CN214011058U

CN214011058U - Micro-droplet three-fluorescence signal detection device

Info

Publication number: CN214011058U
Application number: CN202021932245.5U
Authority: CN
Inventors: 于海侠; 夏雷; 周跃; 黄海旺; 梁欢迎; 白宇; 杨文军
Original assignee: Xinyi Manufacturing Technology Beijing Co ltd
Current assignee: Xinyi Manufacturing Technology Beijing Co ltd
Priority date: 2020-09-07
Filing date: 2020-09-07
Publication date: 2021-08-20
Anticipated expiration: 2030-09-07

Abstract

The utility model relates to a micro-droplet three-fluorescence signal detection device, which comprises a light combination module, a light source module and a light source module, wherein the light combination module is used for combining first laser with a first wavelength, second laser with a second wavelength and third laser with a third wavelength into mixed excitation light; the objective lens is used for confocal focusing the mixed excitation light on the micro-droplet to be detected so as to excite and generate first fluorescence corresponding to the first laser, second fluorescence corresponding to the second laser and third fluorescence corresponding to the third laser; the light processing module is used for reflecting the mixed exciting light to the first dichroic mirror of the objective lens through the light combination module and separating the first fluorescent light, the second fluorescent light and the third fluorescent light. The utility model discloses a three fluorescence signal detection devices of micro-droplet can acquire three fluorescence detection signals of micro-droplet through closing optical module and light processing module, and the testing result is abundanter, suitable scene and field are wider, save the experiment cost, improve detection work efficiency.

Description

Micro-droplet three-fluorescence signal detection device

Technical Field

The utility model relates to a micro-fluidic chip technical field, concretely relates to three fluorescence signal detection devices of micro-droplet.

Background

The biochip has wide application in new medicine development, disease diagnosis, gene expression analysis, etc. The technology of the micro-fluidic chip is mature day by day and becomes a focus of people. There are various biological and chemical processes in the microfluidic detection chip, and the processes are usually completed in the micro-scale flow channel space, wherein some devices capable of detecting the reaction process are also required. The existing detection means can be divided into CCD scanning and laser confocal scanning, the CCD scanning system has a simpler structure and a higher detection speed compared with the laser confocal scanning system, but has a lower transverse resolution, if the transverse resolution is required to be improved, the amplification factor of the imaging system needs to be improved, and the corresponding field of view is reduced (namely the area of a chip to be measured at one time is smaller), when the area of the chip to be measured is larger, the chip can be spliced after being measured in blocks for many times, and because the block scanning actually makes the chip and the imaging system move relatively in a mechanical movement mode, the mechanical positioning error forms the splicing error of a scanned image, so the method is not suitable for high-precision high-density biochip detection.

The biochip detection system constructed based on the laser confocal principle scans the biochip point by point, and the biochip is always positioned on a focal plane, so that the spot size of the exciting light is very small, and the transverse resolution is high. Because the laser confocal detection mode has the characteristics of high resolution and high sensitivity, clear digital fluorescence images and quantitative analysis results of antibodies and the like marked by fluorescence on the biochip can be obtained, and the method can become a new detection means mainly adopted by high-density biochip scanning.

SUMMERY OF THE UTILITY MODEL

Therefore, the to-be-solved technical problem of the utility model is to provide a three fluorescence signal detection devices of micro-droplet can acquire the three fluorescence detection signals of micro-droplet through closing optical module and light processing module, and testing result is abundanter, suitable scene and field are wider, save the experiment cost, improve detection work efficiency.

In order to solve the above problem, the utility model provides a three fluorescence signal detection devices of micro-droplet, include:

the light combining module is used for combining first laser with a first wavelength, second laser with a second wavelength and third laser with a third wavelength into mixed excitation light;

the objective lens is positioned on a light conduction path of the mixed exciting light and is used for confocal the mixed exciting light on the micro-droplet to be detected so as to excite and generate first fluorescence corresponding to the first laser, second fluorescence corresponding to the second laser and third fluorescence corresponding to the third laser;

a light processing module including a first photomultiplier tube for obtaining first fluorescence, a second photomultiplier tube for obtaining second fluorescence, a third photomultiplier tube for obtaining third fluorescence, a first dichroic mirror for reflecting the mixed excitation light from the light combining module to the objective lens, a second dichroic mirror for separating the first fluorescence from the second fluorescence, and a third dichroic mirror for separating the first fluorescence from the second fluorescence

Preferably, the light processing module further comprises a convex lens between the first dichroic mirror and the third dichroic mirror to refract the scattered light therethrough into parallel light.

