CN104502255B - Three-dimensional imaging flow cytometry device - Google Patents
Three-dimensional imaging flow cytometry device Download PDFInfo
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- CN104502255B CN104502255B CN201410831263.7A CN201410831263A CN104502255B CN 104502255 B CN104502255 B CN 104502255B CN 201410831263 A CN201410831263 A CN 201410831263A CN 104502255 B CN104502255 B CN 104502255B
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Abstract
Three-dimensional imaging flow cytometry device, is related to the optical instrument field of biology and medical science, when the existing imaging flow cytometry device of solution is scanned to cell, there is the efficiency that flow stream velocity is slower, affects streaming to test.In addition, its sample stream needs and microcobjective directly contact, sample stream is easy to bring microcobjective pollution, the problems such as affect its life-span.The synchronous cell imaging of both direction is carried out to the cell for moving by the imaging streaming systems of two vertical direction, the relative position distribution of cell interior structure is extracted, and then is obtained the three dimensional structure image of cell.The LASER Light Source of lateral scattering and fluorescence excitation is irradiated in 45 ° of directions, and two covering devices of test the speed focusing unit and image-generating unit share lateral scattering and fluorescence excitation laser light source.Three-dimensional cell imaging keeps original positional information of cell interior structure, imaging flow cytometer can be caused to obtain more, more meaningful biomedical informations to morphologic description verity more preferably.
Description
Technical field
The present invention relates to the optical instrument field of biology and medical science, and in particular to a kind of three-dimensional imaging flow cytometer dress
Put.
Background technology
Flow cytometry be a kind of cell or other biological particles to defiled in liquid stream (such as microsphere, antibacterial,
Small scale mode biology etc.) fast quantitative analysis and the technology for sorting are carried out one by one.In biology and medical domain, when needs are to big
When the cell of amount is scanned, flow cytometer sacrifices spatial resolution completely, it is possible to obtain high detection speed, tens of thousand
Cell is per second.Imaging flow cytometer can not only obtain the group analytic data of a large amount of cells, but also can see in real time
Cell image, the analysis result of each step can be confirmed by image.When needing to obtain cellular morphology and internal structure
During information, relative to traditional flow cytometer, imaging flow cytometer has bigger advantage.
At present, it is imaged flow cytometer and has obtained greatly attention in the world, with U.S. Merck Millipore
Under Amnis companies be that representative has been made the pretty good imaging flow cytometer of performance, model has Image Stream
Mark II and Flow Sight etc., can captured in real time each flow cell can at most reach 12 panel height image in different resolution, detect
Speed is up to 5000 cells/second, and has higher fluorescence sensitivity.
Imaging flow cytometer carries out micro-imaging to the cell of quick flowing, the cell X-Y scheme for obtaining under normal circumstances
Seem that intracellular all of structure is projected to a plane, lose original positional information of cell interior structure, to form
Description lacks verity, therefore, the three-dimensional imaging for being imaged flow cytometer has great Research Significance.Chinese patent
CN201310202769.7 reports a kind of streaming fluorescent microscopic imaging device and method, and it is mating plate microscope in fluidic cell
One kind application in instrument., to meet mating plate direction to cell tomoscan, its flow stream velocity is slower for the device, affects streaming test
Efficiency.In addition, its sample stream needs and microcobjective directly contact, sample stream is easy to bring microcobjective pollution, shadow
Ring its life-span.
The content of the invention
When the present invention is scanned to cell to solve existing imaging flow cytometry device, it is slower to there is flow stream velocity,
Affect the efficiency of streaming test.In addition, its sample stream needs and microcobjective directly contact, sample stream is easy to microcobjective
Pollution is brought, the problems such as affect its life-span, there is provided a kind of three-dimensional imaging flow cytometer.
