Single cell bright field fluorescence imaging, capturing and separation extraction integrated device

Abstract

The utility model discloses a single-cell bright field fluorescence imaging, capturing and separating extraction integrated device, which comprises a microscope objective, an objective lens focusing component, a light source module, a light source module, a light source module and a light source module, wherein the microscope objective moves up and down along the vertical direction through the objective lens focusing component; the dichroscope and the reflector are sequentially arranged on a light path above the microscope objective; the illumination light source is arranged on a light path below the microscope objective; the optical tweezer unit is used for emitting an optical tweezer optical path and coupling the optical tweezer optical path to an upper optical path of the microscope objective through the dichroscope; the image acquisition unit is used for imaging display and is coupled to a light path above the microscope objective through a reflector; the fluorescent unit is arranged on the imaging light path between the image acquisition unit and the reflecting mirror, and the fluorescent unit passes through or does not pass through the imaging light path through a fluorescent unit driving mechanism; and the sample micro-motion platform and the microwell plate three-dimensional mobile platform are respectively used for placing the micro-fluidic chip and the collection pore plate and are arranged between the microscope objective and the illumination light source. Compared with the prior art, the single cell sorting device has the beneficial effects that the overall structural design of the device is more compact by optimizing the overall structural layout, so that the flattening design is realized, multiple functions of bright field imaging, fluorescence imaging and optical tweezers capturing can be simultaneously met, and the requirement on single cell sorting is further met.

Description

Technical Field

[0001] The utility model belongs to the technical field of single cell sorting, and specifically relates to a single cell bright field fluorescence imaging, capturing and separating and extracting integrated device. Background Art

[0002] Single-cell analysis can reveal the diversity and differences in the material composition and physiological behavior of cells, the basic unit of life, and is the mainstream cutting-edge technology for life analysis today. The first technical problem to be solved in single-cell analysis is single-cell imaging, capture, and separation and extraction. The biggest challenge is that the cells are tiny in size and distributed at the micron or even submicron scale. The capture and separation and extraction of single cells at this scale places extremely high demands on instrument resolution and sensitivity.

[0003] Optical microscopes can observe the morphology and fluorescence characteristics of single cells, and realize qualitative and semi-quantitative research on single cells. They are one of the commonly used tools for single-cell research and detection. Patents CN114350474B and CN212077049U, based on the commercially mature microscope structure, realize the capture, manipulation and separation and extraction of single cells through the coupling of optical tweezers modules and the cooperation of microfluidic chips, but they have great limitations. First, in the upper part of the microscope, due to the insertion of the optical tweezers module, the optical path of the microscope imaging light path is greatly increased, so that the tube lens exceeds the normal working distance. Therefore, the imaging of the microscope cannot reach the diffraction limit and increases the aberration, thereby making the imaging quality worse, which has an adverse effect on the analysis of micron and submicron biological samples; then, in the lower part of the microscope, due to the insertion of the two-dimensional motion platform and the collection component, in order to avoid interference, the condenser in the Köhler illumination assembly at the bottom of the microscope cannot reach the working position, the focusing effect is deteriorated, and the overall lighting efficiency is deteriorated. Therefore, the exposure time of the camera must be very large (more than 50ms), The frame rate of imaging is greatly limited, not only will the video appear to be stuck subjectively, but it is also impossible to quickly and quickly capture sample changes with high-speed imaging, which limits the application of the microscope. Secondly, since module coupling is added to the upper and lower parts of the microscope, the overall height of the microscope is greatly increased, and it cannot be used in some space-constrained environments (such as biosafety cabinets, anaerobic workstations, etc.). Finally, the collection component in this structure collects single cells by installing collection containers on its blind holes. The collection containers need to be installed one by one, which is cumbersome. In addition, due to the limited diameter of the turntable, the number of collection containers is also limited (generally ≤30), so the flux that can be collected in a single operation is relatively small.

[0004] In order to solve the above problems, it is urgent to develop an integrated device for bright field / fluorescence imaging, capture and separation and extraction of flat-structured single cells suitable for the use of biological samples. Utility Model Content

[0005] Details of one or more embodiments of the present invention are set forth in the following drawings and description to make other features, objects, and advantages of the present application more readily apparent.