Preferably, the light processing module further comprises a first color filter between the first photomultiplier tube and the second dichroic mirror. The light processing module further includes a second color filter between the second photomultiplier tube and the second dichroic mirror. The light processing module further includes a third color filter between the third photomultiplier tube and the third dichroic mirror.

Preferably, the light processing module further has a mounting housing configured with a light conducting channel having a first aperture corresponding to the first photomultiplier tube, a second aperture corresponding to the second photomultiplier tube, and a third aperture corresponding to the third photomultiplier tube. The first small hole, the second small hole and the third small hole are arranged in a conjugate mode. The mixed excitation light of the conjugated arrangement, namely the first laser, the second laser and the third laser, is reflected to the objective lens through a first dichroic mirror to be confocal on the micro-droplet to be detected, so as to form the mixed fluorescence, wherein the mixed fluorescence comprises the first fluorescence, the second fluorescence and the third fluorescence; the mixed fluorescence passes through the objective lens to form parallel light, then passes through the first dichroic mirror, and is respectively focused on the first small hole, the second small hole and the third small hole through the convex lens; the third fluorescent light is reflected to a third color filter through a third dichroic mirror, reaches a third small hole and is transmitted into a third photomultiplier; the first fluorescent light passes through the third dichroic mirror, is reflected to the first color filter by the second dichroic mirror, reaches the first small hole and is transmitted into the first photomultiplier; and the second fluorescent light passes through the third dichroic mirror, is reflected by the second dichroic mirror to the second color filter, reaches the second small hole and is guided into the second photomultiplier.

Preferably, the mounting housing includes a fixing member, a first connecting member, a second connecting member, and a third connecting member, wherein the fixing member has a first light ray injection opening, a second light ray injection opening, and a third light ray injection opening. The first light ray injection opening is perpendicular to the first light ray injection opening, the first light ray injection opening is coaxial with the second light ray injection opening, and the first light ray injection opening is perpendicular to the third light ray injection opening; the second connecting piece is connected to the first light ray incidence opening, and the convex lens is clamped between the second connecting piece and the first light ray incidence opening; the first light ray outlet is connected with a first photomultiplier fixing seat, and the first color filter and the first small hole piece are sequentially clamped between the second fixing piece and the first photomultiplier fixing seat along a light ray conducting path; the third connecting piece is connected to the second light ray emitting opening, the second dichroic mirror is clamped between the third connecting piece and the second light ray emitting opening, a second photomultiplier fixing seat is arranged at one end, away from the second fixing piece, of the third connecting piece, and a second color filter and a second small hole piece are sequentially clamped between the third connecting piece and the second photomultiplier fixing seat along a light ray conducting path; the third color filter and the third small-hole piece are sequentially clamped between the first fixing piece and the third photomultiplier fixing seat along a light conducting path; the first fixing piece is connected between the second fixing piece and the third dichroic mirror in a clamping mode. The first connecting piece is connected to one side, far away from the first fixing piece, of the second connecting piece, and the first dichroic mirror is clamped between the first connecting piece and the second connecting piece.

Preferably, a joint between the first connecting piece and the second connecting piece, a joint between the second connecting piece and the first fixing piece, a joint between the third connecting piece and the first fixing piece, a joint between the first photomultiplier tube fixing seat and the second fixing piece, a joint between the second photomultiplier tube fixing seat and the third connecting piece, and a joint between the third photomultiplier tube and the first fixing piece are provided with a sealing shock absorbing piece.

Preferably, the objective lens is movably connected to a side of the first connecting piece, which is away from the second connecting piece.

Preferably, the light combining module includes a first laser, a second laser, a third laser, a reflective mirror, a fourth dichroic mirror, and a fifth dichroic mirror. The third laser light emitted by the third laser (10) is reflected by the reflective mirror (143) and then combined with the second laser light emitted by the second laser (12) into the mixed excitation light by the fourth dichroic mirror (142), and the first laser light emitted by the first laser (11) is combined into the mixed excitation light again by the fifth dichroic mirror (141) through the third laser light and the second laser light.

Preferably, the light combining module further includes a fixing plate and a light combining cassette, the first laser, the second laser and the light combining cassette are fixedly connected to the fixing plate, the reflective mirror, the fourth dichroic mirror and the fifth dichroic mirror are disposed in the light combining cassette, and the light combining cassette has a mixed excitation light exit hole.