Three-dimensional imaging flow cytometry device, described device include sample feeding unit, the first light source, secondary light source and
Three light sources, carry out the imaging of both direction by testing sample of two sets of imaging streaming systems synchronously to moving, obtain final
3-D view；Two sets of imagings streaming systems structure is identical, and often set imaging streaming systems include test the speed-focus unit and imaging
Unit；
One direction imaging streaming systems imaging process be：Sample feeding unit keep testing sample it is at the uniform velocity single,
Side by side by image checking region, secondary light source side lighting testing sample, side scattered light is in the first image-generating unit
The first microcobjective test the speed-focus unit into first, obtain the movement velocity and defocusing amount of testing sample, realize to first
The feedback control of TDI CCD and microcobjective；Meanwhile, secondary light source is used as details in a play not acted out on stage, but told through dialogues or fluorescence excitation light source, the 3rd light source conduct
Bright field light source, the scattered light and fluorescence signal of testing sample test the speed-focus in unit first through the first microcobjective, first
Dichroic mirror enters the first image-generating unit, obtains light field, details in a play not acted out on stage, but told through dialogues and the fluoroscopic image of testing sample；
Another direction imaging streaming systems imaging process be：Secondary light source side lighting testing sample, it is lateral to dissipate
Penetrate second microcobjective of the light in the second image-generating unit to test the speed-focus unit into second, obtain the motion of testing sample
Speed and defocusing amount, realize to the 2nd TDI CCD and the feedback control of the second microcobjective；Meanwhile, secondary light source is used as details in a play not acted out on stage, but told through dialogues
Or fluorescence excitation light source, used as bright field light source, the scattered light and fluorescence signal of testing sample is through the second micro- thing for the first light source
Mirror, the second the second dichroic mirror for testing the speed-focusing in unit enter the second image-generating unit, obtain light field, the details in a play not acted out on stage, but told through dialogues of testing sample
And fluoroscopic image；To testing sample, the light field of synchronization gain, details in a play not acted out on stage, but told through dialogues and fluoroscopic image are processed in the two directions, obtain three
Dimension image.
Beneficial effects of the present invention：The present invention is adopted by the imaging streaming systems of two mutually perpendicular directions to motion
Cell carries out the synchronous cell imaging of both direction, extracts the relative position distribution of cell interior structure, and then obtains cell
Three dimensional structure image.The LASER Light Source of lateral scattering and fluorescence excitation is irradiated in 45 ° of directions, and test the speed-focus unit and imaging list
Two covering devices of unit share lateral scattering and fluorescence excitation laser light source.Three-dimensional cell imaging keeps the original of cell interior structure
There is positional information, imaging flow cytometer can be caused to obtain more, more meaningful to morphologic description verity more preferably
Biomedical information.
Description of the drawings
Fig. 1 is the optical principle schematic diagram of three-dimensional imaging flow cytometry device of the present invention；
Fig. 2 is to obtain cell using three-dimensional imaging flow cytometry device of the present invention to be mutually perpendicular to both direction
Plane graph.
Specific embodiment
Specific embodiment one, with reference to Fig. 1 and Fig. 2 explanation present embodiments, three-dimensional imaging flow cytometry device passes through
Two sets of imaging streaming systems synchronously carry out the imaging of both direction to the testing sample of motion, obtain final 3-D view；Should
Device includes that sample feeding unit 100,200, two sets of light source test the speed-focus unit and two sets of image-generating units；Two sets of imagings
Streaming systems structure and working principle is identical, and often set imaging streaming systems include test the speed-focus unit and image-generating unit；
Sample feeding unit 100 ensure the testing samples such as virus, cell, microsphere or small scale mode biology it is single at high speed,
Abreast pass through image checking region.
Light source 200 includes the first light source 201, secondary light source 202 and the 3rd light source 203, and the secondary light source 202 sends side
To scattering and fluorescence excitation light source, centre wavelength 488nm, power 150mw.First light source 201 and the 3rd light source 203 are two bright
Field light source.Bright field light source is LED, power 2W, centre wavelength 830nm.Simultaneously secondary light source 202 can test the speed as two sets-it is right
The lighting source of burnt cell mesh.
The described two sets units that test the speed-focus include identical two parts, respectively as the auxiliary of two sets of image-generating units
Unit.With a set of test the speed-focus unit and a set of image-generating unit therein, (first tests the speed-focuses unit 300a present embodiment
With the first image-generating unit 400a) as a example by the specific embodiment is discussed in detail.
