CN104266955A - High content image flow biological microscopic analysis system - Google Patents
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- CN104266955A CN104266955A CN201410443148.2A CN201410443148A CN104266955A CN 104266955 A CN104266955 A CN 104266955A CN 201410443148 A CN201410443148 A CN 201410443148A CN 104266955 A CN104266955 A CN 104266955A
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Abstract
The invention provides a high content image flow biological microscopic analysis system. The system comprises a liquid flow system and an optical system, wherein the liquid flow system enables cells in sample cell suspension to be focused in the center of a liquid flow under the constraint of the system sheath liquid flow and to flow through a detection window of a flow chamber one by one; the optical system comprises optical sources, an optical path system and an optical filter stack; the optical sources include a halogen lamp and a laser; through the optical path system and the optical filter stack formed by a plurality of dichroscopes, the optical system generates a bright field cell image, a six-channel fluorescent cell image and six PMT (photomultiplier tube) optical intensity signals with different wave bands. The high content image flow biological microscopic analysis system integrates flow cytometry and fluorescence microscopic imaging, has a plurality of detection channels, can collect the cell images of the cells passing through the flow chamber one by one and can carry out quantitative analysis on each cell image by utilizing analysis software, thus providing the statistical data of a cell group and the information of the cell morphology, cell structures and subcellular signal distribution.
Description
Technical field
The present invention relates to flow cytometry, particularly, relate to high intension image streaming biology microscope analytic system.
Background technology
Flow cytometry is the technology that a kind of cell to defiled in liquid stream or other biological particle (as microballoon, bacterium, small scale mode biology etc.) carry out fast quantitative analysis and sorting one by one.Use traditional FCM analysis technology, researchist can analyze thousands of cells, obtains the scattered light signal of each cell and the numerical value of fluorescence signal, thus obtains the various statisticss of cell colony, and can find rare cell subsets.
But conventional flow Cell Measurement Technique still exists limitation, it is very limited that is exactly the cellular informatics obtained.Cell is concerning researchist, and a point just on scatter diagram, instead of real cell image, lack the relevant information of cytomorphology, eucaryotic cell structure and subcellsular level signal distributions.Want to obtain cell image, researchist just must use microscope to observe, but the cell quantity that microscope can be observed is very limited, is difficult to the quantification and the statistics that provide cell colony.Therefore, use traditional cell analysis technology, we just in the face of such dilemma, can only not have a kind of technology both can provide the statistics of cell colony, obtain cell image again.
Summary of the invention
For the defect that flow cytometer single in currently available technology exists, the object of the invention is design rational optical imaging system, wherein, can high speed acquisition be the emphasis of design through the individual cells light field of sheath liquid chamber and fluoroscopic image, bright field image so both can have been utilized to carry out the morphological feature of observation of cell, fluorescence can be utilized again to measure the key component in cell, realize the combination of flow cytometry and micro-imaging.
According to one provided by the invention high intension image streaming biology microscope analytic system, comprising: liquid fluid system, optical system;
Liquid fluid system comprises flow chamber, and flow chamber for receiving sample cell suspension and the system sheath fluid of injection, and makes the cell in sample cell suspension under the constraint of system sheath fluid stream, focus on the center of liquid stream, flows through the detection window of flow chamber one by one;
Optical system comprises light source, light path system, optical filter storehouse;
Described light source, for irradiating the cell by detection window, thus produces light signal; Light source comprises for generation of the Halogen lamp LED of light field cell image, the laser instrument for generation of fluorecyte image;
The light signal that light source irradiation cell produces is collected by object lens, forward light signal, lateral light signal is collected respectively in two TDI cameras by two groups of wedge shape gratings, then be delivered to the optical filter storehouse formed primarily of multiple dichroic mirror by light path system, light signal is divided into different-waveband and projects on a Hexamermis spp PMT array at optical filter storehouse place; And then described optical system produces a light field cell image, the PMT light intensity signal of a Hexamermis spp fluorecyte image and six different-wavebands.
Preferably, also comprise laser light compensating apparatus, wherein, described laser light compensating apparatus is used for by adjusting light intensity at cell side direction light filling, reduce the time of TDI capture delay integration, the too short defect causing light intensity on the weak side integral time that the flow at high speed reducing cell is brought.
