arrangement of the emission light coupling lens and the emission light collimating lens group and the excitation light collimating lens group and the emission light coupling lens on the fixed disk, the pulse output of the excitation light, and the rotation scanning of the color filter wheel, thereby realizing the scanning output of multi-band excitation light and the scanning detection of multiple reaction holes.

[0039] (6) The fluorescence detection system and optical calibration method proposed in this invention, in which different reaction holes are coupled to optical systems through optical fibers, and the absolute light intensity is calibrated between holes, greatly reduces the difference between different reaction holes and improves the consistency between holes for the same optical system.

[0040] (7) The fluorescence detection optical system and its scanning and calibration methods proposed in this invention, through the dye-based optical calibration method, can eliminate the inconsistencies caused by the inconsistencies of light sources, detectors, optical components and assembly of different instruments, reduce the inter-instrument difference between instruments, and greatly improve the consistency between instruments.

[0041] (8) The fluorescence detection optical system proposed in this invention can reduce the wavelength shift of light in the filter by collimating the excitation light and the emission light through the filter, thereby reducing the crosstalk of light emission of fluorescent substances in different wavelength bands.

[0042] (9) The fluorescence detection optical system proposed in this invention emits light in a sequential manner through the excitation light coupled into the optical system corresponding to the corresponding reaction hole. The excitation light coupled into the optical system at the corresponding positions of different excitation filters outputs excitation light in sequence, thereby reducing the interference of excitation stray light between different bands. Attached Figure Description

[0043] Figure 1 This is a diagram of the fluorescence detection optical system of Embodiment 1 of the present invention; Figure 2 This is a diagram showing the excitation light coupling optical system and ray tracing of Embodiment 1 of the present invention; Figure 3 This is a light field energy distribution diagram of the side of the color filter wheel near the excitation light source in Embodiment 1 of the present invention; Figure 4 This is a diagram showing the optical field energy distribution at the coupling surface of the excitation light of the coupled optical fiber in Embodiment 1 of the present invention. Figure 5 This is a diagram showing the emission light coupling optical system and ray tracing of Embodiment 1 of the present invention; Figure 6 This is a light field energy distribution diagram of the side of the color filter wheel near the coupled optical fiber in Embodiment 1 of the present invention, showing the side from which light is emitted. Figure 7 This is a diagram showing the light field energy distribution on the detection surface of the detector in Embodiment 1 of the present invention. Figure 8 This is a diagram showing the composition of the color filter wheel in Embodiment 1 of the present invention; Figure 9 This is a schematic diagram showing the distribution of the surface of the first fixed disk in Embodiment 1 of the present invention; Figure 10 This is a schematic diagram of the distribution of the second fixed disk surface in Embodiment 1 of the present invention. Figure 11 This is a diagram of the fluorescence detection optical system of Embodiment 2 of the present invention; Figure 12 This is a diagram showing the excitation light coupling optical system and ray tracing of Embodiment 2 of the present invention; Figure 13 This is a light field energy distribution diagram of the side of the color filter wheel near the excitation light source in Embodiment 2 of the present invention; Figure 14 This is an optical field energy distribution diagram of the excitation optical coupling surface of the coupled optical fiber in Embodiment 2 of the present invention; Figure 15 This is a diagram showing the emission light coupling optical system and ray tracing of Embodiment 2 of the present invention; Figure 16 This is a light field energy distribution diagram of the side of the color filter wheel near the coupling optical fiber in Embodiment 2 of the present invention, showing the light emanating from it. Figure 17 This is a light field energy distribution diagram of the detector surface of the detector in Embodiment 2 of the present invention; Figure 18 This is a diagram of the fluorescence detection optical system of Embodiment 3 of the present invention; Figure 19 This is a diagram showing the excitation light coupling optical system and ray tracing of Embodiment 3 of the present invention; Figure 20 This is a light field energy distribution diagram of the side of the color filter wheel near the excitation light source in Embodiment 3 of the present invention; Figure 21 This is an energy distribution diagram of the excitation light coupling surface of the coupled optical fiber in Embodiment 3 of the present invention; Figure 22 This is a diagram showing the emission light coupling optical system and ray tracing of Embodiment 3 of the present invention; Figure 23 This is a light field energy distribution diagram of the side of the color filter wheel near the coupling optical fiber in Embodiment 3 of the present invention, showing the side from which light is emitted. Figure 24 This is a light field energy distribution diagram of the detector surface of the detector in Embodiment 3 of the present invention; Figure 25 is A diagram of the fluorescence detection optical system of Embodiment 4 of the present invention; Figure 26 This is a diagram showing the excitation light coupling optical system and ray tracing of Embodiment 4 of the present invention; Figure 27 This is a light field energy distribution diagram of the side of the color filter wheel near the excitation light source in Embodiment 4 of the present invention; Figure 28 This is an energy distribution diagram of the excitation light coupling surface of the coupled optical fiber in Embodiment 4 of the present invention; Figure 29 This is a diagram showing the emission light coupling optical system and ray tracing of Embodiment 4 of the present invention; Figure 30 This is a light field energy distribution diagram of the side of the color filter wheel near the coupling fiber in Embodiment 4 of the present invention, showing the light emanating from it. Figure 31 This is a light field energy distribution diagram of the detector surface of the detector in Embodiment 4 of the present invention.