First test the speed-focus unit 300a include the first dichroic mirror 301a, the first spectroscope 302a, first focus on microscope group
303a and second focuses on microscope group 306a, the first grating 304a and the second grating 307a, the first photodetector 305a and the second light
Electric explorer 308a.First dichroic mirror 301a is that long wave leads to dichroic mirror, centre wavelength 488nm.First spectroscope 302a's
Centre wavelength is 488nm.First grating 304a is positive grating, and its position is after intermediate image plane.Second grating 307a is negative light
Grid, its position is before intermediate image plane.First photodetector 305a and the second photodetector 308a is two centre wavelengths
The high sensitivity photomultiplier tube of 488nm.
First image-generating unit 400a includes the first microcobjective 401a, the first multispectral light splitting microscope group 402a, the first imaging
Object lens 403a, a TDI CCD (Time Delay Integration CCD) 404a.The first microcobjective 401a conducts
Cell high-resolution imaging optical system, focal length 6mm, vertical aperture 0.5,100X200 μm of visual field.The first multispectral light splitting
Microscope group 402a is made up of the logical dichroic mirror of 6 long waves, and light splitting wave band is respectively 420-480nm, 480-560nm, 560-600nm,
600-640nm, 640-745nm, 745-800nm.
The course of work of present embodiment：Sample feeding unit 100 keep testing sample with certain stable speed it is single,
Abreast pass through image checking region.Secondary light source 202 sends the laser beam side lighting testing sample of 488nm, LASER Light Source
In 45 ° of direction irradiations.Side scattered light is through the first microcobjective 401a, the first dichroic mirror 301a and the first spectroscope
302a, first focus on microscope group 303a and second and focus on microscope group 306a and the first grating 304a and the second grating 307a, by first
Photodetector 305a and the second photodetector 308a obtain the cell movement speed needed for the first image-generating unit 400a systems
With defocusing amount information.In addition, by secondary light source 202 as details in a play not acted out on stage, but told through dialogues, fluorescence excitation light source, the 3rd light source 203 as bright field light source,
The scattering of cell and fluorescence signal are through the first microcobjective 401a, the first multispectral light splitting microscope group 402a and the first image-forming objective lens
403a, a TDI CCD 404a obtain the bright of cell, details in a play not acted out on stage, but told through dialogues and fluoroscopic image.
At the same time, second test the speed-focus unit 300b and the second one-tenth what first set image-generating unit vertical direction was placed
As unit 400b synchronously obtains the cell image of the direction, wherein secondary light source 202 is still as details in a play not acted out on stage, but told through dialogues, fluorescent exciting
Source, bright field light source of first light source 201 as the part.Synchronous exposure is obtained eventually through both direction, two sides of cell are obtained
To projected image, the 3-D view of cell is finally given.
The imaging streaming systems of the vertical direction are same by the second unit 300b and the second image-generating unit 400b that tests the speed-focus
Step ground obtains the cell image of the direction, the second image-generating unit 400b also include the second multispectral light splitting microscope group 402b, the
Two image-forming objective lens 403b and the 2nd TDI CCD 404b；Second test the speed-focus unit 300b also including the second spectroscope 302b, the
Three focus on microscope group 303b, the 3rd grating 304b, the 3rd photodetector 305b, the 4th focusing microscope group 306b, the 4th grating 307b
With the 4th photodetector 308b；
The vertical direction imaging streaming systems imaging process be：The lateral scattering that the secondary light source 202 sends
Light is divided to for two beam scattered lights, a beam spreading through the second microcobjective 401b, the second dichroic mirror 301b and the second spectroscope 302b
Penetrate light to be received by the 3rd photodetector 305b Jing after tertiary focusing microscope group 303b, the 3rd grating 304b；Another beam scattered light Jing
Received by the 4th electric explorer 308b of light after 4th focusing microscope group 306b and the 4th grating 307b, according to the 3rd photodetector
305b and the 4th photodetector 308b receives the intensity and frequency of scattered light, obtains the movement velocity and out of focus of testing sample
Amount, realizes to TDI cameras and the feedback control of microcobjective；Meanwhile, secondary light source 202 as details in a play not acted out on stage, but told through dialogues or fluorescence excitation light source,
First light source 201 as bright field light source, the scattered light and fluorescence signal of testing sample through the second microcobjective 401b, more than second
Spectrum microscope group 402b and the second image-forming objective lens 403b, the 2nd TDI CCD404b obtain the light field of testing sample, details in a play not acted out on stage, but told through dialogues and glimmering
Light image.
