CN102998239A - Flow cytometry device for no-flow type cytometry box and flow cytometry method thereof - Google Patents
Flow cytometry device for no-flow type cytometry box and flow cytometry method thereof Download PDFInfo
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Abstract
The invention discloses a flow cytometry device for a no-flow type cytometry box and a flow cytometry method for the no-flow type cytometry box. The device adopts a unique kurt microporous direct coaxial illumination method, so that the necessary expensive flow cytometry box and the complex liquid focusing in the existing mainstream flow cytometry can be thoroughly abandoned, the structure can be simplified, the cost can be reduced, the interaction time between the cell and the excited laser can be prolonged by tens of times, the sensitivity of the apparatus can be improved by orders of magnitude, the backward scattering light which is most sensitive to the structures and the components in the cells can be firstly and successfully applied to the actual commercial flow cytometry due to the unique structure, and the identifying capability of the apparatus to the different cell subsets can be improved. A multi-wavelength excited laser combiner and a multi-wavelength fluorescent detection system of a new device all adopt a single dispersing element, so that the loss of the apparatus can be reduced to the lowest level, and the overlap of the wavelengths can be reduced. Compared with the flow type cytometry which is universally used in the world, the flow cytometry device is obvious in cost performance and market advantage.
Description
Technical field:
The present invention relates to a kind of medical detecting Instrument class flow cytometer apparatus and method, particularly a kind of flow cytometer apparatus and method without the fluidic cell box.
Background technology:
1, flow cytometry (Flow Cytometry, FCM) and flow cytometer
Flow cytometry (FCM) be the fields such as microscopy, dyeing chemistry, electronics technology and computing machine combine and progressive combination under grown up to being in single celled characteristic and the composition thereof in the quick streamlined flow, or other various molecules (such as bacterium) and loaded article thereof the technology of carrying out multi parameter analysis and sorting, it not only can measure the proterties of cell size, internal particle, also can detect DNA, rna content etc. in cell surface and cytoplasmic antigen, the cell, can analyze in unicellular level colony's cell.Detect at short notice and analyze a large amount of cells, and collection, storage and deal with data, carry out the multiparameter quantitative test; Can a certain cell subset of categorised collection (sorting).In the subjects such as hematology, immunology, oncology, materia medica, Genetics Clinic ecsomatics, molecular biology, cyto-dynamics, environmental microorganism analysis, have a wide range of applications.
Flow cytometer is the novel high-tech instrument that the subjects such as collector mechanics, optics and laser technology, electronic engineering, bioinformatics, biophysics, fluorescence chemical, labelling technique, computing machine are integrated; Be used for cell or biological particle that high-speed straight-line flows are carried out quantitative determination, analysis and sorting.Nearly all can the detection with flow cytometer by the composition of fluorochrome label or certain variation in the cell.Be characterized in that detection speed is fast, measurement parameter is many, the Information Monitoring amount large, it is comprehensive to analyze, method is flexible, can also sub-elect the specific cells group and further investigate.
2, flow cytometer Development History
Structure since nineteen thirty Sweden scientist Caspersson and Thorell usefulness microscopic study staining cell, to the whole process of the various flow cytometer of day by day perfect large-scale analytic type and desk-top clinical type now, be full of for more than half a century countless studying intensively and arduous work.In 1934, Canadians Andrew, Moldavan first proposed the suspension of a single red blood cells flowing through glass capillaries, etc., under the bright field microscope counting and measuring with optical recording device as envisaged in the static microscope (static, Microscopy) Conversion into the flow system (flowing, system), since then, taken from microscope quiescent cells first step towards the development of flow cytometry.1936, Caspersson etc. introduced microphotometry.1940, American Albert Coons proposed to remove the interior specific protein of labeled cell with the antibody of combined with fluorescent element.1949, American Walance H.Coulter has submitted patent of invention to, and obtained this patent U.S.Pat.No.2656508, " MEANS FOR COUNTING PARTICLES SUSPENDED IN FLUID " (" particle counting method ") that floats on a liquid in nineteen fifty-three.1958, Walance H.Coulter and his younger brother