[0044] Figure label: 1-Excitation light coupling optical system; 11-Excitation source; 12-Excitation light coupling lens group; 121-First lens; 122-Second lens; 123-Excitation light coupling lens; 13-Coupling fiber; 2-Emitting light coupling optical system; 21-Emitting light coupling lens group; 211-Fourth lens; 212-Fifth lens; 213-Emitting light coupling lens; 22-Coupling fiber; 23-Detector; 3-Reaction aperture; 4-Color filter wheel; 421-First excitation filter; 422-Second excitation filter; 423-Third excitation filter; 424-Fourth excitation filter; 425-Fifth excitation filter; 411-First emission filter; 412-Second emission filter; 413-Third emission filter; 414-Fourth emission filter; 415-Fifth emission filter; 5 - First fixed plate; 521 - First position; 522 - Second position; 523 - Third position; 511 - Fourth position; 512 - Fifth position; 513 - Sixth position; 6 - Second fixed plate; 621 - First position; 622 - Second position; 623 - Third position; 611 - Fourth position; 612 - Fifth position; 613 - Sixth position. Detailed Implementation

[0045] The technical solution of the present invention will be clearly described below with reference to the accompanying drawings. Obviously, the described embodiments are not all embodiments of the present invention. All other embodiments obtained by those skilled in the art without creative effort are within the protection scope of the present invention.

[0046] Example 1: Please see Figure 1 The fluorescence detection optical system consists of an excitation light coupling optical system 1 and an emission light coupling optical system 2. The excitation light coupling optical system 1 consists of an excitation light source 11, an excitation light coupling lens group 12, a coupling fiber 13, and a reaction aperture 3. The emission light coupling optical system 2 consists of an emission light coupling lens group 21, an emission fiber 22, a detector 23, and a reaction aperture 3.

[0047] The excitation light coupling lens group 12 consists of an excitation light collimating lens group, a color filter wheel 4, and an excitation light coupling lens 123. The excitation light collimating lens group consists of a first lens 121 and a second lens 122.

[0048] The light-emitting coupling lens group 21 comprises a light-emitting collimating lens group, another color filter wheel 4, and a light-emitting output lens 213. The light-emitting collimating lens group consists of a fourth lens 211 and a fifth lens 212.

[0049] The excitation light collimating lens group and the emission light coupling lens 213 are fixed on a fixed plate, which connects the detector 23 and the excitation light source 11; the excitation light coupling lens 123 and the emission light collimating lens group are fixed on another fixed plate, which connects the coupling fiber 13 and the coupling fiber 22; the other end of the coupling fiber 13 and the coupling fiber 22 is fixed to the side of the reaction hole 3 or on the reaction hole 3.

[0050] The light emitted by the excitation source 11 is collimated by the excitation light collimating lens group composed of the first lens 121 and the second lens 122. The collimated light passes through the filter in the color filter wheel 4 to obtain the excitation light of the corresponding wavelength. The excitation light of the corresponding wavelength is coupled into the coupling fiber 13 through the excitation light coupling lens 123. The excitation light is transmitted to the reaction hole 3 through the coupling fiber 13. The fluorescent material in the reaction hole 3 is affected by the excitation light and generates emission light of the corresponding wavelength. The emitted light is guided through the coupling fiber 22 into the emission light collimating lens group composed of the fourth lens 211 and the fifth lens 212. After being collimated by the emission light collimating lens group, the collimated light passes through the filter in the color filter wheel 4 to obtain the emission light of the corresponding wavelength band. The emission light of the corresponding wavelength band is coupled into the detector 23 through the emission light coupling lens 213.

[0051] The inserted optical fiber 13 and the output optical fiber 22 are preferably connected to the side of the reaction hole 3, or they can be connected to the top of the reaction hole 3.

[0052] The color filter wheel 4 of the excitation light coupling lens group 12 and the emission light coupling lens group 21 is composed of filters arranged at uniform intervals. The color filter wheel 4 is evenly distributed with N types of excitation filters and N types of emission filters, and the excitation filters and emission filters are arranged alternately in sequence.

[0053] The excitation light coupling optical system 1 and ray tracing are described in [reference]. Figure 2 S0 is the emitting surface of the excitation light source 11; S1 and S2 are two surfaces of the first lens 121, with S1 being the surface closer to the excitation light source 11 and S2 being the surface farther from the excitation light source 11; S3 and S4 are two surfaces of the second lens 121, with S3 being the surface closer to the excitation light source 11 and S4 being the surface farther from the excitation light source 11; S5 and S6 are two surfaces of the filter of the color filter wheel 4, with S5 being the surface closer to the excitation light source 11 and S6 being the surface farther from the excitation light source 11; S7 and S8 are two surfaces of the excitation light coupling lens 123, with S7 being the surface closer to the excitation light source 11 and S8 being the surface farther from the excitation light source 11; and S9 is the excitation light coupling surface of the coupled optical fiber 13. The light path is from S0 to S9. In Table 1, the direction along the light path is positive, and the thickness represents the distance between the surface and the next surface along the light path.

