CN104502255A - Three-dimensional imaging flow cytometer device - Google Patents

Abstract

The invention relates to a three-dimensional imaging flow cytometer device, relating to the field of biological and medical optical apparatuses and aiming at solving the problems that the flow testing efficiency is influenced by low speed of a liquid flow when cells are scanned by an existing imaging flow cytometer device and a micro-objective is easily polluted by a sample flow and the service life of the micro-objective is influenced since the sample flow needs to directly contact the micro-objective, etc. A moving cell is synchronously imaged in two directions through two vertical imaging flow systems; the relative position distribution of the internal structure of the cell is extracted; and therefore, the three-dimensional structural image of the cell is obtained. A lateral scattering and fluorescence exciting laser light source illuminates at 45 DEG and is shared by a speed-measuring and focusing unit and an imaging unit. By means of three-dimensional cell imaging, the original position information of the internal structure of the cell is kept; the morphological description authenticity is better; and thus, the imaging flow cytometer is capable of obtaining more meaningful biomedical information.

Description

3D Imaging Flow Cytometry Device

technical field

The invention relates to the field of biological and medical optical instruments, in particular to a three-dimensional imaging flow cytometer device.

Background technique

Flow cytometry is a technique for rapid quantitative analysis and sorting of cells or other biological particles (such as microspheres, bacteria, small model organisms, etc.) arranged in a single row in a liquid flow. In the fields of biology and medicine, when a large number of cells need to be scanned, the flow cytometer completely sacrifices the spatial resolution to obtain a high detection speed, tens of thousands of cells per second. Imaging flow cytometry can not only obtain population analysis data of a large number of cells, but also can see cell images in real time, and the analysis results of each step can be confirmed by images. Compared with traditional flow cytometers, imaging flow cytometers have greater advantages when it is necessary to obtain cell morphology and internal structure information.

At present, imaging flow cytometer has gained great attention in the world, represented by Amnis, a subsidiary of MerckMillipore in the United States, has made imaging flow cytometer with good performance, models include Image Stream Mark II and Flow Sight, etc. , can capture up to 12 high-resolution images of each flow cell in real time, the detection rate can reach 5000 cells/second, and has stronger fluorescence sensitivity.

Imaging flow cytometry performs microscopic imaging of fast-flowing cells. Usually, the two-dimensional image of the cell obtained is to project all the structures in the cell onto a plane, which loses the original position information of the internal structure of the cell and affects the morphology. The description lacks authenticity, therefore, the three-dimensional imaging of imaging flow cytometry has great research significance. Chinese patent CN201310202769.7 reports a flow fluorescence microscopy imaging device and method, which is an application of light sheet microscopy in flow cytometry. In order to meet the tomographic scanning of cells in the direction of the light sheet, the device has a relatively slow liquid flow velocity, which affects the efficiency of the flow test. In addition, the sample flow needs to be in direct contact with the microscope objective lens, and the sample flow can easily pollute the microscope objective lens and affect its life.

Contents of the invention

The invention aims to solve the problem that the existing imaging flow cytometer device scans the cells due to the slow liquid flow speed, which affects the efficiency of the flow test. In addition, the sample flow needs to be in direct contact with the microscopic objective lens, and the sample flow can easily pollute the microscopic objective lens and affect its life. A three-dimensional imaging flow cytometer is provided.

A three-dimensional imaging flow cytometer device, the device includes a sample sampling unit, a first light source, a second light source and a third light source, and two sets of imaging flow cytometry systems are used to simultaneously image the moving sample to be tested in two directions, Obtaining the final three-dimensional image; the two sets of imaging flow systems have the same structure, and each set of imaging flow systems includes a speed measurement-focusing unit and an imaging unit;

The imaging process of the imaging flow system in one direction is as follows: the sample sampling unit keeps the samples to be tested single and side by side through the imaging detection area at a uniform speed, the second light source illuminates the sample to be tested sideways, and the side scattered light passes through the first imaging unit. The first microscopic objective lens enters the first speed measurement-focusing unit to obtain the moving speed and defocus amount of the sample to be measured, and realizes the feedback control of the first TDI CCD and the microscopic objective lens; at the same time, the second light source acts as a dark field or fluorescence The excitation light source, the third light source as a bright field light source, the scattered light and fluorescence signals of the sample to be measured enter the first imaging unit through the first microscopic objective lens and the first dichroic mirror in the first speed measuring-focusing unit, and obtain the Brightfield, darkfield and fluorescence images of samples;

The imaging process of the imaging flow system in the other direction is as follows: the second light source illuminates the sample to be tested sideways, and the side scattered light enters the second velocity measuring-focusing unit through the second microscopic objective lens in the second imaging unit to obtain the sample to be tested. The movement speed and defocus of the sample realize the feedback control of the second TDI CCD and the second microscope objective lens; at the same time, the second light source is used as a dark field or fluorescence excitation light source, and the first light source is used as a bright field light source. Scattered light and fluorescence signals enter the second imaging unit through the second microscopic objective lens and the second dichroic mirror in the second speed measurement-focusing unit to obtain bright field, dark field and fluorescence images of the sample to be tested; Brightfield, darkfield and fluorescence images obtained simultaneously in two directions are processed to obtain a three-dimensional image.