Preferably, also comprise automatic focusing mechanism, wherein, described automatic focusing mechanism is used for utilizing laser spot method, is calculated the focal position of cell by the edge and size measuring disc of confusion.
Preferably, also comprise velocity measuring device, wherein, described velocity measuring device to be fixed time the move distance of inner cell for utilizing laser ranging to carry out determination limit, converts out the movement rate of cell.Described TDI camera is captured in the single celled image in flow chamber according to the movement rate of cell.
Preferably, also comprise analytic system, wherein, described analytic system adopts the image registration mode of fluorescence and light field, correct the imaging deviation of fluorescence and light field, spot center is overlapped, and carrys out the fluorescence intensity of correcting image by multispectral PMT light signal and multispectral fluoroscopic image, the fluorescence intensity of output and PMT are consistent.
Preferably, described analytic system regulates the segmentation threshold of fluoroscopic image by PMT light signal, and determines the abnormal cell in a large amount of cell by the analysis of image micromechanism.
Preferably, the Numerical Aperture NA of described object lens is greater than 0.75.
Compared with prior art, the present invention has following beneficial effect:
1, the present invention can provide the statistics of cell mass, can obtain again the image of individual cells, thus provides the information of cytomorphology, eucaryotic cell structure and subcellular fraction signal distributions.
2, flow cytometry and fluorescent microscopic imaging are incorporated into all over the body by the present invention, it has multiple sense channel, the cell image that can gather one by one the cell by flow chamber, and utilize analysis software to carry out quantitative analysis to each cell image, thus provide the statistics of cell mass and the information of cytomorphology, eucaryotic cell structure and subcellular fraction signal distributions.
3, the present invention may be used for cell signalling/path analysis, cell-cell interaction, molecule and locates altogether and molecular transport, cytomorphology, fluorescence in situ hybridization, gene expression analysis, Apoptosis, cell cycle analysis, WBC sub-population analysis in born of the same parents.
Accompanying drawing explanation
By reading the detailed description done non-limiting example with reference to the following drawings, other features, objects and advantages of the present invention will become more obvious:
Fig. 1 is light path design principle schematic of the present invention;
Fig. 2 is liquid fluid system structural representation of the present invention.
In figure:
1 is multi-channel laser bundle;
2 is cell focusing device;
3 is condenser;
4 is spectroscope;
5 is optical filter;
6 is PMT;
7 is wedge shape grating;
8 is TDI camera;
9 is a point CD;
10 is two look reflective mirrors.
Embodiment
Below in conjunction with specific embodiment, the present invention is described in detail.Following examples will contribute to those skilled in the art and understand the present invention further, but not limit the present invention in any form.It should be pointed out that to those skilled in the art, without departing from the inventive concept of the premise, some distortion and improvement can also be made.These all belong to protection scope of the present invention.
According to high intension image streaming biology microscope analytic system provided by the invention, primarily of a few major part composition such as liquid fluid system, optical system, electronic system, flow chamber.
Liquid fluid system comprises flow chamber, and sample cell suspension and system sheath fluid are injected flow chamber by liquid fluid system, and flow chamber, for the center making cell focus on liquid stream under the constraint of sheath fluid stream, flows through the detection window of flow chamber one by one.Flow chamber has sorting function, and when setting the characterisitic parameter being sorted cell in experimental design, this type of cell can be charged when forming drop, makes it with positive charge or negative charge, is not set the cell of sorting parameter and blank drop neutral.Charged drop is falling into the high-voltage electrostatic field of electrode deflection plate, and electrically charged according to institute is just or negative and occur to the right or road deflection left, to fall into the gatherer of specifying, complete the object of cell sorting.
Optical system comprises light source, light path system, optical filter storehouse.
Light source irradiation by the cell of detection window, thus produces light signal; Light source is divided into two kinds, and a kind of is Halogen lamp LED for generation of light field cell image, and another kind is the laser instrument for generation of fluorecyte image.