Joseph Coulter, Jr. has created jointly " Coulter Electronics,c. ", start selling the Ku Erte blood-counter system.From then on blood count and analysis automated new era have been opened.Nineteen fifty-nine, Type B Coulter Counter comes out, it is exactly the predecessor of streaming Fine born of the same parents instrument today, and it possesses all characteristics of streaming Fine born of the same parents instrument because of As: can make one by one Kuai Su Tong Over hole of Single one Fine born of the same parents, can survey Fine born of the same parents and have the robotization of signal analysis with electronic signal Come Surveillance.Nineteen fifty-three Crosland-Taylor is according to the research of Reynolds to Newtonian fluid flowing law in round tube, designed a fluid system: make cell suspending liquid to be analyzed all be collected at the pipe near axis and flow through its skin and then be wrapped in sheath fluid, cell suspension and sheath fluid are all keeping high speed laminar flow motion state, Fine born of the same parents can be flowed one by one at the central Inner of fluid column, and principle design has gone out red blood cell optics auto-counter, the basis of having established Fluid focus technology in the modern flow cytometry according to this.Nineteen sixty-five, Kamemtsky etc. propose two imaginations, namely with the quantitative cell component of spectrophotometer with in conjunction with measured value cell are classified.1967, Kamemtsky and Melamed proposed the method for cell sorting on the method basis of Moldaven.Fulwyler Knot has closed the Spray China ink technology (ink jet technology) of Coulter technology and the use of RG Sweet Give computer Spray Spray China ink printer and has developed the streaming Fine born of the same parents screening instrument that First Ju You Sieve screens (sorting) function, i.e. the predecessor of present cellsorter.The ultimate principle of Spray China ink technology namely is to utilize the Spray mouth of high-frequency Zhen Swing (vibration of nozzle), fluid column (stream) is split into drop (drop) And make electric charge on the drop band of planning to screen, the recycling high-voltage electric field makes drop produce deviation and is imported in the collection tube.Hou Come Fulwyler is Jia Yi Gai Jin again, according to the Coulter apparatus measures to electronic signal ' coulter volume ’ Come Decision is Dinged Give and is given the corresponding electric charge of drop band that contains specific Fine born of the same parents' size, then utilize electric field to make it deviation, so can isolate specific Fine born of the same parents' kind.1969, Van Dilla invention First fluoroscopic examination cytometer.The technology that the Xian Wei Mirror lamp source Yi Ji Hair fluorescent material And Knot that this instrument utilizes argon laser to replace Chuan System closes hydrodynamic focusing and Fulwyler ' s sorter, the predecessor who has developed streaming Fine born of the same parents instrument FACS today (fluorescence activated cell sorter).Soon, team development with the Herzenberg of Stanford university professor As head goes out similar instrument, Hai Knot closes the function of multi-parameter fluorescence, light scatter, coulter volume and cell sorting except These characteristics, has established the framework of the FACS that widely uses now.The manufacturer of the current large FACS cytometer of Two, the Coulter company (Beckman Coulter) of the beautiful Country East of total position what bank, the Becton Dickinson (BD) of west bank, namely be combined by Coulter Technology and with the technology of Stanford university research respectively derive Come's.
The two laser four look fluorescence flow cytometers of the 1980's First and First commercialization sorting type flow cytometer come out.2000 high-precision digital color compensating technology ADC (Advanced Digital Compensation) be applied to the streaming platform.Single laser five colors flow cytometer FC500/MCL and have the EV parameter, the flow cytometer Quanta SC that carries out absolute counting and Accurate Measurement cell volume simultaneously came out one after another later in 2003.Beckman Coulter Inc. introduces flow cytometer with the theory of clinical labororatory's automation equipment and has released flow cytometer auto injection (multiple Plate Loading, MPL), 24 holes, the common and deep-well plates in 96 holes have been realized, 24 and 40 test tube auto injections.Moflo and Cyan that Dako company creates, energy high speed, Accurate Analysis and sorting target protein and cell.The analytic type flow cytometer Gallios/Navios of First three laser ten look fluorescence in 2009 comes out, and there has been again the Gallios with temperature control and long-distance service system next year.The high speed sorting type flow cytometer FACSAria III that BD releases, the excitation laser of 6 different wave lengths of use can be surveyed 18 look fluorescence simultaneously.