Beneficial effects of the present invention: the present invention uses two mutually perpendicular imaging flow systems to perform cell imaging in two directions synchronously on moving cells, extracts the relative position distribution of the internal structure of the cells, and then obtains the three-dimensional structure image of the cells. The laser light source for side scattering and fluorescence excitation is irradiated in a direction of 45°, and the two sets of devices of the speed measuring-focusing unit and the imaging unit share the side scattering and fluorescence excitation laser light source. Three-dimensional cell imaging maintains the original position information of the internal structure of cells, and the description of morphology is more authentic, which can enable imaging flow cytometry to obtain more and more meaningful biomedical information.

Description of drawings

Fig. 1 is a schematic diagram of the optical principle of the three-dimensional imaging flow cytometer device of the present invention;

Fig. 2 is a plan view of cells in two directions perpendicular to each other obtained by using the three-dimensional imaging flow cytometer device of the present invention.

Detailed ways

Specific Embodiments 1. This embodiment is described in conjunction with Fig. 1 and Fig. 2. The three-dimensional imaging flow cytometer device performs two-direction imaging on the moving sample to be tested synchronously through two sets of imaging flow cytometry systems to obtain the final three-dimensional image. The device includes a sample sampling unit 100, a light source 200, two sets of speed measurement-focus units and two sets of imaging units; the structure and working principle of the two sets of imaging flow systems are exactly the same, and each set of imaging flow systems includes speed measurement-focus unit and imaging unit;

The sample injection unit 100 ensures that samples such as viruses, cells, microspheres or small model organisms pass through the imaging detection area individually and side by side at high speed.

The light source 200 includes a first light source 201, a second light source 202 and a third light source 203. The first light source 202 emits side scattering and fluorescence excitation light sources with a central wavelength of 488nm and a power of 150mw. The first light source 201 and the third light source 203 are two bright field light sources. The bright field light source is LED, the power is 2W, and the center wavelength is 830nm. At the same time, the second light source 202 can be used as an illumination light source for two sets of speed measurement-focusing units.

The two sets of speed measuring-focusing units include two completely identical parts, which are respectively used as auxiliary units of the two sets of imaging units. In this embodiment, a set of speed measuring-focusing units and a set of imaging units (the first speed measuring-focusing unit 300a and the first imaging unit 400a ) are taken as examples to describe this specific embodiment in detail.

The first speed measurement-focusing unit 300a includes a first dichroic mirror 301a, a first beam splitter 302a, a first focusing mirror group 303a and a second focusing mirror group 306a, a first grating 304a and a second grating 307a, a first photodetector device 305a and a second photodetector 308a. The first dichroic mirror 301a is a long-pass dichroic mirror with a central wavelength of 488nm. The center wavelength of the first beam splitter 302a is 488nm. The first grating 304a is a positive grating, and its position is behind the intermediate image plane. The second grating 307a is a negative grating, and its position is before the intermediate image plane. The first photodetector 305a and the second photodetector 308a are two high-sensitivity photomultiplier tubes with a center wavelength of 488nm.

The first imaging unit 400a includes a first microscope objective lens 401a, a first multispectral beam splitter group 402a, a first imaging objective lens 403a, and a first TDI CCD (Time Delay Integration CCD) camera 404a. The first microscope objective lens 401a is used as a high-resolution imaging optical system for cells, with a focal length of 6 mm, a vertical aperture of 0.5, and a field of view of 100×200 μm. The first multi-spectral beam splitter group 402a is composed of 6 long-pass dichroic mirrors, and the beam splitting bands are 420-480nm, 480-560nm, 560-600nm, 600-640nm, 640-745nm, 745-800nm.

The working process of this embodiment: the sample sampling unit 100 keeps the samples to be tested passing through the imaging detection area individually and side by side at a certain stable speed. The second light source 202 emits a 488nm laser beam to illuminate the sample to be tested sideways, and the laser light source irradiates in a 45° direction. The side scattered light passes through the first microscope objective lens 401a, the first dichroic mirror 301a and the first beam splitter 302a, the first focusing lens group 303a and the second focusing lens group 306a and the first grating 304a and the second grating 307a, The cell movement speed and defocus information required by the first imaging unit 400a system are obtained from the first photodetector 305a and the second photodetector 308a. In addition, the second light source 202 is used as a dark field and fluorescence excitation light source, and the third light source 203 is used as a bright field light source. The scattering and fluorescence signals of cells pass through the first microscope objective lens 401a, the first multi-spectral beam splitter group 402a and the first imaging The objective lens 403a and the first TDI camera 404a obtain bright, dark field and fluorescence images of cells.

