Fluorescence detection optical system and scanning method and calibration method thereof

Abstract

The invention belongs to the technical field of fluorescence signal detection, and particularly relates to a fluorescence detection optical system and a scanning method and a calibration method thereof. The system comprises an excitation light coupling-in optical system and an emission light coupling-out optical system. The exciting light coupling optical system comprises an exciting light source, an exciting light coupling lens group, a coupling optical fiber and a reaction hole; the exciting light coupling lens group comprises an exciting light collimating lens group, a color filter wheel and an exciting light coupling lens; the emitted light out-coupling optical system comprises an emitted light coupling lens group, an out-coupling optical fiber, a detector and a reaction hole; the emitted light coupling lens group comprises an emitted light collimating lens group, a color filter wheel and an emitted light coupling-out lens. According to the fluorescence detection optical system and the scanning method and the calibration method thereof, the optical detection efficiency can be improved, the stability of optical signal output is improved, and the system size is reduced; the scanning output of multi-band exciting light and the scanning detection of multiple reaction holes are realized; the consistency between holes is improved; and the inter-platform difference between the instruments is reduced.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of fluorescence signal detection technology, and particularly relates to a fluorescence detection optical system and a scanning method and a calibration method thereof. BACKGROUND

[0002] Fluorescence is a photoluminescence phenomenon. After some substances absorb light of a specific wavelength, electrons are excited from the ground state to a high-energy state, and then energy is released in the form of light of a longer wavelength when returning to the ground state. In fluorescence quantitative polymerase chain reaction (PCR), the hydrolysis probe contains a fluorescence reporter group. When PCR is extended, the taq enzyme hydrolyzes the probe, and under the action of excitation light, emits emission light of the corresponding waveband. The DNA amplification process is monitored by the emission light intensity to achieve high-sensitivity and quantitative analysis.

[0003] To detect the fluorescence signal generated in the polymerase chain reaction (PCR) and achieve high-sensitivity and quantitative analysis, it is required that the optical system increases the light power of the coupled-in light as much as possible at the illumination end, increases the light power of the coupled-out light as much as possible at the detection end, increases the coupled-in and coupled-out optical efficiency, and enables extremely weak light to be detected. During optical scanning, the excitation light forms stable coupled-in light into the reaction hole through the coupled-in optical system, and forms stable coupled-out light from the detection hole through the coupled-out optical system. During the scanning process, the optical system needs to ensure the consistency of the coupled-in light and the coupled-out light in each reaction hole. Moreover, in order to meet the needs of current miniaturized medical devices, the optical system, as an important component of the PCR fluorescence device, needs to meet the miniaturization requirements.

[0004] There are mainly three kinds of current polymerase chain reaction (PCR) optical systems and their implementation methods: The first kind is a reaction hole top or side scanning system composed of a photodiode (PD) or a silicon photodiode (SIPM) or a photomultiplier tube (PMT), a dichroic mirror, an excitation filter, an emission filter, and a coupling lens. This kind of system has the problems of stability of the coupled-in detection hole excitation light and the coupled-out emission light from the detection hole during scanning, and it is difficult for the excitation light and the emission light efficiency of this kind of system to achieve a relatively high coupling efficiency, which affects the sensitivity of the system. The second kind is a reaction hole top scanning system composed of a complementary metal oxide (CMOS) camera or a charge-coupled device (CCD), a dichroic mirror, an excitation filter, an emission filter, an illumination optical system, and an imaging optical system. This kind of system has edge effects, and there is a high inconsistency between the excitation light and the emission light at the edge and the excitation light and the emission light at the center, which leads to the problem of overall consistency. Moreover, this kind of system is large in size and cannot meet the current miniaturized medical devices. The third kind: based on photodiode (PD) or silicon photodiode (SIPM) or photomultiplier tube (PMT) through excitation filter and excitation light coupling-in optical system, emission filter and emission light coupling-out optical system and optical fiber to form an optical fiber scanning system, the optical fiber light guide port can be on the side of the reaction hole or on the top of the reaction hole, and such a system has the problem of alignment between the coupling-in and coupling-out optical spots of the optical system and the optical fiber during scanning, which can cause stability to decrease. At present, all polymerase chain reaction (PCR) fluorescence detection systems do not consider the calibration of absolute light intensity between instruments, which also increases the inter-bench difference between instruments. SUMMARY

[0005] In order to solve at least one of the technical problems of how to increase the light detection efficiency, how to improve the stability of the light signal output, how to reduce the volume of the overall fluorescence detection optical system, and how to reduce the inter-bench difference between instruments, the present application provides the following technical solutions: In a first aspect, the present application provides a fluorescence detection optical system, comprising 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, another color filter wheel and an emission light coupling-out lens. The excitation light collimating lens group and the emission light coupling-out lens are fixed on a fixed disc, and the fixed disc is connected with the detector and the excitation light source; the excitation light coupling-in lens and the emission light collimating lens group are fixed on another fixed disc, and the fixed disc is connected with the coupling-in optical fiber and the coupling-out optical fiber; the other ends of the coupling-in optical fiber and the coupling-out optical fiber are fixed on the reaction hole.

[0006] Further, the excitation light collimating lens group comprises 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.

[0007] Further, the emission light collimating lens group comprises 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] Further, the excitation light collimating lens group satisfies the following conditions: D1≤5mm, D2≤7.5mm, wherein D1 is the aperture of the first lens, and D2 is the aperture of the second lens. 0.43≤fexcitation collimate / f1≤0.63, 0.56≤fexcitation collimate / f2≤0.76, wherein fexcitation collimate 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, wherein n1 and n2 are the refractive indices of the first lens and the second lens, respectively; 55≤v1≤70, 55≤v2≤70, wherein v1 and v2 are the Abbe numbers of the first lens and the second lens, respectively.

[0009] Further, the excitation light coupling-in lens satisfies the following conditions: D3≤7.5mm, 0.75≤fexcitation coupling / f3≤1, wherein D3 is the aperture of the excitation light coupling-in lens, f3 is the focal length of the excitation light coupling-in lens, and fexcitation coupling is the total focal length of the excitation coupling lens group; 1.47≤n3≤1.56, 55≤v3≤70, wherein n3 is the refractive index of the excitation light coupling-in lens, and v3 is the Abbe number of the excitation light coupling-in lens.

[0010] Further, the emission collimation lens group satisfies the following conditions: D4≤7.5mm, D5≤12mm, wherein D4 is the aperture of the fourth lens, and D5 is the aperture of the fifth lens; 0.68≤femission collimate / f4≤0.88, 0.29≤femission collimate / f5≤0.49, wherein femission collimate is the total focal length of the emission collimation lens group, and f4 and f5 are the focal lengths of the fourth lens and the fifth lens, respectively; 1.47≤n4≤1.56, 1.47≤n5≤1.56, wherein n4 and n5 are the refractive indices of the fourth lens and the fifth lens, respectively; 55≤v4≤70, 55≤v5≤70, wherein v4 and v5 are the Abbe numbers of the fourth lens and the fifth lens, respectively.