Preferably, the first laser is a 473nm laser; the second laser is a 532nm laser; the third laser is a 633nm laser.

The utility model provides a pair of three fluorescence signal detection devices of micro-droplet, first laser, second laser and third laser through the wavelength difference are in the synthesis formation treat behind the mixed exciting light and detect the micro-droplet and arouse and form first fluorescence, second fluorescence and third fluorescence, and pass through first photomultiplier, second photomultiplier and third photomultiplier acquire the back respectively and reach and carry out corresponding processing (for example count) to the data processing equipment (such as computer etc.) that corresponds to realized that the single detects and to have acquireed multinomial detection parameter, make the testing result abundanter, it is more extensive to be suitable for scene and field, this reduction experiment cost that can the very big degree improves and detects work efficiency.

Drawings

Fig. 1 is a schematic view of an exploded structure of a micro-droplet dual fluorescence signal detection device according to an embodiment of the present invention;

fig. 2 is a schematic diagram of a light transmission path of the micro-droplet dual fluorescence signal detection device according to an embodiment of the present invention;

FIG. 3 is an exploded view of the optical processing module of FIG. 1;

fig. 4 is an exploded structural diagram of the light combining module in fig. 1.

The reference signs are:

1. a light combining module; 11. a first laser; 12. a second laser; 10. a third laser; 13. a fixing plate; 14. a light-combining cassette; 143. a reflective mirror; 142. a fourth dichroic mirror; 141. a fifth dichroic mirror; 2. an objective lens; 3. a light processing module; 32. a first photomultiplier tube; 31. a second photomultiplier tube; 30. a third photomultiplier tube; 33. a first dichroic mirror; 34. a second dichroic mirror; 39. a third dichroic mirror; 35. a first color filter; 36. a second color filter; 37. a third color filter; 371. a first fixing member; 379. a second fixing member; 372. a first connecting member; 373. a second connecting member; 374. a third connecting member; 377. a first photomultiplier tube holder; 378. a first orifice member; 375. a second photomultiplier tube holder; 376. a second orifice member; 396. a third photomultiplier tube holder; 380. a third orifice member; 38. a convex lens; 391. a shock pad; 392. a gasket; 393. a light-tight pad; 100. micro-droplets are detected.

Detailed Description

Referring to fig. 1 to 4 in combination, according to an embodiment of the present invention, there is provided a micro-droplet three-fluorescence signal detection apparatus, including: the light combining module 1 is used for combining first laser light with a first wavelength, second laser light with a second wavelength and third laser light with a third wavelength into mixed excitation light; the objective lens 2 is located on a light conduction path of the mixed excitation light, and is used for confocal focusing the mixed excitation light on the microdroplet 100 to be detected so as to excite and generate first fluorescence corresponding to the first laser, second fluorescence corresponding to the second laser, and third fluorescence corresponding to the third laser; the light processing module 3 includes a first photomultiplier tube (PMT)32 for acquiring the first fluorescence, a second PMT 31 for acquiring the second fluorescence, a third PMT 30 for acquiring the third fluorescence, a first dichroic mirror 33 for reflecting the mixed excitation light from the light combining module to the objective lens 2, a second dichroic mirror 34 for separating the first fluorescence from the second fluorescence, and a third dichroic mirror 39 for separating the first fluorescence from the third fluorescence. In the technical scheme, the mixed excitation light is synthesized by the first laser, the second laser and the third laser with different wavelengths to excite the micro-droplet 100 to be detected and form the first fluorescence, the second fluorescence and the third fluorescence, and the first photomultiplier tube 32, the second photomultiplier tube 31 and the third photomultiplier tube 30 are used for acquiring and then transmitting the acquired light to corresponding data processing equipment (such as a computer and the like) to perform corresponding processing (such as counting), so that multiple detection parameters can be acquired by single detection, the detection result is richer, the application scenes and the application fields are wider, the experiment cost can be greatly reduced, and the detection work efficiency is improved.

Preferably, the light processing module 3 further includes a convex lens 38, and the convex lens 38 is located between the first dichroic mirror 33 and the third dichroic mirror 39, so that the scattered light passing through the convex lens 38 is refracted into parallel light, so that the light passing through the convex lens 38 can be emitted to the third dichroic mirror 39 more regularly, and the detection accuracy of the subsequent fluorescent signal is ensured.