[0054] Table 1

[0055] See Figure 3 To ensure the light field energy distribution on the side S5 of the filter of the color filter wheel 4 near the excitation light source 11, the light spot is circular with a diameter of less than 6.35 mm. The diameter of the excitation filter of the color filter wheel 4 is 8.4 mm. During motion scanning, there is a motion alignment tolerance of 2.05 mm, which can ensure the stability of the excitation light output during the scanning process.

[0056] See Figure 4 To release the optical field energy at the excitation light coupling surface S9 of the coupled fiber 13, the light spot is square with a side length of less than 2.7 mm, which can couple into optical fibers of 1 mm to 3 mm.

[0057] See Figure 2 The total optical length of the excitation light source 11 and the excitation light coupling lens group 12 is 20.4 mm, and the optical diameter is 6.3 mm. It belongs to a miniature optical system to reduce the overall system size.

[0058] The emitted light-coupled optical system 2 and ray tracing are described in [reference]. Figure 5 S10 is the light-emitting surface of the coupling fiber 22. S11 and S12 are the two surfaces of the fourth lens 211, with S11 being the surface closer to the coupling fiber 22 and S12 being the surface farther from the coupling fiber 22. S13 and S14 are the two surfaces of the fifth lens 212, with S13 being the surface closer to the coupling fiber 22 and S14 being the surface farther from the coupling fiber 22. S15 and S16 are the two surfaces of the filter of the color filter wheel 4, with S15 being the surface closer to the coupling fiber 22 and S16 being the surface farther from the coupling fiber 22. S17 and S18 are the two surfaces of the emitting light coupling lens 213, with S17 being the surface closer to the coupling fiber 22 and S18 being the surface farther from the coupling fiber 22. S19 is the detection surface of the detector 23, and the light path is from S10 to S19. In Table 2, the path of light is the positive direction, and the thickness represents the distance between the surface and the next surface along the path of light.

[0059] Table 2

[0060] See Figure 6 To ensure the light field energy distribution on the side S15 where the light is emitted, the light spot is circular with a diameter of less than 7.26 mm. The excitation filter of the color filter wheel 4 has a diameter of 9.4 mm. During motion scanning, there is a motion alignment tolerance of 2.14 mm, which can ensure the stability of the excitation light output during the scanning process.

[0061] See Figure 7 To ensure the energy distribution of the light field on the detection surface S19 of detector 23, the light spot is circular with a diameter of less than 2.5 mm. For detectors with a detection surface diameter of less than 2.6 mm, the energy can be completely coupled in. Furthermore, for the coupled light emitted from S10, through the emitted light coupling optical system 2, it can be completely coupled into detectors with a detection surface diameter greater than 2.6 mm, greatly increasing the coupling efficiency of the emitted light.

[0062] See Figure 5 The total optical length of the light-emitting coupling lens group 21 and the detector 23 is 28.4 mm, and the optical diameter is 10 mm. It belongs to a miniature optical system to reduce the overall system size.

[0063] See Figure 8 In this embodiment, the color filter wheel 4 is composed of five excitation filters and five emission filters alternating in a circular direction.

[0064] The five excitation filters, arranged in circumferential direction, are: first excitation filter 421, second excitation filter 422, third excitation filter 423, fourth excitation filter 424, and fifth excitation filter 425. The angle between adjacent excitation filters is 72°; The corresponding excitation light filter bands are as follows: 455nm-480nm, 510nm-540nm, 575nm-595nm, 620nm-640nm, and 672nm-688nm.

[0065] The five emission filters, arranged in circumferential direction, are: first emission filter 411, second emission filter 412, third emission filter 413, fourth emission filter 414, and fifth emission filter 415. The angle between adjacent emission filters is 72°; The corresponding emission light filtering bands are: 515nm-535nm, 555nm-575nm, 610nm-630nm, 655nm-675nm, and 705nm-745nm.

[0066] The two fixed plates are designated as the first fixed plate 5 and the second fixed plate 6.

[0067] See Figure 9In this embodiment, the excitation light coupling lens 123 and the fourth lens 211 and fifth lens 212 of the emission light collimating lens group are fixed to a first fixed disk 5. The first fixed disk 5 has a first position 521, a second position 522, and a third position 523, and an excitation light coupling lens 123 is provided at each of the first position 521, the second position 522, and the third position 523. The first fixed disk 5 also has a fourth position 511, a fifth position 512, and a sixth position 513, and an emission light collimating lens group is provided at each of the fourth position 511, the fifth position 512, and the sixth position 513, each including the fourth lens 211 and the fifth lens 212. The angle between the first position 521 and the second position 522 is 72°, and the angle between the first position 521 and the third position 523 is 144°. The angle between the fourth position 511 and the fifth position 512 is 72°, and the angle between the fourth position 511 and the sixth position 513 is 144°. The angle between the fourth position 511 and the fifth position 512 is the same as the angle between the first emission filter 411 and the second emission filter 412.