[0011] Further, the emission light coupling-out lens satisfies the following conditions: D6≤12mm, 0.55≤femission coupling / f6≤0.75, wherein D6 is the aperture of the emission light coupling-out lens, f6 is the focal length of the emission light coupling-out lens, and femission coupling is the total focal length of the emission coupling lens group; 1.47≤n6≤1.56, 55≤v6≤70, wherein n6 is the refractive index of the excitation light coupling-in lens, and v6 is the Abbe number of the excitation light coupling-in lens.

[0012] Further, the excitation light source wavelength band ∈ [450nm-750nm].

[0013] Further, the light source type of the excitation light source is not limited to light-emitting diode, laser diode, xenon lamp.

[0014] Further, the detection wavelength band of the detector is [500nm-800nm].

[0015] Further, the detector type is not limited to photodiode, silicon photodiode, avalanche photodiode, photomultiplier tube.

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

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

[0018] Through excitation light collimation and emission light collimation through filters, the wavelength shift of light in the filters can be reduced, and the light emission crosstalk of different wavelength bands of fluorescent substances can be reduced.

[0019] Further, the color filter wheel is uniformly arranged with N kinds of excitation filters and N kinds of emission filters, the excitation filters and the emission filters are alternately arranged, N is an integer and N≥1; M reaction holes are provided, M is an integer and M≥1; each reaction hole is matched with an excitation light coupling-in optical system and an emission light coupling-out optical system, the interval angles of each excitation filter are consistent, the distances from each excitation filter to the center of the color filter wheel are consistent, the interval angles of each emission filter are consistent, and the distances from each emission filter to the center of the color filter wheel are consistent.

[0020] The N kinds of excitation filters and the N kinds of emission filters respectively represent N kinds of excitation filters and emission filters with different optical specifications.

[0021] The excitation light coupling-in optical system corresponding to the corresponding reaction hole is sequentially excited light output at the position corresponding to the different excitation filters, and the excitation stray light interference between different wavelength bands is reduced.

[0022] Further, the arrangement of the excitation light coupling-in optical system and the emission light coupling-out optical system on the fixed disc is based on the color filter wheel, If N≤M, the interval angle between the excitation light collimating lens group and the emission light coupling-out lens on the fixed disc is 360 / N°, the distance to the center of the fixed disc is consistent with the distance from the excitation filter to the center of the color filter wheel; the interval angle between the excitation light coupling-in lens and the emission light collimating lens group on the fixed disc is 360 / N°, the distance to the center of the fixed disc is consistent with the distance from the emission filter to the center of the color filter wheel.

[0023] Further, if N>M, u is the quotient of M / N, u is rounded down, the interval angle between the excitation light collimating lens group and the emission light coupling-out lens on the fixed disc is 360 / (N×(u+1))°, the distance to the center of the fixed disc is consistent with the distance from the excitation filter to the center of the color filter wheel; the interval angle between the excitation light coupling-in lens and the emission light collimating lens group on the fixed disc is 360 / (N×(u+1))°, the distance to the center of the fixed disc is consistent with the distance from the emission filter to the center of the color filter wheel.

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

[0025] The overall aperture of the excitation light coupling-in optical system is less than 7.5mm, and the overall working length is less than 22mm, which belongs to a miniature optical system, thereby reducing the volume of the overall fluorescence detection optical system.

[0026] The fluorescent substance in the reaction hole of the emission light coupling-out optical system is excited by the excitation light to emit emission light different from the wave band of the excitation light, and the emission light is conducted into the emission light coupling-in lens group of the emission light coupling-out optical system through the coupling-out optical fiber, wherein the emission light is collimated through the emission light collimating lens group of the emission light coupling-in lens group to generate collimated light with an aperture less than 8.5 mm and a divergence less than ±3°, the collimated emission light is incident into the color filter wheel, the light spot of the collimated emission light at the filter position of the color filter wheel is less than the size of the filter, and the filter has a large movement range to receive the collimated emission light during the movement of the color filter wheel, thereby improving the stability during scanning. After the collimated emission light passes through the color filter wheel, it is incident into the emission light coupling-out lens of the emission light coupling-in lens group, the emission light coupling-in lens focuses the collimated emission light into a detection coupling light spot at the detector position, and the real-time optical power is output through the detector. The overall aperture of the emission light coupling-out optical system is less than 12 mm, and the overall working length is less than 30 mm, which belongs to a miniature optical system, thereby reducing the volume of the overall fluorescence detection optical system.

[0027] In a second aspect, the present application provides a fluorescence detection optical scanning method using the fluorescence detection optical system described above, and the method comprises: The excitation light is output into the reaction hole through the excitation light coupling-in optical system in different wave bands, specifically: through the rotation of the color filter wheel and the pulsed excitation light output of the excitation light source, the color filter wheel is rotated to the position of the corresponding excitation filter, which is consistent with the aperture position of the collimated excitation light of the excitation light collimating lens group; And the emission light is output into the detector through the emission light coupling-out optical system in different wave bands, specifically: through the rotation of the color filter wheel and the pulsed emission light output generated by the fluorescent substance in the reaction hole affected by the pulsed excitation light, the color filter wheel is rotated to the position of the corresponding emission filter, which is consistent with the aperture position of the collimated emission light of the emission light collimating lens group.

[0028] Through the rotation of the color filter wheel, the positions of the corresponding excitation filter and emission filter are moved to the aperture positions of the collimated excitation light and emission light, the rotation is at different positions at different times, and the pulsed light is generated at the corresponding time by cooperating with the excitation light source, so as to realize the scanning output of excitation light and emission light in different wave bands and detect fluorescence in different wave bands.

[0029] Through the fluorescence detection optical scanning method provided by the present application, during the optical scanning process, the size of the light spot at the filter position of the excitation light coupling-in optical system and the emission light coupling-out optical system is controlled, so that the color filter wheel has a large movement scanning range to ensure the stability of the excitation light coupling-in efficiency and the emission light coupling-out efficiency, and the stability of the optical signal output is improved.

[0030] Further, the scanning output of the N waveband excitation light and the N waveband emission light and the scanning detection of the M reaction wells are also included, and the implementation method is as follows: When the positions of the corresponding excitation filter and emission filter of the color filter wheel move to the aperture positions of the collimated excitation light and emission light, the corresponding excitation light of the reaction well is sequentially emitted in time sequence through the excitation light coupling-in optical system, the excitation light at the corresponding positions of the N excitation filters and the N emission filters is sequentially output through the excitation light coupling-in optical system, the excitation light passes through the excitation light coupling-in optical system to make the fluorescent substance in the reaction well generate emission light, and the emission light intensity is recorded by the detector through the emission light coupling-out optical system.

[0031] The N waveband excitation light and the N waveband emission light correspond to the N excitation filters and the N emission filters.

