Multicolor fluorescence analyzer for liquid drop detection

Abstract

The utility model provides a multicolor fluorescence analyzer for droplet detection, and belongs to the technical field of droplet detection and identification in droplet microfluidics, the multicolor fluorescence analyzer comprises a multiband excitation module, an objective lens module, a light processing module and a camera imaging module, and is characterized in that the multiband excitation module is connected with the objective lens module, and the light processing module is connected with the camera imaging module. Comprising multi-band lasers, a multi-band band-pass optical filter and a multi-band dichroscope, the lasers with multiple wavelengths are coupled together to synthesize mixed excitation light, and the multi-band band-pass optical filter filters excitation fluorescence; and the objective lens module comprises an objective lens, and the filtered mixed exciting light irradiates the liquid drop to be detected from the objective lens and is focused on the liquid drop on the objective table. The problems that in the prior art, monochromatic fluorescence detection results are limited, and application scenes and fields are narrow are solved, and the problems that a multi-color fluorescence analyzer is poor in stability, large in size and low in detection sensitivity are solved. And meanwhile, the problems of poor resolution and poor imaging quality of the CCD camera are solved.

Description

Technical Field

[0001] This utility model relates to the field of droplet detection and identification in droplet microfluidics, and more specifically, to a multicolor fluorescence analyzer for droplet detection. Background Technology

[0002] Droplet microfluidics is a technology based on microfluidic chips to process and manipulate droplet systems. It is widely used in biomedicine, chemical analysis, biomolecular detection and other fields, and has important scientific research and application value.

[0003] Droplet microfluidics generates water-in-oil droplets, which emit light of a specific wavelength upon excitation by light. Currently, laser confocal microscopy is commonly used to acquire the fluorescence signal of the droplets on the chip. However, most fluorescence detection analyzers currently use a single fluorescence detection signal, resulting in limited and restrictive detection results, limiting their applicability to specific scenarios and fields. A limited number of multicolor fluorescence analyzers used for droplet detection suffer from poor stability, large size, and low detection sensitivity. Furthermore, CCD cameras in this ultra-high-throughput detection system also exhibit poor resolution and image quality, failing to meet the requirements for tracking droplet motion trajectories.

[0004] Therefore, we have made improvements to this and proposed a multicolor fluorescence analyzer for droplet detection. Utility Model Content

[0005] The purpose of this invention is to address the limitations of existing monochromatic fluorescence detection technologies, such as narrow applicability and limited application scenarios, as well as the problems of multicolor fluorescence analyzers, including poor stability, large size, and low detection sensitivity. It also solves the problems of poor resolution and image quality associated with CCD cameras.

[0006] To achieve the above-mentioned objectives, this utility model provides the following technical solution:

[0007] A multicolor fluorescence analyzer for droplet detection was developed to address the aforementioned issues.

[0008] The application is as follows:

[0009] The device comprises a multi-band excitation module, an objective lens module, an optical processing module, and a camera imaging module. The multi-band excitation module includes a multi-band laser, a multi-band bandpass filter, and a multi-band dichroic mirror. Lasers with multiple wavelengths are coupled together to synthesize a mixed excitation light, and the multi-band bandpass filter filters the excitation fluorescence. The objective lens module includes an objective lens through which the filtered mixed excitation light is projected onto the droplet under test and focused onto the droplet on the stage. The droplet under test generates different fluorescence wavelengths after being irradiated by the excitation light, which are then collimated into parallel light by the objective lens.

[0010] As a preferred technical solution of this application, the light processing module consists of four groups. The first group includes acquiring a first fluorescent photomultiplier tube (PMT), a first fluorescent aperture, a first convex lens, a first single-band filter, and a second dichroic mirror that separates the first fluorescence and the second fluorescence. The second group includes acquiring a second fluorescent photomultiplier tube (PMT), a second fluorescent aperture, a second convex lens, a second single-band filter, and a third dichroic mirror that separates the second fluorescence and the third fluorescence. The third group includes acquiring a third fluorescent photomultiplier tube (PMT), a third fluorescent aperture, a third convex lens, a third single-band filter, and a fourth dichroic mirror that separates the third fluorescence and the fourth fluorescence. The fourth group includes acquiring a fourth fluorescent photomultiplier tube (PMT), a fourth fluorescent aperture, a fourth convex lens, and a fourth single-band filter.

[0011] As a preferred technical solution of this application, the camera imaging module includes a CMOS camera, a reflector, an imaging lens, a first dichroic mirror, and an LED illumination source.

[0012] Compared with the prior art, the beneficial effects of this utility model are as follows:

[0013] In the scheme of this application:

[0014] Droplets are excited by mixed laser light of different wavelengths to form first, second, third, and fourth fluorescence, which are then acquired by corresponding photomultiplier tubes and transmitted to corresponding data processing devices for processing. This allows for the acquisition of multiple detection parameters in a single detection, broadening its application scenarios and significantly improving detection efficiency. Furthermore, this invention exhibits good stability, high detection sensitivity, and a small size; its light processing modules can be independently configured, providing flexibility in spatial arrangement. It also solves the problems of poor resolution and image quality associated with CCD cameras. Attached Figure Description

[0015] Figure 1 This is a schematic diagram of the optical path of this utility model;

[0016] Figure 2This is a schematic diagram of the overall structure of this utility model.