[0006] The utility model discloses a single cell bright field fluorescence imaging, capture and separation extraction integrated device, comprising:

[0007] The microscope objective lens is adjusted up and down in the vertical direction through the objective lens focusing assembly;

[0008] A dichroic mirror and a reflector are sequentially arranged in the optical path above the microscope objective lens;

[0009] An illumination light source is arranged in the optical path below the microscope objective lens;

[0010] An optical tweezers unit, used for emitting an optical tweezers light path and coupling it into an upper light path of the microscope objective lens through the dichroic mirror;

[0011] An image acquisition unit, used for imaging display and coupled to the upper optical path of the microscope objective lens through the reflector;

[0012] A fluorescent unit is provided on the imaging optical path between the image acquisition unit and the reflector, and the fluorescent unit is made to pass through or not pass through the imaging optical path through a fluorescent unit driving mechanism;

[0013] The sample micro-motion platform and the micro-well plate three-dimensional moving platform are used to place the microfluidic chip and the collection well plate respectively, and are arranged between the microscope objective lens and the illumination light source.

[0014] In some embodiments, the movement direction of the fluorescent unit is arranged at 90° to the direction of the imaging light path.

[0015] In some embodiments, the fluorescent unit is composed of a plurality of fluorescent cubes of different colors arranged side by side.

[0016] In some embodiments, the fluorescent unit driving mechanism includes:

[0017] A fluorescent unit sliding seat, used for installing the fluorescent unit;

[0018] The lead screw motor is threadedly connected to the fluorescent unit sliding seat.

[0019] In some embodiments, the image acquisition unit includes a color camera and a focusing lens.

[0020] In some embodiments, the optical tweezers unit includes an optical tweezers laser, an optical shutter, and a variable-focus Galilean beam expander.

[0021] In some embodiments, it further comprises:

[0022] The first reflecting mirror and the second reflecting mirror are arranged on both sides of the optical shutter and are used to change the direction of the optical path of the optical tweezers.

[0023] In some embodiments, the sample micro-motion platform is a horizontal two-axis motion platform; and the microplate three-dimensional motion platform is a three-axis motion platform.

[0024] In some embodiments, the sample micro-motion platform is located above the micro-well plate three-dimensional movement platform.

[0025] In some embodiments, the optical tweezers unit and the image acquisition unit are respectively located on two sides of the microscope objective lens.

[0026] Compared with the prior art, the beneficial effects of the utility model are as follows:

[0027] 1. By optimizing the overall structural layout, the overall structural design of the device is made more compact, thereby achieving a flat design, and can simultaneously meet multiple functions of bright field imaging, fluorescence imaging, and optical tweezers capture, thereby meeting the needs for single cell sorting.

[0028] 2. The capture light path of the optical tweezers is adjusted by a dichroic mirror, and the imaging light path (bright field imaging and fluorescence imaging) is adjusted by a reflector, thereby ensuring the convenience and speed of light path calibration. At the same time, the layout of the two tweezers on both sides of the microscope objective lens also greatly reduces the space occupied by the equipment.

[0029] 3. In combination with the demand for single-cell sorting, the collection method of cell sorting has been improved. A high-precision two-axis sample micro-motion platform is used to realize the capture and sorting of optical tweezers, and a low-precision three-axis microplate three-dimensional moving platform is used to achieve the high-throughput collection demand for target cell sorting. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] The drawings described herein are used to provide a further understanding of the present invention and constitute a part of the present invention. The illustrative embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation on the present invention.

[0031] Figure 1 It is a three-dimensional structural schematic diagram of the pin seat and the rotating seat of the utility model when they are closed.

[0032] Description of the drawings: sample micro-motion platform 1, microplate three-dimensional moving platform 2, microscope objective lens 3, objective lens focusing assembly 4, illumination light source 5, dichroic mirror 6, variable-focus Galilean beam expansion assembly 7, optical tweezers laser 8, optical shutter 9, multi-fluorescence excitation module 10, reflector 11, focusing lens group 12, color camera 13. DETAILED DESCRIPTION

[0033] In order to make the purpose, technical solution and advantages of the utility model clearer, the utility model is described and illustrated in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the utility model and are not used to limit the utility model. Based on the embodiments provided by the utility model, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the utility model.

[0034] Obviously, the drawings described below are only some examples or embodiments of the utility model. For ordinary technicians in this field, the utility model can also be applied to other similar scenarios based on these drawings without creative work. In addition, it can be understood that although the efforts made in this development process may be complicated and lengthy, for ordinary technicians in this field related to the content disclosed by the utility model, some changes in design, manufacturing or production based on the technical content disclosed by the utility model are just conventional technical means, and should not be understood as the content disclosed by the utility model is insufficient.