[0032] Through the time sequence generation of the excitation light of different wavebands, the specific arrangement of different filters on the color filter wheel, the specific arrangement of the emission light coupling-out lens, the emission light collimating lens group and the excitation light collimating lens group on the fixed disc, the pulse output of the excitation light and the rotation scanning of the color filter wheel, the crosstalk of the excitation stray light between different wavebands is reduced, and the scanning output of the multi-waveband excitation light and the scanning detection of the multi-reaction well are realized.

[0033] In a third aspect, the present application provides a fluorescence detection optical calibration method, characterized in that, The first reaction well of the mother machine is used as a hole position for measuring the concentration of the luminescent substance, is a mother reaction well, and the slope formula of the emission light intensity of the mother reaction well with respect to the concentration of the luminescent substance is calibrated; Different luminescent substances with high and low concentrations are used to calibrate the slope formula of the emission light intensity of different hole positions of different instruments with respect to the concentration of the luminescent substance; The slope formula of the emission light intensity of the preset reaction well with respect to the concentration of the substance is calibrated to the slope formula of the mother reaction well, so that the two slope formulas are consistent; Under the condition that the concentration of the fluorescent substance is the same, the emission light intensity of the mother reaction well is consistent with the emission light intensity of the preset reaction well after calibration, and the emission light intensity of the preset reaction well after calibration is obtained.

[0034] Compared with the prior art, the present application has the following beneficial effects: (1) The fluorescence detection optical system provided by the present application can couple out the light rays of the full-aperture full-numerical-aperture optical fiber into the detector through the coupling-out end of the reaction well of the emission light coupling-out optical system, thereby greatly increasing the light detection efficiency.

[0035] (2) The fluorescence detection optical system and the scanning method thereof have the advantages that, in the optical scanning process, the coupling light collimation and the spot size of the excitation light coupling-in optical system and the emission light coupling-out optical system in the color filter wheel are controlled, the color filter wheel has a larger scanning position range, the coupling-in efficiency of the excitation light and the coupling-out efficiency of the emission light are stable, and the stability of the optical signal output is improved.

[0036] (3) The fluorescence detection optical system has the advantages that the excitation light coupling-in optical system and the emission light coupling-out optical system are both micro optical systems, the coupling-in of the excitation light and the coupling-out of the emission light are realized through a smaller volume, the volume of the whole fluorescence detection optical system is reduced, and the demand for small fluorescence detection equipment and medical equipment is met.

[0037] (4) The fluorescence detection optical scanning method has the advantages that, through the rotation of the color filter wheel, the positions of the corresponding excitation filter and the emission filter are moved to the aperture positions of the collimated excitation light and the emission light, the color filter wheel is rotated at different positions at different times, the excitation light source generates pulsed excitation light at the corresponding time, the scanning output of different waveband excitation light and emission light is realized, and different waveband fluorescence is detected.

[0038] (5) The fluorescence detection optical scanning method has the advantages that, through the time sequence generation of different waveband excitation light, the specific arrangement of different filters on the color filter wheel, the specific arrangement of the emission light coupling-out lens and the emission light collimating lens group on the fixed disc, the excitation light collimating lens group and the emission light coupling-out lens, the pulsed output of the excitation light, and the rotation scanning of the color filter wheel, the crosstalk of excitation stray light between different wavebands is reduced, the scanning output of multi-waveband excitation light is realized, and multi-reaction hole scanning detection is realized.

[0039] (6) The fluorescence detection system and the optical calibration method have the advantages that, different reaction holes are coupled with the optical system through optical fibers, the method for calibrating the absolute light intensity between holes greatly reduces the difference between different reaction holes for the same optical system, and the consistency between holes is improved.

[0040] (7) The fluorescence detection optical system, the scanning method thereof, and the calibration method have the advantages that, through the dye-based optical calibration method, the inconsistency caused by the inconsistency of light sources, the inconsistency of detectors, and the inconsistency of optical elements and assembly of different instruments is eliminated, the inter-instrument difference is reduced, and the inter-instrument consistency is greatly improved.

[0041] (8) The fluorescence detection optical system has the advantages that, through the excitation light collimation and the emission light collimation through the filter, the wavelength shift of light in the filter is reduced, and the light emission crosstalk of different waveband fluorescent substances is reduced.

[0042] (9) The fluorescence detection optical system provided by the present application carries out time sequence light emission through the excitation light coupling-in optical system corresponding to the corresponding reaction hole, and the excitation light coupling-in optical system at the position corresponding to different excitation filters sequentially outputs excitation light, so that the excitation stray light interference between different wave bands is reduced. BRIEF DESCRIPTION OF DRAWINGS

[0043] Figure 1 It is a fluorescence detection optical system diagram of the embodiment 1 of the present application; Figure 2 It is an excitation light coupling-in optical system and light ray tracing diagram of the embodiment 1 of the present application; Figure 3 It is a light field energy distribution diagram of the filter close to the excitation light source of the filter wheel of the embodiment 1 of the present application; Figure 4 It is a light field energy distribution diagram of the excitation light coupling-in surface of the coupling-in optical fiber of the embodiment 1 of the present application; Figure 5 It is an emission light coupling-out optical system and light ray tracing diagram of the embodiment 1 of the present application; Figure 6 It is a light field energy distribution diagram of the filter close to the coupling-out optical fiber and the light ray emission surface of the filter wheel of the embodiment 1 of the present application; Figure 7 It is a light field energy distribution diagram of the detection surface of the detector of the embodiment 1 of the present application; Figure 8 It is a composition diagram of the filter wheel of the embodiment 1 of the present application; Figure 9 It is a distribution diagram of the first fixed disc surface of the embodiment 1 of the present application; Figure 10 It is a distribution diagram of the second fixed disc surface of the embodiment 1 of the present application Figure 11 It is a fluorescence detection optical system diagram of the embodiment 2 of the present application; Figure 12 It is an excitation light coupling-in optical system and light ray tracing diagram of the embodiment 2 of the present application; Figure 13 It is a light field energy distribution diagram of the filter close to the excitation light source of the filter wheel of the embodiment 2 of the present application; Figure 14 It is a light field energy distribution diagram of the excitation light coupling-in surface of the coupling-in optical fiber of the embodiment 2 of the present application; Figure 15 It is an emission light coupling-out optical system and light ray tracing diagram of the embodiment 2 of the present application; Figure 16 It is a light field energy distribution diagram of the filter close to the coupling-out optical fiber and the light ray emission surface of the filter wheel of the embodiment 2 of the present application; Figure 17 Light field energy distribution map of the detection surface of the detector of the embodiment 2 of the present application; Figure 18 Fluorescence detection optical system diagram of the embodiment 3 of the present application; Figure 19 Excitation light coupling-in optical system and ray tracing diagram of the embodiment 3 of the present application; Figure 20 Light field energy distribution map of the filter surface of the color filter wheel of the embodiment 3 of the present application, which is close to the excitation light source; Figure 21 Light field energy distribution map of the excitation light coupling-in surface of the coupling-in optical fiber of the embodiment 3 of the present application; Figure 22 Emission light coupling-out optical system and ray tracing diagram of the embodiment 3 of the present application; Figure 23 Light field energy distribution map of the filter surface of the color filter wheel of the embodiment 3 of the present application, which is close to the coupling-out optical fiber and the light ray exits; Figure 24 Light field energy distribution map of the detection surface of the detector of the embodiment 3 of the present application; Figure 25 is Fluorescence detection optical system diagram of the embodiment 4 of the present application; Figure 26 Excitation light coupling-in optical system and ray tracing diagram of the embodiment 4 of the present application; Figure 27 Light field energy distribution map of the filter surface of the color filter wheel of the embodiment 4 of the present application, which is close to the excitation light source; Figure 28 Light field energy distribution map of the excitation light coupling-in surface of the coupling-in optical fiber of the embodiment 4 of the present application; Figure 29 Emission light coupling-out optical system and ray tracing diagram of the embodiment 4 of the present application; Figure 30 Light field energy distribution map of the filter surface of the color filter wheel of the embodiment 4 of the present application, which is close to the coupling-out optical fiber and the light ray exits; Figure 31 Light field energy distribution map of the detection surface of the detector of the embodiment 4 of the present application.