[0068] See Figure 10 In this embodiment, the light emitting lens 213 and the first lens 121 and the second lens 122 of the excitation light collimating lens group are fixed on the second fixed disk 6. The second fixed disk 6 has a first position 621, a second position 622 and a third position 623. An excitation light collimating lens group is set at each of the first position 621, the second position 622 and the third position 623, and each position includes the first lens 121 and the second lens 122. The second fixed disk 6 has a fourth position 611, a fifth position 612, and a sixth position 613, with an emitting light coupling lens 213 set at each position. The angle between the first position 621 and the second position 622 is 72°, the angle between the first position 621 and the third position 623 is 144°, the angle between the fourth position 611 and the fifth position 612 is 72°, and the angle between the fourth position 611 and the sixth position 613 is 144°. The angle between the fourth position 611 and the fifth position 612 is the same as the angle between the first emitting filter 411 and the second emitting filter 412.

[0069] During the scanning process, the color filter wheel 4 rotates to the position of the corresponding excitation filter and emission filter. This position is consistent with the aperture position of the excitation light collimating lens group in the excitation light coupling optical system 1, and also consistent with the aperture position of the emission light collimating lens group in the emission light coupling optical system 2. Through the rotation of the color filter wheel 4, the position of the corresponding excitation filter and emission filter is continuously moved to the aperture position of the collimated excitation light and emission light. The rotation time is different at different positions, and the excitation light source 11 generates pulse light at the corresponding time. The excitation light optical system corresponding to the corresponding reaction aperture 3 emits light in sequence. The excitation light coupling optical system 1 at the corresponding positions of the five excitation and emission filters outputs excitation light in sequence, reducing crosstalk of excitation stray light between different bands and realizing scanning detection of five bands and three reaction apertures 3.

[0070] Example 2: The fluorescence detection optical system shown in the second embodiment of the present invention is basically the same as the fluorescence detection optical system shown in the first embodiment, and will not be described again here. The differences are as follows: Please see Figure 11 In the second embodiment of the present invention, the two surfaces of the first lens 121 of the excitation light collimating lens group are spherical, the two surfaces of the second lens 122 are both spherical, and the two surfaces of the excitation light coupling lens 123 are both spherical. The two surfaces of the first lens 211 of the emission light collimating lens group are spherical, the two surfaces of the second lens 212 are both spherical, and the two surfaces of the emission light coupling lens 213 are both spherical.

[0071] In this embodiment, the excitation light coupling optical system 1 and the ray tracing are described in detail below. Figure 12 .

[0072] In this embodiment, the data of the first lens 121, the second lens 122, and the excitation light coupling lens 123 in the excitation light coupling optical system 1, including the radius of curvature, thickness, refractive index, dispersion coefficient, etc. of each lens, are adopted from Table 3.

[0073] Table 3

[0074] See Figure 13 To ensure the light field energy distribution on the side S5 of the filter of the color filter wheel 4 near the excitation light source 11, the light spot is circular with a diameter of less than 6.8 mm. The diameter of the excitation filter of the color filter wheel 4 is 8.4 mm. During motion scanning, there is a motion alignment tolerance of 1.6 mm, which can ensure the stability of the excitation light output during the scanning process.

[0075] See Figure 14To release the light field energy at the excitation light coupling surface S9 of the coupled fiber 13, the light spot is square with a side length of less than 2.4 mm, which can couple to optical fibers of 1 mm to 3 mm. Compared with Example 1, the coupled fiber 13 has more energy.

[0076] See Figure 12 The total optical length of the excitation light source 11 and the excitation light coupling lens group 12 is 17.2 mm, and the optical diameter is 6.3 mm. It belongs to a miniature optical system and is smaller in size than that of Example 1.

[0077] In this embodiment, the emitted light coupling optical system 2 and the ray tracing are described in detail below. Figure 15 .

[0078] In this embodiment, the data of the fourth lens 211, the fifth lens 212, and the emitting light coupling lens 213 in the coupling optical system 2, including the radius of curvature, thickness, refractive index, dispersion coefficient, etc. of each lens, are adopted from Table 4.

[0079] Table 4

[0080] See Figure 16 To ensure the light field energy distribution on the side S15 where the light is emitted, the light spot is circular with a diameter of less than 7.14 mm. The excitation filter of the color filter wheel 4 has a diameter of 9.4 mm. During motion scanning, there is a motion alignment tolerance of 2.26 mm, which can ensure the stability of the excitation light output during the scanning process.

[0081] See Figure 17 To achieve the desired light field energy distribution on the detection surface S19 of detector 23, the light spot is circular with a diameter less than 2.4 mm. For detectors with a detection surface diameter less than 2.5 mm, the energy can be completely coupled in. Furthermore, the coupled light emitted from S10 can be fully coupled into detectors with a detection surface diameter greater than 2.6 mm through the emitted light coupling optical system 2. Compared to Example 1, the coupled light spot is smaller, allowing for the selection of detectors with a smaller detection area.

[0082] See Figure 15 The total optical length of the light-emitting coupling lens group 21 and the detector 23 is 23.3 mm, and the optical diameter is 10 mm. It belongs to a miniature optical system and is smaller in size than that of Example 1.