Further, the light processing module 3 further includes a first color filter 36, the first color filter 36 is located between the first photomultiplier tube 32 and the second dichroic mirror 34, and the light processing module 3 further includes a second color filter 35, the second color filter 35 is located between the second photomultiplier tube 31 and the second dichroic mirror 34. The light processing module 3 further comprises a third color filter 37, the third color filter 37 being located between the third photomultiplier tube 30 and the third dichroic mirror 39.

Specifically, the light processing module 3 further has an installation housing, the installation housing is configured with a light conduction channel, the light conduction channel has a first small hole corresponding to the first photomultiplier 32, a second small hole corresponding to the second photomultiplier 31, and a third small hole corresponding to the third photomultiplier 30, and the first small hole, the second small hole and the third small hole are arranged in a conjugate manner. The mixed excitation light of the conjugated arrangement, that is, the first laser, the second laser and the third laser, is reflected to the objective lens 2 through the first dichroic mirror 33 to be confocal on the micro-droplet 100 to be detected, so as to form the mixed fluorescence, wherein the mixed fluorescence includes the first fluorescence, the second fluorescence and the third fluorescence; the mixed fluorescence passes through the objective lens 2 to form parallel light, then passes through the first dichroic mirror 33, and is respectively focused on the first small hole, the second small hole and the third small hole through the convex lens 38; the third fluorescence is reflected by a third dichroic mirror 39 to a third color filter 37 to reach a third small hole and is transmitted into a third photomultiplier 30; the first fluorescence is reflected by the second dichroic mirror 34 through the third dichroic mirror 39 to the first color filter 36 to reach the first small hole, and then is introduced into the first photomultiplier 32; the second fluorescence is reflected by third dichroic mirror 39 and second dichroic mirror 34 to second color filter 35, reaches the second small hole, and is guided into second photomultiplier 31. As can be seen from fig. 3, the three paths of fluorescence are reflected by the same starting point and the dichroic mirror, then separated from each other, and form two pairs of mutually perpendicular optical paths, and pass through the corresponding small holes, and finally reach the corresponding photomultiplier tubes.

More specifically, the mounting housing includes a first fixing member 371, a second fixing member 379, a first connecting member 372, a second connecting member 373, and a third connecting member 374, wherein the first fixing member 371 has a first light emitting opening, a second light emitting opening, and a third light emitting opening, the first light emitting opening is perpendicular to the first light emitting opening, the first light emitting opening is coaxial with the second light emitting opening, and the first light emitting opening is perpendicular to the third light emitting opening. The second connecting member 373 is connected to the first light entrance, and the convex lens 38 is sandwiched between the second connecting member 373 and the first light entrance; the third connector 374 is connected to the second light emitting port, and the second dichroic mirror 34 is sandwiched therebetween, a first photomultiplier tube holder 377 is disposed at an end of the third connector 374 away from the first fixing part 371, and the first color filter 36 and a first small hole member 378 (on which the first small hole is configured) are sequentially sandwiched between the third connector 374 and the first photomultiplier tube holder 377 along a light conducting path; the second light exit hole is connected to a second photomultiplier tube holder 375, and the second color filter 35 and a second small hole member 376 (on which the second small hole is configured) are sequentially interposed between the third connector 374 and the second photomultiplier tube holder 375 along a light conducting path; and; the third light exit is connected to a second third photomultiplier tube holder 396, and the third color filter 37 and the third small hole 380 (on which the third small hole is configured) are sequentially interposed between the first fixing 371 and the third photomultiplier tube holder 396 along a light conducting path; the first connecting member 372 is connected to a side of the second connecting member 373 away from the first fixing member 371, and the first dichroic mirror 33 is sandwiched between the first connecting member and the second connecting member. In this technical solution, a specific implementation manner of the light processing module 3 is provided, which is formed by mutually assembling and assembling each relatively independent connecting component and parts, and it can be understood that, in order to ensure the rationality of the light conducting channel, in particular, the design of the light conducting channel is in accordance with the light processing path shown in fig. 2 of the present application, that is, although the foregoing does not specifically define the corresponding light conducting channels in the first connecting component 372, the second connecting component 373, and the third connecting component 374, it is clear that, in order to ensure the light conducting requirements of fig. 2 and the compactness of the overall structure of the light processing module 3, the first connecting component 372, the second connecting component 373, the first fixing component 371, the second fixing component 379, and the third connecting component 374 are sequentially on the light conducting path and satisfy the light conducting requirements shown in fig. 2, the first connector 372, the second connector 373, and the third connector 374 are all isosceles right triangles in shape, that is, they all have a 45 ° slope, the first fastener 371 also has a 45 ° slope, and the second fastener 379 also has a 45 ° slope.