[0044] Reference signs: 1-Excitation light coupling-in optical system; 11-Excitation light source; 12-Excitation light coupling lens group; 121-First lens; 122-Second lens; 123-Excitation light coupling-in lens; 13-Coupling-in optical fiber; 2 - emission light coupling-out optical system; 21 - emission light coupling lens group; 211 - fourth lens; 212 - fifth lens; 213 - emission light coupling-out lens; 22 - coupling-out optical fiber; 23 - detector; 3 - reaction hole; 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 disc; 521 - first position; 522 - second position; 523 - third position; 511 - fourth position; 512 - fifth position; 513 - sixth position; 6 - second fixed disc; 621 - first position; 622 - second position; 623 - third position; 611 - fourth position; 612 - fifth position; 613 - sixth position. DETAILED DESCRIPTION

[0045] The technical solutions of the present application will be described in detail below with reference to the accompanying drawings. Obviously, the described embodiments are not all the embodiments of the present application, and all other embodiments obtained by those of ordinary skill in the art without creative labor fall within the protection scope of the present application.

[0046] Embodiment 1: Please refer to Figure 1 The fluorescence detection optical system is composed of an excitation light coupling-in optical system 1 and an emission light coupling-out optical system 2. The excitation light coupling-in optical system 1 is composed of an excitation light source 11, an excitation light coupling lens group 12, a coupling-in optical fiber 13, and a reaction hole 3. The emission light coupling-out optical system 2 is composed of an emission light coupling lens group 21, a coupling-out optical fiber 22, a detector 23, and a reaction hole 3.

[0047] The excitation light coupling lens group 12 is composed of an excitation light collimation lens group, a color filter wheel 4, and an excitation light coupling-in lens 123. The excitation light collimation lens group is composed of a first lens 121 and a second lens 122.

[0048] The emission light coupling lens group 21 includes an emission light collimation lens group, another color filter wheel 4, and an emission light coupling-out lens 213. The emission light collimation lens group is composed of a fourth lens 211 and a fifth lens 212.

[0049] The excitation light collimating lens group and the emission light coupling-out lens 213 are fixed on a fixed disc, which is connected with the detector 23 and the excitation light source 11; the excitation light coupling-in lens 123 and the emission light collimating lens group are fixed on another fixed disc, which is connected with the coupling-in optical fiber 13 and the coupling-out optical fiber 22; the coupling-in optical fiber 13 and the coupling-out optical fiber 22 are fixed on the side of the reaction hole 3 or on the reaction hole 3.

[0050] The light emitted by the excitation light source 11 is collimated by the excitation light collimating lens group composed of the first lens 121 and the second lens 122, and the collimated light passes through the filter in the color filter wheel 4 to obtain excitation light of a corresponding wave band, the excitation light of the corresponding wave band is coupled into the coupling-in optical fiber 13 through the excitation light coupling-in lens 123, and the excitation light is conducted to the reaction hole 3 through the coupling-in optical fiber 13, the fluorescent substance in the reaction hole 3 is affected by the excitation light to generate emission light of a corresponding wave band; The emission light is conducted into the emission light collimating lens group composed of the fourth lens 211 and the fifth lens 212 through the coupling-out optical fiber 22, and the emission light is collimated by the emission light collimating lens group, and the collimated light passes through the filter in the color filter wheel 4 to obtain emission light of a corresponding wave band, and the emission light of the corresponding wave band is coupled into the detector 23 through the emission light coupling-out lens 213.

[0051] The coupling-in optical fiber 13 and the coupling-out optical fiber 22 are preferably connected with the side of the reaction hole 3, and can also be connected with 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 uniformly spaced filters. The color filter wheel 4 is uniformly arranged with N excitation filters and N emission filters, and the excitation filters and the emission filters are arranged alternately.

[0053] The excitation light coupling optical system 1 and the ray tracing are described in Figure 2 S0 is the light emitting surface of the excitation light source 11, S1 and S2 are two surfaces of the first lens 121, S1 is the surface close to the excitation light source 11, S2 is the surface away from the excitation light source 11, S3 and S4 are two surfaces of the second lens 121, S3 is the surface close to the excitation light source 11, S4 is the surface away from the excitation light source 11, S5 and S6 are two surfaces of the filter in the color filter wheel 4, S5 is the surface close to the excitation light source 11, S6 is the surface away from the excitation light source 11, S7 and S8 are two surfaces of the excitation light coupling-in lens 123, S7 is the surface close to the excitation light source 11, S8 is the surface away from the excitation light source 11. S9 is the excitation light coupling-in surface of the coupling-in optical fiber 13, and the light path is 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] Referring to Figure 3 For the light field energy distribution of the excitation light source 11, the light spot is circular, the light spot diameter is less than 6.35 mm, the excitation filter diameter of the filter wheel 4 is 8.4 mm, and there is a movement alignment tolerance of 2.05 mm in the movement scanning, which can ensure the stability of the excitation light output in the scanning process.

[0056] Referring to Figure 4 For the light field energy distribution of the excitation light source 11, the light spot is circular, the light spot diameter is less than 6.35 mm, the excitation filter diameter of the filter wheel 4 is 8.4 mm, and there is a movement alignment tolerance of 2.05 mm in the movement scanning, which can ensure the stability of the excitation light output in the scanning process.

[0057] Referring to Figure 2 The optical total 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, which belongs to a miniature optical system, so as to reduce the volume of the whole system.

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

[0059] Table 2

[0060] Referring to Figure 6 For the light field energy distribution of the excitation light source 11, the light spot is circular, the light spot diameter is less than 6.35 mm, the excitation filter diameter of the filter wheel 4 is 8.4 mm, and there is a movement alignment tolerance of 2.05 mm in the movement scanning, which can ensure the stability of the excitation light output in the scanning process.