[0017] In the diagram: 1. LED lighting source; 2. Stage; 3. Multi-band laser; 4. Objective lens; 5. First dichroic mirror; 7. Second dichroic mirror; 8. First convex lens; 9. First single-band filter; 10. First fluorescent pinhole; 11. First fluorescent photomultiplier tube (PMT); 12. Multi-band dichroic mirror; 13. Multi-band bandpass filter; 14. Third dichroic mirror; 15. Second convex lens; 16. Second single-band filter; 17. Second... 18. Fluorescent aperture; 19. Second fluorescent photomultiplier tube (PMT); 20. Third fluorescent photomultiplier tube (PMT); 21. Third fluorescent aperture; 22. Third single-band filter; 23. Third convex lens; 24. Fourth dichroic mirror; 25. Fourth single-band filter; 26. Fourth fluorescent aperture; 27. Fourth fluorescent photomultiplier tube (PMT); 28. Achromatic lens; 29. ​​Reflector; 30. CMOS camera. Detailed Implementation

[0018] To make the objectives, technical solutions, and advantages of the embodiments of this utility model clearer, the technical solutions of the embodiments of this utility model will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this utility model.

[0019] Therefore, the following detailed description of the embodiments of this utility model is not intended to limit the scope of the claimed utility model, but merely to illustrate some embodiments of the utility model. All other embodiments obtained by those skilled in the art based on the embodiments of this utility model without inventive effort are within the scope of protection of this utility model.

[0020] It should be noted that, unless otherwise specified, the embodiments and features and technical solutions in the present invention can be combined with each other.

[0021] It should be noted that similar labels and letters in the following figures indicate similar items. Therefore, once an item is defined in one figure, it does not need to be further defined and explained in subsequent figures.

[0022] In the description of this utility model, it should be noted that the terms "upper," "lower," etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings, or the orientation or positional relationship commonly used when the product is in use, or the orientation or positional relationship commonly understood by those skilled in the art. These terms are only for the convenience of describing this utility model and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation, and therefore should not be construed as a limitation on this utility model. In addition, the terms "first," "second," etc., are only used to distinguish descriptions and should not be construed as indicating or implying relative importance.

[0023] Example 1:

[0024] like Figure 1 and Figure 2 As shown, this embodiment proposes a multicolor fluorescence analyzer for droplet detection, including a multi-band excitation module, an objective lens module, an optical processing module, and a camera imaging module. The multi-band excitation module includes a multi-band laser 3, a multi-band bandpass filter 13, and a multi-band dichroic mirror 12. Lasers with multiple wavelengths are coupled together to synthesize mixed excitation light, and the multi-band bandpass filter 13 filters the excitation fluorescence.

[0025] The objective module includes an objective lens 4. The filtered mixed excitation light is irradiated onto the droplet to be tested through the objective lens 4 and focused onto the droplet on the stage 2 to achieve confocalization. After being irradiated by the excitation light, the droplet to be tested generates different fluorescence wavelengths, which are collimated into parallel light through the objective lens 4.

[0026] Example 2:

[0027] The solution in Example 1 will be further described below with reference to its specific working method.

[0028] like Figure 1As shown, in a preferred embodiment, based on the above method, a further light processing module is provided, comprising four groups. The first group includes a first fluorescence photomultiplier tube (PMT) 11, a first fluorescence aperture 10, a first convex lens 8, a first single-band filter 9, and a second dichroic mirror 7 that separates the first and second fluorescence. The second group includes a second fluorescence photomultiplier tube (PMT) 18, a second fluorescence aperture 17, a second convex lens 15, a second single-band filter 16, and a third dichroic mirror 14 that separates the second and third fluorescence. The third group includes a third fluorescence photomultiplier tube (PMT) 18. T)19, third fluorescent aperture 20, third convex lens 22, third single-band filter 21, and fourth dichroic mirror 23 that separates the third and fourth fluorescence; the fourth group includes a fourth fluorescence photomultiplier tube (PMT) 27, fourth fluorescent aperture 26, fourth convex lens 24, and fourth single-band filter 25; the four fluorescence paths start from the same point, are separated after reflection by the dichroic mirror, form two perpendicular light paths, and pass through their respective apertures, finally reaching their respective photomultiplier tubes (PMTs). The second dichroic mirror 7 divides the light into two different parts according to the wavelength, and the first single-band filter 9 extracts the light of a specific wavelength.