At the same time, the second speed-measuring-focus unit 300b and the second imaging unit 400b placed in the vertical direction of the first set of imaging units synchronously obtain cell images in this direction, wherein the second light source 202 is still used as a dark field and fluorescence excitation light source, The first light source 201 serves as a bright field light source for this part. Finally, through simultaneous exposure in two directions, the projection images of cells in two directions are obtained, and finally a three-dimensional image of cells is obtained.

The imaging flow system in the vertical direction obtains cell images in this direction synchronously by the second speed measurement-focus unit 300b and the second imaging unit 400b, and the second imaging unit 400b also includes a second multi-spectral beam splitter group 402b, Second imaging objective lens 403b and second photoelectric imaging sensor 404b; Mirror group 306b, fourth grating 307b and fourth photodetector 308b;

The imaging process of the imaging flow system in the vertical direction is as follows: the side scattered light emitted by the second light source 202 is divided into two parts by the second microscope objective lens 401b, the second dichroic mirror 301b and the second beam splitter 302b. One beam of scattered light is received by the third photodetector 305b after passing through the third focusing lens group 303b and the third grating 304b; The fourth electrical detector 308b receives, according to the intensity and frequency of the scattered light received by the third photodetector 305b and the fourth photodetector 308b, the movement speed and defocus of the sample to be measured are obtained, and the TDI camera and the microscope objective lens At the same time, the second light source 202 is used as a dark field or fluorescence excitation light source, and the first light source 201 is used as a bright field light source, and the scattered light and fluorescence signals of the sample to be measured pass through the second microscope objective lens 401b, the second multi-spectral beam splitter The group 402b and the second imaging objective lens 403b, and the second TDI CCD404b obtain bright field, dark field and fluorescence images of the sample to be tested.

This embodiment is described in conjunction with FIG. 2. FIG. 2 shows plan views 1 and 2 of cells in two directions perpendicular to each other obtained by using the device described in this embodiment. It can be seen that the distribution of cell organelles a and b in two directions , the spatial distribution of organelles a and b can be reconstructed by algorithm. For example, the left figure shows the projection of the organelle a in one direction to obtain the two-dimensional coordinates in the XZ direction, and the right figure shows the projection in the other direction to obtain the two-dimensional coordinates in the YZ direction. Finally, it is easy to obtain the three-dimensional coordinates of the organelle a spatial coordinates. Similarly, the three-dimensional space coordinates of the organelle b can be obtained, and the spatial distribution of the organelle in the internal structure of the cell can be determined through the spatial relative position between the two.

The three-dimensional cell imaging of the present invention maintains the original position information of the internal structure of the cell, and the description of the morphology is more authentic, enabling the imaging flow cytometer to obtain more and more meaningful biomedical information.

Claims (6)

1. three-dimensional imaging flow cytometry device, described device comprises sample feeding unit (100), the first light source (201), secondary light source (202) and the 3rd light source (203), it is characterized in that, synchronously the testing sample of motion is carried out to the imaging of both direction by two cover imaging streaming systems, obtain final 3-D view;

Described two cover imaging streaming systems structures are identical, often overlap imaging streaming systems and comprise test the speed-focus unit and image-generating unit;

The imaging process of the imaging streaming systems in a direction is: sample feeding unit (100) keep testing sample at the uniform velocity single, side by side by image checking region, secondary light source (202) side lighting testing sample, first microcobjective (401a) of side scattered light in the first image-generating unit (400a) enters first and to test the speed-focus unit (300a), obtain movement velocity and the defocusing amount of testing sample, realize the FEEDBACK CONTROL to a TDI CCD (404a) and the first microcobjective (401a); Simultaneously, secondary light source (202) is as details in a play not acted out on stage, but told through dialogues or fluorescence excitation light source, 3rd light source (203) is as bright field light source, (301a enters the first image-generating unit (400a) to the first dichroic mirror that the scattered light of testing sample and fluorescence signal test the speed-focus through the first microcobjective (401a), first in unit (300a), obtains the light field of testing sample, details in a play not acted out on stage, but told through dialogues and fluoroscopic image;