[0035] Reference to "embodiments" in the present invention means that a particular feature, structure, or characteristic described in conjunction with the embodiments may be included in at least one embodiment of the present invention. The appearance of the phrase in various locations in the specification does not necessarily refer to the same embodiment, nor is it an independent or alternative embodiment that is mutually exclusive with other embodiments. It is explicitly and implicitly understood by those skilled in the art that the embodiments described in the present invention may be combined with other embodiments without conflict.

[0036] An integrated device for single-cell bright-field fluorescence imaging, capture, and separation and extraction, comprising:

[0037] The microscope objective lens 3 is adjusted and moved up and down in the vertical direction through the objective lens focusing assembly 4; specifically, the structural form of the objective lens focusing assembly 4 can be various, such as a screw motor, a lifting electric cylinder, a lifting gas / liquid cylinder, etc., which can specifically meet the lifting and lowering adjustment requirements of the microscope objective lens 3.

[0038] The dichroic mirror 6 and the reflector 11 are sequentially arranged in the optical path above the microscope objective 3; the dichroic mirror 14 is high-reflection and low-pass, so it can reflect the long-wavelength light beam and transmit the short-wavelength light beam. Specifically, the light beam of the optical tweezers optical path is a long-wavelength light beam that can be reflected into the microscope objective 3, while the short-wavelength light beam can pass through the dichroic mirror 6 and enter the image acquisition unit after being refracted by the reflector 11. By separately arranging the dichroic mirror 6 and the reflector 11, it is also convenient to adjust the corresponding optical path, so that the accuracy of the optical path is higher.

[0039] The illumination light source 5 is arranged in the optical path below the microscope objective lens 3 and is used to emit the illumination light path. A light source in the form of a structure such as an LED can be used.

[0040] The optical tweezers unit is used to launch the optical tweezers light path and couple it to the upper light path of the microscope objective 3 through the dichroic mirror 6; specifically, the optical tweezers unit includes an optical tweezers laser 8, an optical shutter 9, and a variable-focus Galilean beam expander assembly 7.

[0041] The image acquisition unit is used for imaging display and is coupled to the upper optical path of the microscope objective lens 3 through the reflector 11 ; specifically, the image acquisition unit includes a color camera 13 and a focusing lens group 12 .

[0042] The fluorescent unit 10 is arranged on the imaging light path between the image acquisition unit and the reflector 11, and the fluorescent unit driving mechanism allows the fluorescent unit 10 to pass through or not pass through the imaging light path; when it is necessary to switch to the fluorescence mode, the fluorescent unit 10 is moved to the imaging light path and started to excite fluorescence. When the fluorescence mode is not needed and only the bright field mode is needed, the fluorescent unit 10 is moved to the imaging light path.

[0043] The sample micro-motion platform 1 and the micro-well plate three-dimensional moving platform 2 are used to place the microfluidic chip and the collection well plate, respectively, and are arranged between the microscope objective 3 and the illumination light source 5. The sample micro-motion platform 1 is moved to cooperate with the optical tweezers to achieve cell sorting, and the micro-well plate three-dimensional moving platform 2 is moved to achieve accurate collection of target cells.

[0044] In some embodiments, the movement direction of the fluorescent unit 10 is set at 90° to the direction of the imaging light path.

[0045] Specifically, the fluorescent unit 10 is composed of a plurality of fluorescent cubes of different colors arranged side by side. For example, fluorescent cubes of three different colors, red, blue and green, can be arranged side by side, and their positions can be moved as a whole to meet the adjustment of their corresponding fluorescent colors according to actual needs.

[0046] In some embodiments, the fluorescent unit driving mechanism includes:

[0047] A fluorescent unit sliding seat, used for installing the fluorescent unit 10;

[0048] The lead screw motor is threadedly connected to the sliding seat of the fluorescent unit.

[0049] In this embodiment, a screw rod is used for overall displacement drive, and a pulley structure or a rack guide structure may also be used.

[0050] In some embodiments, it also includes:

[0051] The first reflector and the second reflector are arranged on both sides of the optical shutter 9 to change the direction of the optical tweezers light path. The first reflector and the second reflector are arranged accordingly to realize the overall flat structure design and small volume structure requirements of the device.