[0029] like Figure 1 As shown, in a preferred embodiment, based on the above method, the camera imaging module further includes a CMOS camera 30, a reflector 29, an imaging lens, a first dichroic mirror 5, and an LED illumination source 1. The droplet under test, illuminated by the illumination source and fluorescence, is dispersed by the first dichroic mirror 5, then passes through the achromatic lens 28, and forms a high-resolution image in the CMOS camera 30, meeting the requirements for tracking the high-speed movement trajectory of the droplet. The sample illuminated by the LED illumination source 1 has its light collected by the objective lens 4, then converged by the achromatic lens 28 to form an image in the CMOS camera 30. The reflector 29 deflects the light path, reducing the vertical length of the light path and minimizing space requirements.

[0030] Specifically, the multicolor fluorescence analyzer originally designed for droplet detection is used in the following situations:

[0031] like Figure 1 and Figure 2As shown, the mixed excitation light emits parallel fluorescence excitation light, which passes through a multi-band bandpass filter 13 to filter stray light, allowing only the excitation band light to pass through. It is then reflected by a multi-band dichroic mirror 12 to the objective lens 4, confocalizing the mixed excitation light onto the microdroplet to be tested placed on the stage 2. This excitation generates mixed fluorescence corresponding to different lasers, including a first fluorescence, a second fluorescence, a third fluorescence, and a fourth fluorescence. The mixed fluorescence is parallelized by the objective lens 4, passes through the multi-band dichroic mirror 12, then through the first dichroic mirror 5, and is confocalized by convex lenses onto the first fluorescence aperture 10, the second fluorescence aperture 17, the third fluorescence aperture 20, and the fourth fluorescence aperture 26, respectively. The fourth fluorescence is reflected by the fourth dichroic mirror 23 to the fourth single-band filter 25, reaches the fourth fluorescence aperture 26, and is transmitted into the fourth fluorescence photomultiplier tube (PMT) 27.

[0032] The first fluorescence is reflected by the second dichroic mirror 7 to the first single-band filter 9, reaching the first fluorescence aperture 10 and being transmitted into the first fluorescence photomultiplier tube (PMT) 11; the second fluorescence is reflected by the third dichroic mirror 14 to the second single-band filter 16, reaching the second fluorescence aperture 17 and being transmitted into the second fluorescence photomultiplier tube (PMT) 18; the third fluorescence is reflected by the fourth dichroic mirror 23 to the third convex lens 22, reaching the third fluorescence aperture 20 and being transmitted into the third fluorescence photomultiplier tube (PMT) 19.

[0033] Finally, the acquired fluorescence signals are transmitted to the corresponding data processing system for processing, thereby obtaining multiple detection parameters. The images are then projected onto the CMOS camera 30 via the reflector 29, thus obtaining the detection results for different droplet fluorescence intensities. Compared to industrial CCD cameras, the CMOS camera 30 offers advantages such as higher resolution, clearer images, more stable performance, higher sensitivity, longer lifespan, resistance to vibration and shock, resistance to strong light, and ease of operation, making it more suitable for integration. The CMOS camera 30 represents existing technology.

[0034] The above embodiments are only used to illustrate the present utility model and are not intended to limit the technical solutions described in the present utility model. Although the present utility model has been described in detail with reference to the above embodiments, the present utility model is not limited to the specific embodiments described above. Therefore, any modifications or equivalent substitutions to the present utility model, and all technical solutions and improvements that do not depart from the spirit and scope of the invention, are covered within the scope of the claims of the present utility model.

Claims

1. A multi-color fluorescence analyzer for droplet detection, comprising a multi-band excitation module, an objective module, a light processing module, and a camera imaging module, characterized in that, The multi-band excitation module includes a multi-band laser (3), a multi-band band-pass filter (13), and a multi-band dichroic mirror (12). The lasers with multiple wavelengths are coupled together to synthesize mixed excitation light, and the multi-band band-pass filter (13) filters the excitation fluorescence.

2. The multi-color fluorescence analyzer for droplet detection according to claim 1, wherein, The light processing module is composed of four groups. The first group includes a first fluorescence photomultiplier tube (PMT) (11), a first fluorescence aperture (10), a first convex lens (8), a first single-band filter (9), and a second dichroic mirror (7) for separating the first fluorescence and the second fluorescence. The second group includes a second fluorescence photomultiplier tube (PMT) (18), a second fluorescence aperture (17), a second convex lens (15), a second single-band filter (16), and a third dichroic mirror (14) for separating the second fluorescence and the third fluorescence. The third group includes a third fluorescence photomultiplier tube (PMT) (19), a third fluorescence aperture (20), a third convex lens (22), a third single-band filter (21), and a fourth dichroic mirror (23) for separating the third fluorescence and the fourth fluorescence. The fourth group includes a fourth fluorescence photomultiplier tube (PMT) (27), a fourth fluorescence aperture (26), a fourth convex lens (24), and a fourth single-band filter (25).

3. The multi-color fluorescence analyzer for droplet detection according to claim 1, wherein, The camera imaging module includes a CMOS camera (30), a mirror (29), an imaging lens, a first dichroic mirror (5), and an LED illumination light source (1).

Images