The imaging process of the imaging streaming systems in another direction is: secondary light source (202) side lighting testing sample, second microcobjective (401b) of side scattered light in the second image-generating unit (400b) enters second and to test the speed-focus unit (300b), obtain movement velocity and the defocusing amount of testing sample, realize the FEEDBACK CONTROL to the 2nd TDI CCD (404b) and the second microcobjective (401b); Simultaneously, secondary light source (202) is as details in a play not acted out on stage, but told through dialogues or fluorescence excitation light source, first light source (201) is as bright field light source, the second dichroic mirror (301b) that the scattered light of testing sample and fluorescence signal test the speed-focus through the second microcobjective (401b), second in unit enters the second image-generating unit (400b), obtains the light field of testing sample, details in a play not acted out on stage, but told through dialogues and fluoroscopic image;

To testing sample in the two directions the light field of synchronization gain, details in a play not acted out on stage, but told through dialogues and fluoroscopic image process, obtain 3-D view.

2. three-dimensional imaging flow cytometry device according to claim 1, it is characterized in that, described first image-generating unit (400a) also comprises the first multispectral spectroscope group (402a), the first image-forming objective lens (403a) and the first photoelectric imaging sensor (404a); First unit (300a) that tests the speed-focus also comprises the first spectroscope (302a), the first focus lamp group (303a), the first grating (304a), the first photodetector (305a), the second focus lamp group (306a), the second grating (307a) and the second photodetector (308a);

Described side scattered light is divided into two bundle scattered lights through the first microcobjective (401a), the first dichroic mirror (301a) and the first spectroscope (302a), and a branch of scattered light is received by the first photodetector (305a) after the first focus lamp group (303a), the first grating (304a); Another bundle scattered light is received by light second electric explorer (308a) after the second focus lamp group (306a) and the second grating (307a), according to intensity and the frequency of the first photodetector (305a) and the second photodetector (308a) receiving scattered light, obtain movement velocity and the defocusing amount of testing sample, realize the FEEDBACK CONTROL to a TDI CCD (404a) and microcobjective; Simultaneously, secondary light source (202) is as details in a play not acted out on stage, but told through dialogues or fluorescence excitation light source, 3rd light source (203) is as bright field light source, the scattered light of testing sample and fluorescence signal are through the first microcobjective (401a), the first multispectral spectroscope group (402a) and the first image-forming objective lens (403a), and a TDI CCD (404a) obtains the light field of testing sample, details in a play not acted out on stage, but told through dialogues and fluoroscopic image.

3. three-dimensional imaging flow cytometry device according to claim 1, it is characterized in that, described second image-generating unit (400b) also comprises the second multispectral spectroscope group (402b), the second image-forming objective lens (403b) and the second photoelectric imaging sensor (404b); Second unit (300b) that tests the speed-focus also comprises the second spectroscope (302b), the 3rd focus lamp group (303b), the 3rd grating (304b), the 3rd photodetector (305b), the 4th focus lamp group (306b), the 4th grating (307b) and the 4th photodetector (308b);

Described side scattered light is divided into two bundle scattered lights through the second microcobjective (401b), the second dichroic mirror (301b) and the second spectroscope (302b), and a branch of scattered light is received by the 3rd photodetector (305b) after the 3rd focus lamp group 303b, the 3rd grating (304b); Another bundle scattered light is received by light the 4th electric explorer (308b) after the 4th focus lamp group (306b) and the 4th grating (307b), according to intensity and the frequency of the 3rd photodetector (305b) and the 4th photodetector (308b) receiving scattered light, obtain movement velocity and the defocusing amount of testing sample, realize the FEEDBACK CONTROL to a TDI CCD (404b) and microcobjective; Simultaneously, secondary light source (202) is as details in a play not acted out on stage, but told through dialogues or fluorescence excitation light source, first light source (201) is as bright field light source, the scattered light of testing sample and fluorescence signal are through the second microcobjective (401b), the second multispectral spectroscope group (402b) and the second image-forming objective lens (403b), and the 2nd TDI CCD (404b) obtains the light field of testing sample, details in a play not acted out on stage, but told through dialogues and fluoroscopic image.

4. the three-dimensional imaging flow cytometry device according to claim 1,2 or 3, it is characterized in that, described secondary light source (202) sends the laser beam side lighting of 488nm for laser instrument, and the LASER Light Source of side scattered light and fluorescence excitation is irradiated in 45 ° of directions.

5. three-dimensional imaging flow cytometry device according to claim 4, is characterized in that, laser light source adopts dichroic mirror to close bundle, adopts the LASER Light Source of multiple wavelength to carry out multi-color illumination and excites.

6. three-dimensional imaging flow cytometry device according to claim 1, is characterized in that, described first light source (201) and the 3rd light source (203) are LED, and the centre wavelength of light source is 830nm.