[0061] Referring toFigure 7 For the light field energy distribution of the detection surface S19 of the detector 23, the light spot is circular, and the light spot size diameter is less than 2.5mm. For the detector with a diameter less than 2.6mm, the energy can be completely coupled in, and for the out-coupled light emitted from S10, through the emission light out-coupling optical system 2, all the out-coupled light can be coupled into the detector with a detection surface diameter greater than 2.6mm, greatly increasing the emission light coupling efficiency.

[0062] Referring to Figure 5 The total optical length of the emission light coupling lens group 21 combined with the detector 23 is 28.4mm, and the optical diameter is 10mm, which belongs to a miniature optical system to reduce the volume of the overall system.

[0063] Referring to Figure 8 The color filter wheel 4 described in the embodiment is composed of five excitation filters and five emission filters alternately arranged along the circumferential direction.

[0064] The five excitation filters along the circumferential direction are a first excitation filter 421, a second excitation filter 422, a third excitation filter 423, a fourth excitation filter 424, and a fifth excitation filter 425, The interval angle between adjacent excitation filters is 72°. The corresponding excitation light filter wavelength bands are 455nm-480nm, 510nm-540nm, 575nm-595nm, 620nm-640nm, and 672nm-688nm, respectively.

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

[0066] The two fixed discs are a first fixed disc 5 and a second fixed disc 6.

[0067] Referring to Figure 9The excitation light coupling-in lens 123 and the fourth lens 211 and the fifth lens 212 of the emission light collimation lens group are fixed on the first fixed disc 5, the first fixed disc 5 has a first position 521, a second position 522 and a third position 523, and one excitation light coupling-in lens 123 is arranged at each position of the first position 521, the second position 522 and the third position 523. The first fixed disc 5 has a fourth position 511, a fifth position 512 and a sixth position 513, and one emission light collimation lens group is arranged at each position of the fourth position 511, the fifth position 512 and the sixth position 513, and each position contains the fourth lens 211 and the fifth lens 212. The included angle between the first position 521 and the second position 522 is 72°, and the included angle between the first position 521 and the third position 523 is 144°. The included angle between the fourth position 511 and the fifth position 512 is 72°, the included angle between the fourth position 511 and the sixth position 513 is 144°, and the included angle between the fourth position 511 and the fifth position 512 is the same as the included angle between the first emission filter 411 and the second emission filter 412.

[0068] Referring to Figure 10 The emission light coupling-out lens 213 and the first lens 121 and the second lens 122 of the excitation light collimation lens group are fixed on the second fixed disc 6, the second fixed disc 6 has a first position 621, a second position 622 and a third position 623, and one excitation light collimation lens group is arranged at each position of the first position 621, the second position 622 and the third position 623, and each position contains the first lens 121 and the second lens 122. The second fixed disc 6 has a fourth position 611, a fifth position 612 and a sixth position 613, and one emission light coupling-out lens 213 is arranged at each position, wherein the included angle between the first position 621 and the second position 622 is 72°, the included angle between the first position 621 and the third position 623 is 144°, the included angle between the fourth position 611 and the fifth position 612 is 72°, the included angle between the fourth position 611 and the sixth position 613 is 144°, and the included angle between the fourth position 611 and the fifth position 612 is the same as the included angle between the first emission filter 411 and the second emission filter 412.

[0069] In the scanning process, the color filter wheel 4 is rotated to the position of the corresponding excitation filter and emission filter, which is consistent with the aperture position of the collimated excitation light in the excitation light coupling-in optical system 1 and also consistent with the aperture position of the collimated emission light in the emission light coupling-out optical system 2; through the rotation of the color filter wheel 4, 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, and the different positions are rotated at different times, which is matched with the pulsed light generated by the excitation light source 11 at the corresponding time, the corresponding excitation light optical system of the reaction well 3 is time-sequenced to emit light, the excitation light coupling-in optical system 1 at the corresponding position of the five excitation and emission filters is sequentially outputted to reduce the crosstalk of excitation stray light between different wavebands, and the scanning detection of the five wavebands and three reaction wells 3 is realized.

[0070] Embodiment 2: The fluorescence detection optical system shown in the second embodiment of the application is basically the same as the fluorescence detection optical system shown in the first embodiment, and details are not repeated here. The differences are as follows: Please refer to Figure 11 In the second embodiment of the application, the two surfaces of the first lens 121 of the excitation light collimating lens group are spherical surfaces, the two surfaces of the second lens 122 are spherical surfaces, and the two surfaces of the excitation light coupling-in lens 123 are spherical surfaces. The two surfaces of the first lens 211 of the emission light collimating lens group are spherical surfaces, the two surfaces of the second lens 212 are spherical surfaces, and the two surfaces of the emission light coupling-out lens 213 are spherical surfaces.

[0071] In this embodiment, the excitation light coupling-in optical system 1 and the ray tracing are described in Table 3. Figure 12 .

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

[0073] Table 3

[0074] Please refer to Figure 13 For the light field energy distribution of the filter on the side S5 of the color filter wheel 4 close to the excitation light source 11, the light spot is circular, and the light spot diameter is less than 6.8 mm. The diameter of the excitation filter of the color filter wheel 4 is 8.4 mm, and there is a movement alignment tolerance of 1.6 mm in the movement scanning, which can ensure the stability of the excitation light output in the scanning process.

[0075] Please refer to Figure 14In order to distribute the light field energy of the excitation light coupled into the excitation light coupling surface S9 of the coupling-in optical fiber 13, the light spot is square, the side length of the light spot size is less than 2.4 mm, and the optical fiber with a diameter of 1 mm to 3 mm can be coupled in. Compared with the first embodiment, more energy can be coupled into the coupling-in optical fiber 13.

[0076] Referring to Figure 12 The total optical length of the combination 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, which belongs to a micro optical system, and the volume is smaller compared with the first embodiment.

[0077] In the embodiment, the light ray tracing of the emission light coupling-out optical system 2 is described with reference to Figure 15 .

[0078] In the embodiment, the data of the fourth lens 211, the fifth lens 212 and the emission light coupling lens 213 in the coupling-out optical system 2, including the curvature radius, thickness, refractive index and dispersion coefficient of each lens, are shown in Table Four.

[0079] Table Four

[0080] Referring to Figure 16 In order to distribute the light field energy of the light ray emitted from the side S15 of the filter of the color filter wheel 4 close to the coupling-out optical fiber 22, the light spot is circular, the light spot diameter is less than 7.14 mm, the diameter of the excitation filter of the color filter wheel 4 is 9.4 mm, and there is a movement alignment tolerance of 2.26 mm in the movement scanning, which can ensure the stability of the excitation light output in the scanning process.

[0081] Referring to Figure 17 In order to distribute the light field energy of the light ray emitted from the side S15 of the filter of the color filter wheel 4 close to the coupling-out optical fiber 22, the light spot is circular, the light spot diameter is less than 7.14 mm, the diameter of the excitation filter of the color filter wheel 4 is 9.4 mm, and there is a movement alignment tolerance of 2.26 mm in the movement scanning, which can ensure the stability of the excitation light output in the scanning process.