3, Coulter principle (Coulter Principle)
＜1 〉(referring to Fig. 1)
Micropore pond (1-1) in container is divided into two parts by microporous pipe (1-2), and two parts are only by a micropore (1-5) binding that communicates.Two electrodes (1-6), (1-7) place respectively two parts of container, so the micropore electric current just forms (1-8) in path.A particle in being suspended in weak electrolyte solution when passing through micropore such as haemocyte (1-4), because the impedance of the impedance ratio electrolyte solution of particle is large, just has an instantaneous impedance to increase at micropore (1-5) two ends.The zone that the micropore impedance changes has formed one " sensitizing range ".The instantaneous variation of impedance produces an electric pulse, is commonly called direct current (Direct Current) pulse, is called for short the DC pulse.The amplitude of this DC pulse is directly proportional with the volume of particle by micropore (1-5).Here it is Coulter principle.Utilize the Coulter principle just can be to count and analyze the size of particle by the particle of micropore, this method be also referred to as dc impedance method or Ku Erte impedance method.Coulter principle can be analyzed various particle, be not only applicable to haemocyte, also can be used for other cells, animal blood cell, other fine particles, there are granularity and inside particles composition that each industrial circle of requirement is arranged to material, such as eating crystalline substance, pharmacy, chemical industry, petrochemical industry etc., equally also be used for detecting the purity of fuel in the aerospace industry.
Coulter principle and Ku Erte impedance method are the most classical principle and the methods of blood count and analysis, use till today from invention always, be that any blood analyser and stream make the requisite ingredient of cell instrument, and its principle and method there is no the change what is basic in decades.
4, blood constituent
＜2 〉(referring to Fig. 2)
Blood is comprised of blood plasma (2-1) (～58%) and tangible cell component (2-2) (～42%).Mainly comprise red blood cell (2-4) (1,000,000 of every microlitre 4-5 tangible being divided into, RBC is the abbreviation of red blood cell Red Blood Cell), leucocyte (2-5) (every microlitre 5～9,000, WBC is the abbreviation of leucocyte White Blood Cell) and blood platelet (2-3) (ten thousand of every microlitre 20-40, PLT are the abbreviation of its Platelet) three major types.
Leucocyte (2-5) can rough segmentation be granulocyte (Granulocyte), lymphocyte (2-9) (Lymphocytes) and monocyte (2-10) (Monocyte).Granulocyte comprises basophil (2-8) (Basophil), eosinophilic granulocyte (2-7) (Eosinophil) and neutrophil leucocyte (2-6) (Neutrophil).So leucocyte can be subdivided into five subcellular fraction groups, i.e. lymphocyte (2-9), monocyte (2-10), neutrophil cell (2-6), acidophil (2-7), basocyte (2-8).
5, three classification and five classification cellanalyzers
Because Coulter principle is the haemocyte that the amplitude of the DC pulse that the variation of electrical impedance produces during by micropore with particle is differentiated different volumes, and the neutrality in the leucocyte, have a liking for acid, three kinds of cells of basophilic volume very approaching, so the cellanalyzer of only making with Coulter principle can only be divided three classes leucocyte, so be called three classifying apparatus, that is: neutrophil leucocyte (GRN), lymphocyte (LYM), and cell,intermediate mass (MID comprises basophil, eosinophilic granulocyte, neutrophil leucocyte).Can differentiate that five kinds of components distinguishing in the leucocyte (be lymph, monokaryon, neutrality, have a liking for acid, basocyte) and the cellanalyzer of differential count are exactly usually five alleged classification cellanalyzers.In order to reach the purpose of five classification, the nineties, the methods such as laser light scattering, radio frequency, chemical staining counting are applied to the analysis to cell characteristics in succession, differential hematology analyzer occurred.
6, based on the laser light scattering method of flow cytometer principle
For utilizing laser light scattering, begin to occur the light scattering cellanalyzer based on the flow cytometer principle nineties, its ultimate principle can illustrate with Fig. 3.The core is the capsule that liquid and particle passage are provided that is commonly referred to as fluidic cell box (3-1) (Flow Cell), as shown in Figure 4.The quartzy rectangular cylinder of about 4 millimeters x4 millimeter x8 millimeters along major axis at the center someways (such as laser) get out the straight-through micropore of 50 microns of diameters, then grind two circular cones (4-3) of relative aligning from the two ends of clear opening, until leave about 70 microns long micropores at the center of square body, be called micro through hole (road) (4-4) (Orifice), Here it is fluidic cell box.Laser beam (4-1) injects and focuses on the center of micro through hole (4-4) from horizontal direction, when particle vertically passes through the center of micro through hole (4-4), will and laser interaction, produce forward direction, light scattering (the Light Scattering of side direction and all directions such as backward, be abbreviated as LS), be forward light scattering (forward light scatter, be abbreviated as FLS), side direction or 90 ° of light scattering (side light scatter, be abbreviated as SLS), and backward scattered light (back light scatter is abbreviated as BLS), and fluorescence signal.Side direction or 90 ° of light scattering can be detected by 90 ° of light scattering or fluorescence detection device (3-9), and (referring to Fig. 3) can be detected to light scattering and axial light loss pick-up unit (3-10) by front and back to light scattering and axial light loss in front and back.(4-2) expression sheath cover a fluid stream, (4-5) expression sample (particle) a fluid stream.