[0083] Example 3: The fluorescence detection optical system shown in the third embodiment of the present invention is basically the same as the fluorescence detection optical system shown in the first embodiment, and will not be described again here. The differences are as follows: Please see Figure 18In the third embodiment of the present invention, the two surfaces of the first lens 121 of the excitation light collimating lens group are spherical near the excitation source 11 and quadratic surfaces away from the excitation source 11. Both surfaces of the second lens 122 are quadratic surfaces, and both surfaces of the excitation light coupling lens 123 are quadratic surfaces. The two surfaces of the first lens 211 of the emission light collimating lens group are spherical, both surfaces of the second lens 212 are quadratic surfaces, and both surfaces of the emission light coupling lens 213 are quadratic surfaces. The quadratic surfaces satisfy the following expression:

[0084] Where z is the surface elevation, r is the vertical distance from the surface vertex to any point on the surface, c is the curvature of the surface vertex, and k is the surface conic coefficient.

[0085] In this embodiment, the excitation light coupling optical system 1 and the ray tracing are described in detail below. Figure 19 .

[0086] In this embodiment, the data of the first lens 121, the second lens 122, and the excitation light coupling lens 123 in the excitation light coupling optical system 1, including the radius of curvature, thickness, refractive index, dispersion coefficient, etc. of each lens, are adopted from Table 5.

[0087] Table 5

[0088] See Figure 20 To ensure the light field energy distribution on the side S5 of the filter of the color filter wheel 4 near the excitation light source 11, the light spot is circular with a diameter of less than 6.8 mm. The diameter of the excitation filter of the color filter wheel 4 is 8.4 mm. During motion scanning, there is a motion alignment tolerance of 1.6 mm, which can ensure the stability of the excitation light output during the scanning process.

[0089] See Figure 21 To release the light field energy at the excitation light coupling surface S9 of the coupled fiber 13, the light spot is square with a side length of less than 2.08 mm, which can couple fiber optic cables of 1 mm to 2.2 mm. Compared with Examples 1 and 2, the coupled fiber optic cable has more energy and the coupled light spot is more uniform.

[0090] See Figure 19 The total optical length of the excitation light source 11 and the excitation light coupling lens group 12 is 15.3 mm, and the optical diameter is 6.3 mm. It belongs to a miniature optical system and is smaller in size than that of Embodiment 1 and Embodiment 2.

[0091] In this embodiment, the emitted light coupling optical system 2 and the ray tracing are described in detail below. Figure 22 .

[0092] In this embodiment, the data of the fourth lens 211, the fifth lens 212, and the light-emitting coupling lens 213 in the light-emitting optical system 2, including the radius of curvature, thickness, refractive index, dispersion coefficient, etc. of each lens, are adopted from Table 6.

[0093] Table 6

[0094] See Figure 23 To ensure the light field energy distribution on the side S15 of the color filter wheel 4 near the light emitted from the coupling fiber 22, the light spot is circular with a diameter less than 5.94 mm. The excitation filter of the color filter wheel 4 has a diameter of 9.4 mm. During motion scanning, there is a motion alignment tolerance of 3.46 mm, which ensures the stability of the excitation light output during scanning. Compared with embodiments 1 and 2, this embodiment has a larger motion alignment tolerance and more stable excitation light output.

[0095] See Figure 24 To achieve the desired light field energy distribution on the detection surface S19 of detector 23, the light spot is circular with a diameter less than 1.48 mm. For detectors with a detection surface diameter less than 1.6 mm, the energy can be completely coupled in. Furthermore, the coupled light emitted from S10 can be fully coupled into detectors with a detection surface diameter greater than 1.6 mm through the emitted light coupling optical system 2. Compared to embodiments 1 and 2, the coupled light spot is smaller, allowing for the selection of detectors with a smaller detection area.

[0096] See Figure 22 The total optical length of the light-emitting coupling lens group 21 and the detector 23 is 19.3 mm and the optical diameter is 10 mm. It belongs to a miniature optical system and is smaller in size compared with embodiments 1 and 2.

[0097] Example 4: The fluorescence detection optical system shown in the fourth embodiment of the present invention is basically the same as the fluorescence detection optical system shown in the first embodiment, and will not be described again here. The differences are as follows: Please see Figure 25, In the fourth embodiment of the present invention, the two surfaces of the first lens 121 of the excitation light collimating lens group are a quadratic surface closer to the excitation light source 11 and an even-order aspherical surface farther from the excitation light source 11. Both surfaces of the second lens 122 are even-order aspherical surfaces. The two surfaces of the excitation light coupling lens 123 are even-order aspherical surfaces closer to the excitation light source 11 and quadratic surfaces farther from the excitation light source 11.

[0098] The first lens 211 of the light-emitting collimating lens group has two spherical surfaces, the second lens 212 has two even-order aspherical surfaces, and the light-emitting coupling lens 213 has two even-order aspherical surfaces.

[0099] Even-order aspherical surfaces satisfy the following expression:

[0100] Where z is the surface elevation, r is the vertical distance from the surface vertex to any point on the surface, c is the curvature of the surface vertex, k is the surface conic coefficient, and α1-α8 are the first to eighth aspherical coefficients, respectively.

[0101] In this embodiment, the excitation light coupling optical system 1 and the ray tracing are described in detail below. Figure 26 .