[0082] Referring to Figure 15 The total optical length of the combination of the emission light coupling lens group 21 and the detector 23 is 23.3 mm, and the optical diameter is 10 mm, which belongs to a micro optical system, and the volume is smaller compared with the first embodiment.

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

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

[0085] In the embodiment, the excitation light coupling-in optical system 1 and the ray tracing are described with reference to Figure 19 .

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

[0087] Table 5

[0088] Referring to Figure 20 For the light field energy distribution on the filter side S5 of the color filter wheel 4 close to the excitation light source 11, the light spot is circular, the light spot diameter is less than 6.8 mm, the excitation filter diameter of the color filter wheel 4 is 8.4 mm, and there is a 1.6 mm motion alignment tolerance in the motion scanning, which can ensure the stability of the excitation light output in the scanning process.

[0089] Referring to Figure 21 For the light field energy distribution on the excitation light coupling-in surface S9 of the coupling-in optical fiber 13, the light spot is square, the light spot size side length is less than 2.08 mm, and a 1 mm to 2.2 mm optical fiber can be coupled in. Compared with Embodiment 1 and Embodiment 2, more excitation light energy can be coupled in, and the coupling-in light spot is more uniform.

[0090] Referring to Figure 19 The total optical length of the combination 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, which belongs to a micro optical system, and the volume is smaller than that of Embodiment 1 and Embodiment 2.

[0091] In the embodiment, the emission light coupling-out optical system 2 and the ray tracing are described with reference to Figure 22 .

[0092] In this embodiment, the fourth lens 211, the fifth lens 212, and the emission light coupling lens 213 in the emission light coupling-out optical system 2 include the data of the curvature radius, thickness, refractive index, dispersion coefficient, and the like of each lens, which are shown in Table 6.

[0093] Table 6

[0094] Referring to Figure 23 For the light field energy distribution of the filter wheel 4 at the side S15 close to the light emission side of the coupling-out optical fiber 22, the light spot is circular, and the light spot diameter is less than 5.94 mm. The diameter of the excitation filter of the filter wheel 4 is 9.4 mm. In the motion scanning, there is a motion alignment tolerance of 3.46 mm, which can ensure the stability of the excitation light output in the scanning process. Compared with the first and second embodiments, the motion alignment tolerance of this embodiment is larger, and the excitation light output is more stable.

[0095] Referring to Figure 24 For the light field energy distribution of the detector 23 at the detection surface S19, the light spot is circular, and the light spot size diameter is less than 1.48 mm. For the detector with a detection surface diameter of less than 1.6 mm, the energy can be completely coupled in, and for the coupling-out light emitted from S10, through the emission light coupling-out optical system 2, it can be completely coupled into the detector with a detection surface diameter greater than 1.6 mm. Compared with the first and second embodiments, the coupling light spot is smaller, and a detector with a smaller detection area can be selected.

[0096] Referring to Figure 22 The total optical length of the emission light coupling lens group 21 and the detector 23 combination is 19.3 mm, and the optical diameter is 10 mm, which belongs to a miniature optical system. Compared with the first and second embodiments, the volume is smaller.

[0097] Embodiment 4 The fluorescence detection optical system shown in the fourth embodiment of the present application is basically the same as the fluorescence detection optical system shown in the first embodiment, and will not be described here. The differences are as follows: Referring to Figure 25, In the fourth embodiment of the present application, the two surfaces of the first lens 121 of the excitation light collimation lens group are a quadric surface close to the excitation light source 11 and an even aspheric surface away from the excitation light source 11. The two surfaces of the second lens 122 are both even aspheric surfaces. The two surfaces of the excitation light coupling-in lens 123 are an even aspheric surface close to the excitation light source 11 and a quadric surface away from the excitation light source 11.

[0098] The two surfaces of the first lens 211 of the emission light collimation lens group are spherical surfaces. The two surfaces of the second lens 212 are both even aspheric surfaces. The two surfaces of the emission light coupling-out lens 213 are both even aspheric surfaces.

[0099] The even-order aspheric surface satisfies the following expression:

[0100] Wherein, z is the surface height, 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 a1-a8 are the first to eighth non-spherical coefficients, respectively.

[0101] In this embodiment, the excitation light coupling optical system 1 and the light ray tracing refer to Figure 26 .

[0102] In this embodiment, the first lens 121, the second lens 122, and the excitation light coupling lens 123 in the excitation light coupling optical system 1 include the radius of curvature, thickness, refractive index, dispersion coefficient, and other data of each lens, which are shown in Table Seven Table Seven

[0103] Referring to Figure 27 The light field energy distribution on the light field energy distribution of the filter of the color filter wheel 4 on the side S5 close to the excitation light source 11 is circular, the light spot diameter is less than 6.14mm, the excitation filter diameter of the color filter wheel 4 is 8.4mm, and there is a movement alignment tolerance of 2.26mm in the movement scanning, which can ensure the stability of the excitation light output in the scanning process.

[0104] Referring to Figure 28 The light field energy distribution on the excitation light coupling surface S9 of the coupling optical fiber 13 is square, the light spot size is less than 2.06mm, and the coupling optical fiber can be coupled to 1mm to 2.2mm, compared with Embodiments 1 and 2, the coupling optical fiber energy is more, and the coupling spot is more uniform.

[0105] Referring to Figure 26 The optical total length of the combination of the excitation light source 11 and the excitation light coupling lens group 12 is 13mm, and the optical diameter is 6.3mm, which belongs to a miniature optical system, and compared with Embodiments 1, 2 and 3, the volume is smaller.

[0106] In this embodiment, the emission light coupling optical system 2 and the light ray tracing refer to Figure 29 .

[0107] In this embodiment, the fourth lens 211, the fifth lens 212, and the emission light coupling lens 213 in the emission light coupling optical system 2 include the radius of curvature, thickness, refractive index, dispersion coefficient, and other data of each lens, which are shown in Table Eight.

[0108] Table Eight

[0109] Referring to Figure 30For the light field energy distribution of the filter wheel 4 on the side S15 of the filter close to the light emission of the coupling-out optical fiber 22, the light spot is circular, the light spot diameter is less than 5.1mm, the excitation filter diameter of the filter wheel 4 is 9.4mm, and in the motion scanning, there is a motion alignment tolerance of 4.3mm, which can ensure the stability of the excitation light output in the scanning process. Compared with embodiments 1, 2 and 3, the motion alignment tolerance of this embodiment is larger, and the excitation light output is more stable.

[0110] Referring to Figure 31 For the light field energy distribution of the detector 23 on the detection surface S19, the light spot is circular, the light spot size diameter is less than 1.58mm, and for the detector with a detection surface diameter of less than 1.7mm, the energy can be completely coupled in, and for the coupling-out light emitted from S10, through the emission light coupling-out optical system 2, all the coupling-out light can be coupled into the detector with a detection surface diameter greater than 1.7mm. Compared with embodiments 1 and 2, the coupling light spot is smaller, and a smaller detection area detector can be selected.