Further, in order to protect the optical lens such as the convex lens 38 provided in the light processing module 3 from damage and to ensure that it is protected from external light, it is preferable that, the joint between the first connecting member 372 and the second connecting member 373, the joint between the second connecting member 373 and the first fixing member 371, the junction between the third connector 374 and the first fastener 371, the junction between the first photomultiplier tube holder 377 and the second fastener 379, the connection between the second photomultiplier tube holder 375 and the third connector 374, a sealing shock-absorbing member is arranged at the joint between the third photomultiplier tube fixing seat 396 and the first fixing member 371, the seal cushion may comprise, for example, one or more of a cushion 391, a seal 392, and a seal 393.

The objective lens 2 is movably connected to a side of the first connecting member 372 away from the second connecting member 373 as a component for performing focus detection on the microdroplets 100 to be detected, as a specific embodiment, it is only required to use a 20-fold microscope, and it can be understood that the movable connection here means that the objective lens 2 has a rotational (around Z axis) degree of freedom and a translational (along Z axis or X axis) degree of freedom, so as to ensure that a detection person can flexibly adjust a focal point (focal plane) of the objective lens 2, and further, it can be understood that the aforementioned movement of the objective lens 2 can be controlled by a power module such as a lead screw motor.

As a specific embodiment of the light combining module 1, preferably, the light combining module 1 includes a first laser 11, a second laser 12, a third laser 10, a reflective mirror 143, a fourth dichroic mirror 142, and a fifth dichroic mirror 141, the third laser light emitted by the third laser (10) is reflected by the reflective mirror (143) and then combined with the second laser light emitted by the second laser (12) into the mixed excitation light at the fourth dichroic mirror (142), and the first laser light emitted by the first laser (11) is combined into the mixed excitation light again at the fifth dichroic mirror (141) by the third laser light and the second laser light. For example, the first laser 11 is a 473nm laser, in which case it is a blue laser; the second laser 12 is a 532nm laser, in this case a green laser; the third laser 10 is a 633nm laser, in this case a red laser. More specifically, the light combining module 1 further includes a fixing plate 13, the first laser 11, the second laser 12, the third laser 10, and a light combining cassette 14 fixedly connected to the fixing plate 13, wherein the reflective mirror 143 and the fourth dichroic mirror 142 are disposed in the light combining cassette 14, and the light combining cassette 14 has a mixed excitation light exit port. At this time, it can be understood that the light combining module 1 is constructed as a whole, so that the micro-droplet dual fluorescence signal detection apparatus is further compact in structure.

It is readily understood by a person skilled in the art that the advantageous ways described above can be freely combined, superimposed without conflict.

The above description is only exemplary of the present invention and should not be construed as limiting the present invention, and any modifications, equivalents and improvements made within the spirit and principles of the present invention are intended to be included within the scope of the present invention. The above is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, a plurality of modifications and variations can be made without departing from the technical principle of the present invention, and these modifications and variations should also be regarded as the protection scope of the present invention.

Claims

1. A microdroplet three-fluorescence signal detection device is characterized by comprising:

the light combining module (1) is used for combining first laser with a first wavelength, second laser with a second wavelength and third laser with a third wavelength into mixed excitation light;

the objective lens (2) is positioned on a light conduction path of the mixed exciting light and is used for focusing the mixed exciting light on the micro-droplet (100) to be detected so as to excite and generate first fluorescence corresponding to the first laser, second fluorescence corresponding to the second laser and third fluorescence corresponding to the third laser;

a light processing module (3) comprising a first photomultiplier (32) for obtaining first fluorescence light, a second photomultiplier (31) for obtaining second fluorescence light, a third photomultiplier (30) for obtaining third fluorescence light, a first dichroic mirror (33) for reflecting the mixed excitation light from the light combining module to the objective lens (2), a second dichroic mirror (34) for separating the first and second fluorescence light and a third dichroic mirror (39) for separating the first, second and third fluorescence light;

the light processing module (3) further has a mounting housing configured with a light conducting channel having a first aperture corresponding to the first photomultiplier tube (32), a second aperture corresponding to the second photomultiplier tube (31), a third aperture corresponding to the third photomultiplier tube (30);

the three paths of fluorescent light are reflected by the dichroic mirror from the same starting point, are separated from each other, form two pairs of mutually perpendicular light paths, pass through the corresponding small holes and finally reach the corresponding photomultiplier tubes.