[0102] In this embodiment, the data of the first lens 121, the second lens 122, and the excitation light coupling lens 123 in the excitation light coupling optical system 1, including the radius of curvature, thickness, refractive index, dispersion coefficient, etc. of each lens, are as shown in Table 7. Table 7

[0103] See Figure 27 To ensure the light field energy distribution on the side S5 of the filter of the color filter wheel 4 near the excitation light source 11, the light spot is circular with a diameter of less than 6.14 mm. The diameter of the excitation filter of the color filter wheel 4 is 8.4 mm. During motion scanning, there is a motion alignment tolerance of 2.26 mm, which can ensure the stability of the excitation light output during the scanning process.

[0104] See Figure 28 To release the light field energy at the excitation light coupling surface S9 of the coupled fiber 13, the light spot is square with a side length of less than 2.06 mm, which can couple fiber optics from 1 mm to 2.2 mm. Compared with Examples 1 and 2, the coupled fiber has more energy and the coupled light spot is more uniform.

[0105] See Figure 26 The total optical length of the excitation light source 11 and the excitation light coupling lens group 12 is 13mm and the optical diameter is 6.3mm. It belongs to a miniature optical system and is smaller in size than embodiments 1, 2 and 3.

[0106] In this embodiment, the emitted light coupling optical system 2 and the ray tracing are described in detail below. Figure 29 .

[0107] In this embodiment, the data of the fourth lens 211, the fifth lens 212, and the light-emitting coupling lens 213 in the light-emitting optical system 2, including the radius of curvature, thickness, refractive index, dispersion coefficient, etc. of each lens, are adopted from Table 8.

[0108] Table 8

[0109] See Figure 30To ensure the light field energy distribution on the side S15 of the color filter wheel 4 near the light emitted from the coupling fiber 22, the light spot is circular with a diameter less than 5.1 mm. The excitation filter of the color filter wheel 4 has a diameter of 9.4 mm. During motion scanning, there is a motion alignment tolerance of 4.3 mm, which ensures the stability of the excitation light output during scanning. Compared with embodiments 1, 2, and 3, this embodiment has a larger motion alignment tolerance and more stable excitation light output.

[0110] See Figure 31 To achieve the desired light field energy distribution on the detection surface S19 of detector 23, the light spot is circular with a diameter less than 1.58 mm. For detectors with a detection surface diameter less than 1.7 mm, the energy can be completely coupled in. Furthermore, the coupled light emitted from S10 can be fully coupled into detectors with a detection surface diameter greater than 1.7 mm through the emitted light coupling optical system 2. Compared to embodiments 1 and 2, the coupled light spot is smaller, allowing for the selection of detectors with a smaller detection area.

[0111] See Figure 29 The total optical length of the light-emitting coupling lens group 21 and the detector 23 is 13.7 mm and the optical diameter is 7 mm. It belongs to a miniature optical system and is smaller in size compared with embodiments 1, 2 and 3.

[0112] Example 5: This embodiment provides a fluorescence detection optical scanning method, employing the fluorescence detection optical system described in claim 12, the method comprising: The excitation light is coupled into the optical system through the excitation light and outputs excitation light of different wavelengths into the reaction aperture. Specifically, by rotating the color filter wheel in conjunction with the pulse excitation light output from the excitation source, the color filter wheel rotates to the position of the corresponding excitation filter, which is consistent with the aperture position of the excitation light collimated by the excitation light collimating lens group. Furthermore, the emitted light is output through the emitted light coupling optical system to emit emitted light of different wavelengths into the detector. Specifically, by rotating the color filter wheel, in conjunction with the pulsed emitted light output generated by the fluorescent material in the reaction aperture under the influence of pulsed excitation light, the color filter wheel rotates to the position of the corresponding emission filter, which is consistent with the aperture position of the emitted light collimated by the emission light collimating lens group.

[0113] Furthermore, it also includes scanning output of N band excitation light and N band emission light, and scanning detection of M reaction holes, which is achieved by: The excitation and emission filters of the color filter wheel move to the aperture positions of the collimated excitation and emission light. The excitation light corresponding to the reaction aperture is coupled into the optical system 1 for sequential emission. The excitation light at the corresponding positions of the N excitation filters and N emission filters is coupled into the optical system 1 in sequence to output excitation light. The excitation light is coupled into the optical system to cause the fluorescent material in the reaction aperture to emit light. The intensity of the emitted light is recorded by the detector through the emission light coupled out of the optical system.

[0114] Example 6: This embodiment provides a fluorescence detection optical calibration method, including: The first reaction orifice of the master prototype is used as the orifice for measuring the concentration of the luminescent material, and is called the master reaction orifice. The slant formula for the emission intensity of the master reaction orifice with respect to the concentration of the luminescent material is calibrated as follows: y_m ...

[0115] The slant formula for the emission intensity of different instruments at different apertures with respect to the concentration of the luminescent material is used to calibrate the emission intensity of different instruments at different apertures by using different concentrations of luminescent material: ynm = knm × xnm + bnm, where ynm is the original emission intensity of the m-th reaction aperture of the n-th prototype, xnm is the concentration of the fluorescent material in the m-th reaction aperture of the n-th prototype, and knm and bnm are the parameters of the slant formula for the emission intensity of the m-th reaction aperture of the n-th prototype with respect to the concentration of the luminescent material.