[0111] Referring to Figure 29 The total optical length of the emission light coupling lens group 21 combined with the detector 23 is 13.7mm, and the optical diameter is 7mm, which belongs to a miniature optical system, and compared with embodiments 1, 2 and 3, the volume is smaller.

[0112] Embodiment 5: This embodiment provides a fluorescence detection optical scanning method, which adopts the fluorescence detection optical system of claim 12, and the method comprises: The excitation light is output by the excitation light coupling-in optical system into the reaction hole in different wavebands, specifically: through the rotation of the filter wheel, cooperating with the pulsed excitation light output of the excitation light source, the filter wheel is rotated to the position of the corresponding excitation filter, which is consistent with the aperture position of the collimated excitation light of the excitation light collimating lens group; And the emission light is output by the emission light coupling-out optical system into the detector in different wavebands, specifically: through the rotation of the filter wheel, cooperating with the pulsed emission light output generated by the pulsed excitation light of the fluorescent substance in the reaction hole, the filter wheel is rotated to the position of the corresponding emission filter, which is consistent with the aperture position of the collimated emission light of the emission light collimating lens group.

[0113] Further, it also includes the scanning output of N waveband excitation light and N waveband emission light and the scanning detection of M reaction holes, and the implementation method is: When the positions of the corresponding excitation filter and emission filter of the color filter wheel move to the aperture positions of the collimated excitation light and emission light, the corresponding excitation light of the reaction hole is coupled into the optical system 1 for time sequence light emission. The excitation light at the corresponding positions of the N excitation filters and N emission filters is sequentially output to the optical system 1. The excitation light passes through the excitation light coupling-in optical system to make the fluorescent substance in the reaction hole generate emission light. The emission light is recorded by the detector through the emission light coupling-out optical system.

[0114] Embodiment 6 The embodiment provides a fluorescent detection optical calibration method, which comprises the following steps: The first reaction hole of the mother machine is taken as a hole position for measuring the concentration of the luminescent substance, as a mother reaction hole, and the slope formula of the emission light intensity of the mother reaction hole with respect to the concentration of the luminescent substance is calibrated: ymother=kmother×xmother+bmother, wherein ymother is the emission light intensity of the mother reaction hole, xmother is the concentration of the fluorescent substance of the mother reaction hole, and kmother and bmother are the slope formula parameters of the emission light intensity of the mother reaction hole with respect to the concentration of the luminescent substance.

[0115] The slope formula of the emission light intensity of different holes of different instruments with respect to the concentration of the luminescent substance is calibrated by using different luminescent substances with high and low concentrations: ynm=knm×xnm+bnm, wherein ynm is the original emission light intensity of the mth reaction hole of the nth machine, xnm is the concentration of the fluorescent substance of the mth reaction hole of the nth machine, and knm and bnm are the slope formula parameters of the emission light intensity of the mth reaction hole of the nth machine with respect to the concentration of the luminescent substance.

[0116] The slope formula of the emission light intensity of the mth reaction hole of the nth instrument with respect to the concentration of the substance is calibrated to the slope formula of the mother reaction hole, so that the two slope formulas are consistent. Specifically, the luminescent substances with high and low concentrations are taken, and the initial concentrations of the luminescent substances with high and low concentrations are recorded. Then, the luminescent substances with high and low concentrations are respectively placed in the preset reaction hole and the mother reaction hole, and the emission light intensities of the luminescent substances with high and low concentrations in the preset reaction hole and the mother reaction hole are recorded. According to the initial concentrations and the emission light intensities of the luminescent substances with high and low concentrations, the intercept and the slope of the two slope formulas are calculated through conversion. Take high and low concentration of luminescent substance, the concentration is xH and Xl respectively, respectively placed in the n number of sample machine m number of holes and the mother reaction hole, record the respective emission intensity y mother H, ynmH, y mother L, ynmL; wherein, y mother H is the emission intensity of high concentration of luminescent substance in the mother reaction hole, ynmH is the emission intensity of high concentration of luminescent substance in the n number of sample machine m number of reaction hole, y mother L is the emission intensity of low concentration of luminescent substance in the mother reaction hole, ynmL is the emission intensity of low concentration of luminescent substance in the n number of sample machine m number of reaction hole; knm and bnm can be converted by ynmH, ynmL, xH and Xl, k mother and b mother can be converted by y mother H, y mother L, xH and Xl.

[0117] In the case of the same concentration of fluorescent substance, the emission intensity of the mother reaction hole is consistent with the preset emission intensity of the reaction hole after calibration, and the preset emission intensity of the reaction hole after calibration is obtained. In the case of the same concentration of fluorescent substance, the emission intensity of the mother reaction hole is consistent with the preset emission intensity of the n number of sample machine m number of reaction hole after calibration, xnm= (ynm-bnm) / knm is substituted into ynm calibration=k mother xnm+b mother, ynm is the emission intensity of the m hole of the n number of instrument before calibration, ynm calibration is the emission intensity of the m hole of the n number of instrument after calibration, and the following can be obtained: ynm calibration= (ynm-ynmH) x (y mother H-y mother L) / (ynmH-ynmL) +y mother H, the above formula shows the relationship between the emission intensity of the m hole of the n number of instrument after calibration and the initial emission intensity.

[0118] The above technical features constitute the best embodiment of the present application, which has strong adaptability and best implementation effect, and unnecessary technical features can be added or reduced 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 explain the technical solutions of the present application, and is not a limitation on the protection scope of the present application. Simple modifications or equivalent replacements of the technical solutions of the present application made by those skilled in the art do not deviate from the essence and scope of the technical solutions of the present application.

Claims

1. A fluorescence detection optical system, characterized in that: It includes an excitation light coupling optical system and an emission light coupling optical system; The excitation light coupling optical system includes an excitation light source, an excitation light coupling lens group, a coupling optical fiber and a reaction hole; the excitation light coupling lens group includes an excitation light collimating lens group, a color filter wheel and an excitation light coupling lens; The emission light outcoupling optical system includes an emission light coupling lens group, an outcoupling optical fiber, a detector and a reaction hole; the emission light coupling lens group includes an emission light collimating lens group, another color filter wheel and an emission light outcoupling 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-in optical fiber and the coupling-out optical fiber; the other ends of the coupling-in optical fiber and the coupling-out optical fiber are fixed on the reaction hole.

2. The fluorescence detection optical system according to claim 1, characterized in that: The excitation light collimating lens group includes a first lens with positive refractive power and both surfaces are spherical or aspherical, and a second lens with positive refractive power and both surfaces are spherical or aspherical.

3. The fluorescence detection optical system according to claim 1, wherein: The emission light collimating lens group includes a fourth lens having positive refractive power and two surfaces of which are spherical or aspherical, and a fifth lens having positive refractive power and two surfaces of which are spherical or aspherical.