[0052] In some embodiments, the sample micro-motion platform 1 is a horizontal two-axis transport platform; the microplate three-dimensional motion platform 2 is a three-axis transport platform. The microplate three-dimensional motion platform 2 adopts a three-axis transport platform structure design and can be used with a corresponding conventional 96-well plate to collect target cells, thereby meeting the needs of high throughput. Compared with the turntable collection method, it has a larger capacity and higher efficiency.

[0053] In some embodiments, the sample micro-motion platform 1 is located above the microplate three-dimensional moving platform 2. The height difference structure design facilitates the collection of target cells.

[0054] In some embodiments, the optical tweezers unit and the image acquisition unit are respectively located on both sides of the microscope objective lens 3, thereby making the overall structure of the device more compact.

[0055] Its working principle is:

[0056] The optical tweezers laser 8 emits an optical tweezers light path to capture the target cells, and the target cells are introduced from the export capillaries of the microfluidic chip into the holes of the 96-well plate on the corresponding microplate three-dimensional moving platform 2 through the movement of the sample micro-motion platform 1.

[0057] In the above process, the illumination light source 5 emits an illumination light which passes through the microscope objective 3, the dichroic mirror 6 and the reflector 11 in sequence and enters the color camera for imaging. The imaging requirement is selected according to the actual situation, and the position of the fluorescent unit 10 is adjusted to achieve bright field imaging or fluorescent imaging under different colors.

[0058] Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A single-cell bright-field fluorescence imaging, capture and separation and extraction integrated device, characterized in that: include: The microscope objective lens is adjusted up and down in the vertical direction through the objective lens focusing assembly; A dichroic mirror and a reflector are sequentially arranged in the optical path above the microscope objective lens; An illumination light source is arranged in the optical path below the microscope objective lens; An optical tweezers unit, used for emitting an optical tweezers light path and coupling it into an upper light path of the microscope objective lens through the dichroic mirror; An image acquisition unit, used for imaging display and coupled to the upper optical path of the microscope objective lens through the reflector; A fluorescent unit is provided on the imaging optical path between the image acquisition unit and the reflector, and the fluorescent unit is made to pass through or not pass through the imaging optical path through a fluorescent unit driving mechanism; The sample micro-motion platform and the micro-well plate three-dimensional moving platform are used to place the microfluidic chip and the collection well plate respectively, and are arranged between the microscope objective lens and the illumination light source.

2. The single-cell bright-field fluorescence imaging, capture and separation and extraction integrated device according to claim 1, characterized in that: The movement direction of the fluorescent unit is arranged at 90° to the direction of the imaging light path.

3. The integrated device for single-cell bright-field fluorescence imaging, capture and separation and extraction according to claim 2, characterized in that: The fluorescent unit is composed of a plurality of fluorescent cubes of different colors arranged side by side.

4. The integrated device for single-cell bright-field fluorescence imaging, capture and separation and extraction according to claim 3, characterized in that: The fluorescent unit driving mechanism comprises: A fluorescent unit sliding seat, used for installing the fluorescent unit; The lead screw motor is threadedly connected to the fluorescent unit sliding seat.

5. The integrated device for single-cell bright-field fluorescence imaging, capture and separation and extraction according to claim 1, characterized in that: The image acquisition unit includes a color camera and a focusing lens group.

6. The single-cell bright-field fluorescence imaging, capture and separation and extraction integrated device according to claim 1, characterized in that: The optical tweezers unit comprises an optical tweezers laser, an optical shutter, and a variable-focus Galileo beam expansion component.

7. The single-cell bright-field fluorescence imaging, capture and separation and extraction integrated device according to claim 6, characterized in that: Also includes: The first reflecting mirror and the second reflecting mirror are arranged on both sides of the optical shutter and are used to change the direction of the optical path of the optical tweezers.

8. The integrated device for single-cell bright-field fluorescence imaging, capture and separation and extraction according to claim 1, characterized in that: The sample micro-motion platform is a horizontal two-axis transport platform; the microplate three-dimensional movement platform is a three-axis transport platform.

9. The integrated device for single-cell bright-field fluorescence imaging, capture and separation and extraction according to claim 8, characterized in that: The sample micro-motion platform is located above the micro-well plate three-dimensional moving platform.

10. The integrated device for single-cell bright-field fluorescence imaging, capture, and separation and extraction according to claim 1, characterized in that: The optical tweezers unit and the image acquisition unit are respectively located on two sides of the microscope objective lens.

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