[0116] The pre-defined slanted formula for the intensity of light emitted from the m-th reaction hole of the n-th instrument with respect to the concentration of the substance is calibrated onto the slanted formula of the parent reaction hole, so that the two slanted formulas are consistent. Specifically, high and low concentration luminescent materials are taken and their initial concentrations are recorded. Then, they are placed in the preset reaction well and the mother reaction well, respectively, and the light emission intensity of the high and low concentration luminescent materials in the preset reaction well and the mother reaction well is recorded. Based on the initial concentration and the light emission intensity of the high and low concentration luminescent materials, the intercept and slope of the two slant formulas are calculated. High and low concentration luminescent materials, with concentrations of xH and Xl respectively, were placed in the m-th orifice of the n-th prototype and the mother reaction orifice, respectively. The emission intensities ymH, ynmH, ymL, and ynmL were recorded. Among them, ymH is the emission intensity of the high-concentration luminescent material in the mother reaction orifice, ynmH is the emission intensity of the high-concentration luminescent material in the m-th reaction orifice of the n-th prototype, ymL is the emission intensity of the low-concentration luminescent material in the mother reaction orifice, and ynmL is the emission intensity of the low-concentration luminescent material in the m-th reaction orifice of the n-th prototype. The nm and bnm can be calculated from ymH, ymL, xH, and Xl, and the km and bm can be calculated from ymH, ymL, xH, and Xl.

[0117] Under the condition of the same fluorescent substance concentration, the emission intensity of the mother reaction well is consistent with the emission intensity of the pre-calibrated reaction well, thus obtaining the pre-calibrated emission intensity of the reaction well. Specifically, To ensure that the emission intensity of the mother reaction well is consistent with the emission intensity of the m-th reaction well of the n-th prototype after calibration, under the condition of the same fluorescent material concentration, substitute xnm = (ynm - bnm) / knm into ynm calibration = k_m mother × xnm + b_m mother, where ynm is the emission intensity of the m-th well of the n-th instrument before calibration, and ynm calibration is the emission intensity of the m-th well of the n-th instrument after calibration, we can obtain: ynm calibration = (ynm - ynmH) × (y_mHy_mL) / (ynmH - ynmL) + y_mH. The above formula explains the relationship between the light intensity of the m-th hole of the n-th instrument after calibration and the initial light intensity.

[0118] The above technical features constitute the preferred embodiment of the present invention, which has strong adaptability and optimal implementation effect. Non-essential technical features can be added or removed according to actual needs to meet the needs of different situations.