The light signal that light source irradiation cell produces is collected by the object lens with very large-numerical aperture (NA:0.75), forward light signals and lateral light signal is collected in two TDI cameras by two groups of wedge shape gratings, then the optical filter storehouse formed primarily of dichroic mirror is delivered to by light path system, light signal is divided into different-waveband here and projects on a Hexamermis spp PMT array, whole imaging system produces a light field cell image, the PMT light intensity signal of a Hexamermis spp fluorecyte image and six different-wavebands.This light path system can automatic focus adjustable, and the real time measure cell movement speed, and cold CCD adopts time delay integration mode to carry out signals collecting, these means ensure that the quality of the cell image that system acquisition arrives.
Further, the innovative point of high intension image streaming biology microscope analytic system is light path design, combines the advantage of flow cytometer and micro-imaging, has fully merged both advantages.In system, be provided with spectroscope, forward scattering light and side scattered light are divided into two, lead up to photomultiplier (PMT) collection signal, wedge shape grating of separately leading up to decomposes, and utilizes time delay integration CCD (TDI) to gather picture signal.Achieve the combination of both macro and micro.
This system innovation devise laser light compensating apparatus, automatic focusing mechanism and velocity measuring device.
Laser light compensating apparatus is that fluorescence excitation is too weak causes imaging unintelligible in order to solve, and by adjusting light intensity at cell side direction light filling, reduces the time postponing integration, the too short defect causing light intensity on the weak side integral time that the flow at high speed reducing cell is brought.Whole light-supplementing system, when using for the first time, needs the Integral correction doing system, in limiting time, makes the intensity of light source of the different-waveband selected consistent.
What automatic focusing mechanism solved is in the flow at high speed of cell, selects best aligned position to trigger and takes pictures, herein be laser spot method, calculate best focal position by the edge and size measuring disc of confusion.
The laser ranging that velocity measuring device utilizes carrys out determination limit and to fix time the move distance of inner cell, converts out the movement rate of cell.Coordinate TDI lagged product split-phase motor, the single celled image in flow chamber can be captured in accurately.
Further, the a set of powerful analysis software of high intension image streaming biology microscope Analytical System Design, this analysis software adopts the image registration techniques of fluorescence and light field, correct the imaging deviation of fluorescence and light field, spot center is overlapped, carry out the fluorescence intensity of correcting image additionally by multispectral PMT light signal and multispectral fluoroscopic image, the fluorescence intensity of output and PMT are consistent.This software adopts fluoroscopic image and PMT light signal convergence analysis method, the segmentation threshold of fluoroscopic image is regulated by PMT light signal, the generation result of fluoroscopic image and the result of flow cytometer are kept consistency, and the abnormal cell in a large amount of cell can be determined by the analysis of image micromechanism.This software can to each cell analysis more than 500 kinds of quantization parameters.These parameters not only comprise scattered light and the fluorescence signal intensity of cells intact, also comprise cellular morphology, the analysis of eucaryotic cell structure and subcellular fraction signal distributions.By adding up these parameters in cell colony, analysis software can the scatter diagram of cellulation colony and histogram, and these statisticss and cell image are fully integrated, such as click the point on scatter diagram, just can see the image of the cell of this some representative intuitively.In addition, user according to the special requirement of self research, can also carry out the setting of custom parameter, carries out more deep analysis.System can generate the vision-mix of hyperchannel fluorescence, also can generate the independent image of single channel fluorescence, and the fluorescence channel needed for can being selected by software.
Whole system may be used for cell signalling/path analysis, cell-cell interaction, molecule and locates altogether and molecular transport, cytomorphology, fluorescence in situ hybridization, gene expression analysis, Apoptosis, cell cycle analysis, WBC sub-population analysis in born of the same parents.
Above specific embodiments of the invention are described.It is to be appreciated that the present invention is not limited to above-mentioned particular implementation, those skilled in the art can make various distortion or amendment within the scope of the claims, and this does not affect flesh and blood of the present invention.
Claims (7)
1. a high intension image streaming biology microscope analytic system, is characterized in that, comprising: liquid fluid system, optical system;
Liquid fluid system comprises flow chamber, and flow chamber for receiving sample cell suspension and the system sheath fluid of injection, and makes the cell in sample cell suspension under the constraint of system sheath fluid stream, focus on the center of liquid stream, flows through the detection window of flow chamber one by one;
Optical system comprises light source, light path system, optical filter storehouse;
Described light source, for irradiating the cell by detection window, thus produces light signal; Light source comprises for generation of the Halogen lamp LED of light field cell image, the laser instrument for generation of fluorecyte image;
The light signal that light source irradiation cell produces is collected by object lens, forward light signal, lateral light signal is collected respectively in two TDI cameras by two groups of wedge shape gratings, then be delivered to the optical filter storehouse formed primarily of multiple dichroic mirror by light path system, light signal is divided into different-waveband and projects on a Hexamermis spp PMT array at optical filter storehouse place; And then described optical system produces a light field cell image, the PMT light intensity signal of a Hexamermis spp fluorecyte image and six different-wavebands.