4. The fluorescence detection optical system according to claim 2, wherein: The excitation light collimating lens group meets the following conditions: D1≤5mm, D2≤7.5mm, where D1 is the first lens aperture and D2 is the second lens aperture; 0.43≤fexcitation light collimation / f1≤0.63, 0.56≤fexcitation light collimation / f2≤0.76, where fexcitation light collimation is the total focal length of the excitation light 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 and second lenses respectively; 55≤v1≤70, 55≤v2≤70, where v1 and v2 are the Abbe numbers of the first lens and the second lens respectively.

5. The fluorescence detection optical system according to claim 4, characterized in that: The excitation light coupling lens meets the following conditions: D3≤7.5mm, 0.75≤fexcitation light coupling / f3≤1, where D3 is the aperture of the excitation light coupling lens, f3 is the focal length of the excitation light coupling lens, and fexcitation light coupling is the total focal length of the excitation light coupling lens group; 1.47≤n3≤1.56, 55≤v3≤70, where n3 is the refractive index of the excitation light coupling lens, and v3 is the Abbe number of the excitation light coupling lens.

6. The fluorescence detection optical system according to claim 3, characterized in that: The emission light collimating lens group meets the following conditions: D4≤7.5mm, D5≤12mm, where D4 is the fourth lens diameter and D5 is the fifth lens diameter; 0.68≤fEmitted Light Collimation / f4≤0.88, 0.29≤fEmitted Light Collimation / f5≤0.49, where fEmitted Light Collimation is the total focal length of the emitted light collimating lens group, and f4 and f5 are the focal lengths of the fourth lens and the fifth lens 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 lens and the fifth lens respectively.

7. The fluorescence detection optical system according to claim 6, characterized in that: The emission light coupling lens meets the following conditions: D6≤12mm, 0.55≤femitting light coupling / f6≤0.75, where D6 is the aperture of the emitting light coupling lens, f6 is the focal length of the emitting light coupling lens, and femitting light coupling is the total focal length of the emitting light coupling lens group; 1.47≤n6≤1.56, 55≤v6≤70, where n6 is the refractive index of the excitation light coupling lens, and v6 is the Abbe number of the excitation light coupling lens.

8. The fluorescence detection optical system according to claim 1, wherein: The excitation light source wavelength band ∈ [450nm-750nm].

9. The fluorescence detection optical system according to claim 8, characterized in that: The detection band of the detector is [500nm-800nm].

10. The fluorescence detection optical system according to claim 9, characterized in that: The coupling-in optical fiber and the coupling-out optical 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.

11. The fluorescence detection optical system according to any one of claims 1 to 10, characterized in that: The color filter wheels of the excitation light coupling lens group and the emission light coupling lens group are composed of filters arranged at even intervals, and the filters meet the following conditions: the excitation light filter band is: 455nm-480nm, 510nm-540nm, 575nm-595nm, 620nm-640nm, 672nm-688nm; the emission light filter band is: 515nm-535nm, 555nm-575nm, 610nm-630nm, 655nm-675nm, 705nm-745nm; the excitation light filter aperture range is 8.4mm to 10mm, and the emission light filter aperture range is 9.2mm to 12mm.

12. The fluorescence detection optical system according to claim 11, characterized in that: The color filter wheel is evenly distributed with N excitation filters and N emission filters, and the excitation filters and the emission filters are arranged alternately in sequence, N is an integer, and N≥1; M reaction wells are provided, M is an integer, and M≥1; each reaction well is matched with an excitation light coupling optical system and an emission light coupling optical system, the interval angle of each excitation filter is consistent, the distance between each excitation filter and the center of the color filter wheel is consistent, the interval angle of each emission filter is consistent, and the distance between each emission filter and the center of the color filter wheel is consistent.

13. The fluorescence detection optical system according to claim 12, wherein: The arrangement of the excitation light coupling optical system and the emission light coupling optical system on the fixed plate is based on the color filter wheel. If N≤M, the angle between the excitation light collimating lens group and the emission light outcoupling lens on their fixed plates is 360 / N°, and the distance to the center of their fixed plates is consistent with the distance from the excitation filter to the center of the color filter wheel; the angle between the excitation light incoupling lens and the emission light collimating lens group on their fixed plates is 360 / N°, and the distance to the center of their fixed plates is consistent with the distance from the emission filter to the center of the color filter wheel.

14. The fluorescence detection optical system according to claim 13, wherein: If N>M, u is the quotient of M / N, u is rounded to the nearest integer, the angle between the excitation light collimating lens group and the emission light coupling lens on their fixed disk is 360 / (N×(u+1))°, and the distance to the center of their fixed disk is consistent with the distance from the excitation filter to the center of the color filter wheel; the angle between the excitation light coupling lens and the emission light collimating lens group on their fixed disk is 360 / (N×(u+1))°, and the distance to the center of their fixed disk is consistent with the distance from the emission filter to the center of the color filter wheel.

15. A fluorescence detection optical scanning method, using the fluorescence detection optical system according to claim 12, characterized in that: The method comprises: The excitation light is coupled into the optical system to output excitation light of different wavelengths into the reaction wells. Specifically, the color filter wheel rotates to the position of the corresponding excitation filter in coordination with the pulsed excitation light output of the excitation light source. The position is consistent with the aperture position of the excitation light collimated by the excitation light collimating lens group. In addition, the emission light outputs emission light of different bands through the emission light coupling optical system and enters the detector. Specifically: through the rotation of the color filter wheel, the pulse emission light output generated by the fluorescent substance in the reaction hole being affected by the pulse excitation light is coordinated, and the color filter wheel rotates to the position of the corresponding emission filter, which is consistent with the aperture position of the emission light collimated by the emission light collimating lens group.

16. The fluorescence detection optical scanning method according to claim 15, characterized in that: It also includes scanning output of N bands of excitation light and N bands of emission light and scanning detection of M reaction wells, and the implementation method is: When the positions of the corresponding excitation filters and emission filters on the color filter wheel move to the aperture positions of the collimated excitation light and emission light, the excitation light coupling optical system corresponding to the corresponding reaction hole emits light in sequence, and the excitation light coupling optical system at the corresponding positions of N types of excitation filters and N types of emission filters outputs excitation light in sequence. The excitation light passes through the excitation light coupling optical system to make the fluorescent substance in the reaction hole generate emission light, and the emission light intensity is recorded by the detector through the emission light coupling optical system.

17. A fluorescence detection optical calibration method, characterized in that: The first reaction well of the master prototype is used as the well for measuring the concentration of the luminescent substance, which is the master reaction well, and the slope formula of the emission light intensity of the master reaction well with respect to the concentration of the luminescent substance is calibrated; The slash formula of the emission intensity of different instruments at different wells is calibrated by using different luminescent substances with high and low concentrations. Calibrate the preset slope formula of the reaction well emission light intensity with respect to the substance concentration to the slope formula of the parent reaction well so that the two slope formulas are consistent; When the concentration of the fluorescent substance is the same, the emission light intensity of the mother reaction well is consistent with the emission light intensity of the reaction well preset after calibration, and the emission light intensity of the reaction well preset after calibration is obtained.

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