[0119] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A fluorescence detection optical scanning method, employing a fluorescence detection optical system, characterized in that, The fluorescence detection optical system includes an excitation light coupling optical system and an emission light coupling optical system; The excitation light coupling optical system includes an excitation source, an excitation light coupling lens group, a coupling fiber, and a reaction aperture; the excitation light coupling lens group includes an excitation light collimating lens group, a color filter wheel, and an excitation light coupling lens. The emitted light coupling optical system includes an emitted light coupling lens group, a coupling fiber, a detector, and a reaction aperture; the emitted light coupling lens group includes an emitted light collimating lens group, another color filter wheel, and an emitted light coupling lens. The excitation light collimating lens group and the emission light coupling lens are fixed on a fixed plate, which connects the detector and the excitation light source; the excitation light coupling lens and the emission light collimating lens group are fixed on another fixed plate, which connects the coupling fiber and the coupling fiber; the other end of the coupling fiber and the coupling fiber is fixed on the reaction hole. The excitation collimating lens group includes a first lens with positive refractive power and both surfaces being spherical or aspherical, and a second lens with positive refractive power and both surfaces being spherical or aspherical. The light-emitting collimating lens group includes a fourth lens with positive refractive power and both surfaces being spherical or aspherical, and a fifth lens with positive refractive power and both surfaces being spherical or aspherical. The excitation light collimating lens group satisfies the following conditions: D1≤5mm, D2≤7.5mm, where D1 is the aperture of the first lens and D2 is the aperture of the second lens; 0.43≤f_excitation_collimation / f1≤0.63, 0.56≤f_excitation_collimation / f2≤0.76, where f_excitation_collimation is the total focal length of the excitation_collimation lens group, and f1 and f2 are the focal lengths of the first lens and the second lens, respectively; 1.47≤n1≤1.56, 1.47≤n2≤1.56, where n1 and n2 are the refractive indices of the first lens and the second lens, respectively; 55≤v1≤70, 55≤v2≤70, where v1 and v2 are the Abbe numbers of the first lens and the second lens, respectively; The excitation optical coupling lens satisfies the following condition: D3≤7.5mm, 0.75≤fexcitationcouple / f3≤1, where D3 is the aperture of the excitationcouple lens, f3 is the focal length of the excitationcouple lens, and fexcitationcouple is the total focal length of the excitationcouple lens group. 1.47≤n3≤1.56,55≤v3≤70,where n3 is the refractive index of the excitation light coupler lens and v3 is the Abbe number of the excitation light coupler lens; The light-emitting collimating lens group satisfies the following conditions: D4≤7.5mm, D5≤12mm, where D4 is the fourth lens diameter and D5 is the fifth lens diameter; 0.68≤f_emitted_beam_collimation / f4≤0.88, 0.29≤f_emitted_beam_collimation / f5≤0.49, where f_emitted_beam_collimation is the total focal length of the emitted_beam_collimation lens group, and f4 and f5 are the focal lengths of the fourth and fifth lenses, respectively; 1.47≤n4≤1.56, 1.47≤n5≤1.56, where n4 and n5 are the refractive indices of the fourth and fifth lenses, respectively; 55≤v4≤70, 55≤v5≤70, where v4 and v5 are the Abbe numbers of the fourth and fifth lenses, respectively; The emitting optical coupler lens satisfies the following condition: D6≤12mm, 0.55≤f_emitting_coupling / f6≤0.75, where D6 is the aperture of the emitting_coupling lens, f6 is the focal length of the emitting_coupling lens, and f_emitting_coupling is the total focal length of the emitting_coupling lens group. 1.47≤n6≤1.56,55≤v6≤70,where n6 is the refractive index of the excitation light coupler lens and v6 is the Abbe number of the excitation light coupler lens; The color filter wheels of the excitation light coupling lens group and the emission light coupling lens group are composed of uniformly spaced filters, which meet the following conditions: the excitation light filtering band is 455nm-480nm, 510nm-540nm, 575nm-595nm, 620nm-640nm, 672nm-688nm; the emission light filtering band is 515nm-535nm, 555nm-575nm, 610nm-630nm, 655nm-675nm, 705nm-745nm; the aperture range of the excitation light filter is 8.4mm to 10mm, and the aperture range of the emission light filter is 9.2mm to 12mm. The color filter wheel is evenly distributed with N types of excitation filters and N types of emission filters, which are arranged alternately in sequence, where N is an integer and N≥1; it is provided with M reaction holes, where M is an integer and M≥1; each reaction hole is matched with an excitation light coupling optical system and an emission light coupling optical system, the spacing angle between each type of excitation filter is consistent, the distance from each type of excitation filter to the center of the color filter wheel is consistent, the spacing angle between each type of emission filter is consistent, and the distance from each type of emission filter to the center of the color filter wheel is consistent; The fluorescence detection optical scanning method includes: The excitation light is coupled into the optical system through the excitation light and outputs excitation light of different wavelengths into the reaction aperture. Specifically, by rotating the color filter wheel in conjunction with the pulse excitation light output from the excitation source, the color filter wheel rotates to the position of the corresponding excitation filter, which is consistent with the aperture position of the excitation light collimated by the excitation light collimating lens group. Furthermore, the emitted light is output through the emitted light coupling optical system to emit emitted light of different wavelengths into the detector. Specifically, by rotating the color filter wheel, in conjunction with the pulse emitted light output generated by the fluorescent material in the reaction aperture under the influence of pulsed excitation light, the color filter wheel rotates to the position of the corresponding emission filter, which is consistent with the aperture position of the emitted light collimated by the emission light collimating lens group. Furthermore, it also includes scanning output of N band excitation light and N band emission light, and scanning detection of M reaction holes, implemented as follows: The excitation and emission filters of the color filter wheel move to the aperture positions of the collimated excitation and emission light, and the excitation light corresponding to the reaction aperture is coupled into the optical system for sequential emission. The excitation light at the corresponding positions of the N excitation filters and N emission filters is coupled into the optical system to output excitation light in sequence. The excitation light is coupled into the optical system to cause the fluorescent material in the reaction aperture to emit light. The intensity of the emitted light is recorded by the detector through the emission light coupled out of the optical system.

2. The fluorescence detection optical scanning method according to claim 1, characterized in that, In the fluorescence detection optical system used, the excitation light source wavelength is ∈ [450nm-750nm].

3. The fluorescence detection optical scanning method according to claim 2, characterized in that, In the fluorescence detection optical system used, the detection band of the detector is [500nm-800nm].

4. The fluorescence detection optical scanning method according to claim 3, characterized in that, In the fluorescence detection optical system used, the coupled fiber and the coupled fiber meet the following conditions: 0.40≤numerical aperture≤0.70, 0.2mm≤fiber core or bundled fiber core diameter≤3mm, and the wavelength range of the transmitted light is 400nm-800nm.

5. The fluorescence detection optical scanning method according to claim 1, characterized in that, In the fluorescence detection optical system used, the excitation light coupling optical system and the emission light coupling optical system are arranged on the fixed disk with the color filter wheel as the reference. If N≤M, the angle between the excitation collimating lens group and the emission excitation lens on their fixed disk is 360 / N°, and the distance to the center of their fixed disk is the same as the distance from the excitation filter to the center of the color filter wheel; the angle between the excitation input lens and the emission collimating lens group on their fixed disk is 360 / N°, and the distance to the center of their fixed disk is the same as the distance from the emission filter to the center of the color filter wheel.

6. The fluorescence detection optical scanning method according to claim 1, characterized in that, The fluorescence detection optical system used, If N > M, u is the quotient of M / N, u is rounded down to the nearest integer, the angle between the excitation collimating lens group and the emission coupling lens on its fixed disk is 360 / (N×(u+1))°, and the distance to the center of its fixed disk is the same as the distance from the excitation filter to the center of the color filter wheel; the angle between the excitation input lens and the emission collimating lens group on its fixed disk is 360 / (N×(u+1))°, and the distance to the center of its fixed disk is the same as the distance from the emission filter to the center of the color filter wheel.

Images