2. high intension image streaming biology microscope analytic system according to claim 1, it is characterized in that, also comprise laser light compensating apparatus, wherein, described laser light compensating apparatus is used for by adjusting light intensity at cell side direction light filling, reduce the time of TDI capture delay integration, the too short defect causing light intensity on the weak side integral time that the flow at high speed reducing cell is brought.
3. high intension image streaming biology microscope analytic system according to claim 1, it is characterized in that, also comprise automatic focusing mechanism, wherein, described automatic focusing mechanism is used for utilizing laser spot method, is calculated the focal position of cell by the edge and size measuring disc of confusion.
4. high intension image streaming biology microscope analytic system according to claim 1, it is characterized in that, also comprise velocity measuring device, wherein, described velocity measuring device to be fixed time the move distance of inner cell for utilizing laser ranging to carry out determination limit, convert out the movement rate of cell, described TDI camera is captured in the single celled image in flow chamber according to the movement rate of cell.
5. high intension image streaming biology microscope analytic system according to claim 1, it is characterized in that, also comprise analytic system, wherein, described analytic system adopts the image registration mode of fluorescence and light field, corrects the imaging deviation of fluorescence and light field, spot center is overlapped, and carried out the fluorescence intensity of correcting image by multispectral PMT light signal and multispectral fluoroscopic image, the fluorescence intensity of output and PMT are consistent.
6. high intension image streaming biology microscope analytic system according to claim 5, it is characterized in that, described analytic system regulates the segmentation threshold of fluoroscopic image by PMT light signal, and determines the abnormal cell in a large amount of cell by the analysis of image micromechanism.
7. high intension image streaming biology microscope analytic system according to claim 1, it is characterized in that, the Numerical Aperture NA of described object lens is greater than 0.75.
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| CN105928913A (en) * | 2016-04-14 | 2016-09-07 | 天津中医药大学 | Cell phenotype based high-content multi-index renal toxicity detection method and applications thereof |
| CN106383082A (en) * | 2015-10-14 | 2017-02-08 | 北京信息科技大学 | Optical path regulating device of flow cytometry under liquid-path-free condition and method |
| 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 |
| CN107995950A (en) * | 2015-08-12 | 2018-05-04 | 生物辐射实验室股份有限公司 | Multispectral wave filter for flow cytometry dissects and quality control |
| CN108475429A (en) * | 2016-01-21 | 2018-08-31 | 分子装置有限公司 | The system and method for the segmentation of three-dimensional MIcrosope image |
| CN109556999A (en) * | 2018-12-13 | 2019-04-02 | 中南大学湘雅医院 | A kind of application method based on high intension technology in poikilocyte detection |
| JP2019525199A (en) * | 2016-06-10 | 2019-09-05 | アムニス・コーポレーションＡｍｎｉｓ Ｃｏｒｐｏｒａｔｉｏｎ | Brightfield and fluorescence channel integration method for cell image segmentation and morphological analysis using images acquired from an imaging flow cytometer (IFC) |
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| CN105928913A (en) * | 2016-04-14 | 2016-09-07 | 天津中医药大学 | Cell phenotype based high-content multi-index renal toxicity detection method and applications thereof |
| US10846509B2 (en) | 2016-06-10 | 2020-11-24 | Luminex Corporation | Method to combine brightfield and fluorescent channels for cell image segmentation and morphological analysis using images obtained from imaging flow cytometer (IFC) |
| JP2019525199A (en) * | 2016-06-10 | 2019-09-05 | アムニス・コーポレーションＡｍｎｉｓ Ｃｏｒｐｏｒａｔｉｏｎ | Brightfield and fluorescence channel integration method for cell image segmentation and morphological analysis using images acquired from an imaging flow cytometer (IFC) |
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Application publication date: 20150107 |