Present embodiment is illustrated with reference to Fig. 2, Fig. 2 is given and mutually hung down using the device acquisition cell described in present embodiment
Plane Fig. 1 and 2 of straight both direction, it can be seen that cell interior organelle a and b distribution situation in the two directions, by calculating
Method can reconstruct the spatial distribution of organelle a and b.For example, left figure provides projections of the organelle a in a direction and obtains XZ
The two-dimensional coordinate in direction, right figure provide the two-dimensional coordinate that the projection in another direction obtains YZ directions, and being finally easy to can be with
Obtain the three dimensional space coordinate of organelle a.Same obtains organelle b three dimensional space coordinates, by space therebetween
Relative position can determine the spatial distribution of organelle portion's structure in the cell.
Three-dimensional cell imaging of the present invention keeps original positional information of cell interior structure, retouches to morphologic
State verity more preferably, imaging flow cytometer can be caused to obtain more, more meaningful biomedical informations.
Claims (6)
1. three-dimensional imaging flow cytometry device, described device include sample feeding unit (100), the first light source (201), second
Light source (202) and the 3rd light source (203), is characterized in that, be entered by testing sample of two sets of imaging streaming systems synchronously to moving
The imaging of row both direction, obtains final 3-D view；
Two sets of imagings streaming systems structure is identical, and often set imaging streaming systems include test the speed-focus unit and image-generating unit；
One direction imaging streaming systems imaging process be：Sample feeding unit (100) keep testing sample it is at the uniform velocity single,
Side by side by image checking region, secondary light source (202) side lighting testing sample, side scattered light are single through the first imaging
The first microcobjective (401a) in first (400a) tests the speed-focuses unit (300a) into first, obtains the motion of testing sample
Speed and defocusing amount, realize to a TDI CCD (404a) and the feedback control of the first microcobjective (401a)；Meanwhile, second
Light source (202) as details in a play not acted out on stage, but told through dialogues or fluorescence excitation light source, the 3rd light source (203) as bright field light source, the scattered light of testing sample and
Fluorescence signal tests the speed-focuses the first dichroic mirror (301a) in unit (300a) through the first microcobjective (401a), first
Into the first image-generating unit (400a), light field, details in a play not acted out on stage, but told through dialogues and the fluoroscopic image of testing sample are obtained；
Another direction imaging streaming systems imaging process be：Secondary light source (202) side lighting testing sample, it is lateral to dissipate
Penetrate second microcobjective (401b) of the light in the second image-generating unit (400b) to test the speed-focus unit (300b) into second,
The movement velocity and defocusing amount of testing sample are obtained, is realized to the 2nd TDI CCD (404b) and the second microcobjective (401b)
Feedback control；Meanwhile, secondary light source (202) as details in a play not acted out on stage, but told through dialogues or fluorescence excitation light source, the first light source (201) as bright field light source,
The scattered light and fluorescence signal of testing sample tests the speed-focuses the in unit the 2nd 2 through the second microcobjective (401b), second
To Look mirror (301b) into the second image-generating unit (400b), light field, details in a play not acted out on stage, but told through dialogues and the fluoroscopic image of testing sample are obtained；
To testing sample, the light field of synchronization gain, details in a play not acted out on stage, but told through dialogues and fluoroscopic image are processed in the two directions, obtain 3-D view.
2. three-dimensional imaging flow cytometry device according to claim 1, it is characterised in that first image-generating unit
(400a) the first multispectral light splitting microscope group (402a), the first image-forming objective lens (403a) and a TDI CCD (404a) are also included；The
One tests the speed-focuses unit (300a) also including the first spectroscope (302a), the first focusing microscope group (303a), the first grating
(304a), the first photodetector (305a), second focus on microscope group (306a), the second grating (307a) and the second photodetector
(308a)；
The side scattered light is through the first microcobjective (401a), the first dichroic mirror (301a) and the first spectroscope (302a)
It is divided into two beam scattered lights, a branch of scattered light is Jing after first focuses on microscope group (303a), the first grating (304a) by the first photodetection
Device (305a) is received；Another beam scattered light is surveyed by the second electrical resistivity survey of light Jing after second focuses on microscope group (306a) and the second grating (307a)
Device (308a) is received, according to the first photodetector (305a) and the second photodetector (308a) receive the intensity of scattered light with
Frequency, obtains the movement velocity and defocusing amount of testing sample, realizes the feedback control to a TDI CCD (404a) and microcobjective
System；Meanwhile, secondary light source (202) treats test sample as bright field light source as details in a play not acted out on stage, but told through dialogues or fluorescence excitation light source, the 3rd light source (203)
The scattered light and fluorescence signal of product is through the first microcobjective (401a), the first multispectral light splitting microscope group (402a) and the first imaging
Object lens (403a), a TDI CCD (404a) obtain light field, details in a play not acted out on stage, but told through dialogues and the fluoroscopic image of testing sample.