2. The detection apparatus according to claim 1, wherein the light processing module (3) further comprises a convex lens (38), the convex lens (38) being between the first dichroic mirror (33) and the third dichroic mirror (39) to refract the scattered light therethrough into parallel light.

3. The detection apparatus according to claim 2, wherein the light processing module (3) comprises a first color filter (36), the first color filter (36) being between the first photomultiplier tube (32) and the second dichroic mirror (34), the light processing module (3) further comprising a second color filter (35), the second color filter (35) being between the second photomultiplier tube (31) and the second dichroic mirror (34), the light processing module (3) further comprising a third color filter (37), the third color filter (37) being between the third photomultiplier tube (30) and the third dichroic mirror (39).

4. The detecting device for detecting the rotation of a motor rotor according to claim 3, wherein said first small hole, said second small hole and said third small hole are arranged in a conjugate manner.

5. The detecting device according to claim 4, wherein the mounting housing includes a first fixing member (371), a second fixing member (379), a first connecting member (372), a second connecting member (373), and a third connecting member (374), the first fixing member (371) has a first light inlet, a first light outlet, a second light outlet, and a third light outlet, the first light inlet is perpendicular to the first light outlet, the first light inlet is coaxial with the second light outlet, and the first light inlet is perpendicular to the third light outlet; the second connecting piece (373) is connected to the first light ray incidence port, and the convex lens (38) is clamped between the second connecting piece and the first light ray incidence port; the third connecting piece (374) is connected to the second light ray emitting opening, the second dichroic mirror (34) is clamped between the second connecting piece and the second light ray emitting opening, the first light ray emitting opening is connected with a first photomultiplier fixing seat (377), and the first color filter (36) and the first small-hole piece (378) are sequentially clamped between the third connecting piece (374) and the first photomultiplier fixing seat (377) along a light ray conducting path; a second photomultiplier fixing seat (375) is arranged at one end, away from the first fixing part (371), of the third connecting part (374), and the second color filter (35) and the second small-hole part (376) are sequentially clamped between the third connecting part (374) and the second photomultiplier fixing seat (375) along a light conducting path; the first connecting piece (372) is connected to one side, away from the first fixing piece (371), of the second connecting piece (373), and the first dichroic mirror (33) is clamped between the first connecting piece and the second connecting piece; the third light ray outlet is connected with a third photomultiplier fixing seat (396), the second fixing piece (379) is connected with the first fixing piece (371), and a third dichroic mirror (39) is clamped between the second fixing piece and the first fixing piece; and the third color filter (37) and the third small-hole piece (380) are sequentially clamped between the third photomultiplier fixing seats (396) along a light conducting path.

6. The detecting device according to claim 5, characterized in that the junction between the first connector (372) and the second connector (373), the junction between the third connector (374) and the second fixture (379), the junction between the first photomultiplier tube holder (377) and the second fixture (379), the junction between the second photomultiplier tube holder (375) and the third connector (374), and the junction between the third photomultiplier tube holder (396) and the first fixture (371) are provided with a sealing shock absorber.

7. The detection apparatus according to claim 6, wherein the objective lens (2) is movably connected to a side of the first connection member (372) facing away from the second connection member (373).

8. The detection device according to any one of claims 1 to 7, wherein the light combining module (1) comprises a first laser (11), a second laser (12), a third laser (10), a reflective mirror (143), a fourth dichroic mirror (142), and a fifth dichroic mirror (141), wherein the third laser light emitted by the third laser (10) is reflected by the reflective mirror (143) and then combined with the second laser light emitted by the second laser (12) into the mixed excitation light at the fourth dichroic mirror (142), and the first laser light emitted by the first laser (11) is combined with the second laser light into the mixed excitation light again at the fifth dichroic mirror (141) through the third laser light and the second laser light combined mixed excitation light.

9. The detecting device according to claim 8, wherein the light combining module (1) further comprises a fixing plate (13), the first laser (11), the second laser (12), the third laser (10) and a light combining cassette (14) fixedly connected to the fixing plate (13), the reflective mirror (143), the fourth dichroic mirror (142) and the fifth dichroic mirror (141) are disposed in the light combining cassette (14), and the light combining cassette (14) has a mixed excitation light exit.

10. A detection device according to claim 8, characterized in that the first laser (11) is a 473nm laser; the second laser (12) is a 532nm laser and the third laser (10) is a 633nm laser.