3. three-dimensional imaging flow cytometry device according to claim 1, it is characterised in that second image-generating unit
(400b) the second multispectral light splitting microscope group (402b), the second image-forming objective lens (403b) and the 2nd TDI CCD (404b) are also included；The
Two test the speed-focus unit (300b) also including the second spectroscope (302b), tertiary focusing microscope group (303b), the 3rd grating
(304b), the 3rd photodetector (305b), the 4th focus on microscope group (306b), the 4th grating (307b) and the 4th photodetector
(308b)；
The side scattered light is through the second microcobjective (401b), the second dichroic mirror (301b) and the second spectroscope (302b)
It is divided into two beam scattered lights, a branch of scattered light is Jing after tertiary focusing microscope group (303b), the 3rd grating (304b) by the 3rd photodetection
Device (305b) is received；Another beam scattered light is surveyed by the 4th electrical resistivity survey of light Jing after the 4th focuses on microscope group (306b) and the 4th grating (307b)
Device (308b) is received, according to the 3rd photodetector (305b) and the 4th photodetector (308b) receive the intensity of scattered light with
Frequency, obtains the movement velocity and defocusing amount of testing sample, realizes the feedback control to the 2nd TDI CCD (404b) and microcobjective
System；Meanwhile, secondary light source (202) treats test sample as bright field light source as details in a play not acted out on stage, but told through dialogues or fluorescence excitation light source, the first light source (201)
The scattered light and fluorescence signal of product is through the second microcobjective (401b), the second multispectral light splitting microscope group (402b) and the second imaging
Object lens (403b), the 2nd TDI CCD (404b) obtain light field, details in a play not acted out on stage, but told through dialogues and the fluoroscopic image of testing sample.
4. the three-dimensional imaging flow cytometry device according to claim 1,2 or 3, it is characterised in that the secondary light source
(202) in 45 ° of direction irradiation testing samples, the secondary light source (202) sends the side scattered light or work of 488nm for laser instrument
For the LASER Light Source of fluorescence excitation.
5. three-dimensional imaging flow cytometry device according to claim 4, it is characterised in that laser light source using two to
Look mirror closes beam, carries out multi-color illumination using the LASER Light Source of multiple wavelength and excites.
6. three-dimensional imaging flow cytometry device according to claim 1, it is characterised in that first light source (201)
It is LED with the 3rd light source (203), the centre wavelength of light source is 830nm.
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| CN106769809A (en) * | 2016-12-29 | 2017-05-31 | 重庆博奥新景医学科技有限公司 | A kind of flow cytometer and its 3 D video monitoring device |
| CN107091800A (en) * | 2017-06-06 | 2017-08-25 | 深圳小孚医疗科技有限公司 | Focusing system and focus method for micro-imaging particle analysis |
| WO2019161406A1 (en) * | 2018-02-16 | 2019-08-22 | The Regents Of The University Of California | Systems, devices and methods for three-dimensional imaging of moving particles |
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| CN114088606B (en) * | 2021-10-23 | 2023-05-09 | 广州市艾贝泰生物科技有限公司 | Cell analysis device |
| CN113959947A (en) * | 2021-10-25 | 2022-01-21 | 山东大学 | Single-particle multi-modal flow imaging detection device and method based on two-dimensional light scattering |
| CN113768472B (en) * | 2021-11-10 | 2022-03-22 | 华中科技大学 | Three-dimensional image acquisition device with fluorescent marker and method |
| CN114993897B (en) * | 2022-07-18 | 2022-11-18 | 广东省麦思科学仪器创新研究院 | Aerosol particle beam width and particle distribution detection device, set and method |
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