In instrument shown in Figure 3, lighting system is that the light beam relay direction (being horizontal direction among the figure) of LASER Light Source (3-7) is mutually orthogonal with the flow direction (being vertical direction among the figure) of particle, here we are referred to as the vertical illumination mode, in order to distinguished with the coaxial LS of being abbreviated as lighting system of the present invention.All light scattering cellanalyzers based on the flow cytometer principle all are with this vertical illumination mode in the world now.In order to make one by one in succession the center of passing through micro through hole of particle, except the solution concentration of suitable dilute suspension particle, also need to surround sample (particle) a fluid stream that contains tested particle with sheath a fluid stream fluid, be commonly referred to as fluid focus method (Hydrodynamic Focusing).In two circular cones an electrode (3-3) is being installed respectively up and down, when particle passes through little (leading to) hole (3-2), the variation that will produce aforesaid micropore electric current (3-4), i.e. Ku Erte DC pulse (DC).Like this, when each particle passes through micro through hole (3-2), just can obtain simultaneously several signals, such as DC, FLS, SLS, BLS and fluorescence signal.As adding high frequency electric source at two above-mentioned electrodes, just can obtain a signal simultaneously, this also is another invention of great significance of Mr. Ku Erte more, and (US Patent 3502974) is called radio frequency method, referred to as (RF), i.e. the abbreviation of Radio Frequency.Among Fig. 3, (3-1) be the fluidic cell box; (3-5) be sheath cover a fluid stream; (3-6) be sample (particle) a fluid stream; (3-8) be the expression condenser lens; (3-9) 90 ° of light scattering or fluorescence detection device; (3-10) be forward light scattering and axial light loss pick-up unit.
Inside particles constituent and structure sensitive method of testing there are 90 degree light scattering (SLS), radio frequency method (RF), fluorescence dye, backward scattered light (BLS) etc.Wherein responsive with backward light scattering again, just because technical bottleneck does not also solve at present, high-end blood analysis instrument and the flow cytometer on market so far do not have a backward scattered light of surveying.They have only utilized other several method, such as the VCS technology such as Coulter MAXM, STKS, i.e. and Volume (DC), Conductivity (RF/DC), and Scattering (FLS).Sysmex SE-9000, XE-2100 etc. have then used DC, LS and fluorescence dye, ABBOTT that the patent of 90 degree light scattering is then arranged, and its instrument is generally used DC, FLS and SLS.
Flow cytometer without the fluidic cell box of the present invention is then used the characteristic fluorescence of DC, FLS and BLS and mark fluorescent element, is for the first time backward scattered light to be applied in the business-like practical flow cytometer in the world so far.
7, backward scattered light
In laser light scattering particle analysis technology, with respect to the scattering of other direction, back scattering has higher susceptibility to the inner structure of particle.1979, Kerker M,
＜3 〉Deng pointing out to only have backward scattered light that cell interior form and structure are had susceptibility most, nineteen eighty-two Kerker is from further pointing out the review of elastic light scattering theoretical foundation
＜4 〉, the light scattering signal from backward that the shape of cells involved and the information of inner structure can be best obtains.1986, Sloot and Figdor
＜5 〉, in its theory essay, point out, for different cell type in the optimized detection nucleated blood cell mixed in together, need to survey simultaneously forward direction, side direction and backward scattered light.Calculating in the literary composition shows that the back scattering light intensity depends on that nucleus is to the variation of cytoplasmic ratio and nucleus and cytoplasmic optical density (OD).Analysis lay special stress in the literary composition the directly related property between back scattering light intensity and the nuclear transparency.Dakota Watson in 2004
＜6 〉Deng also the light scattering intensity of all directions and the relation of cellular morphology are done more detailed discussion, and done corresponding experiment.On the basis of above these work, some are also arranged in recent years about the United States Patent (USP) of backward scattered light, such as US Patent 6743634 B2 (Kramer, June 1,2004) and US Patent 6869569B2 (Kramer, March 22,2005).The method that these patents are given all is the detection of making the back scattering light signal with multifiber and a plurality of photomultiplier, its structure and cost all with the commercial instrument of practicality require far apartly, can only in the laboratory, pursue fundamental studies.
8, existing main flow flow cytometer structure
Fig. 5 is the index path of BD FACSVantage SE sorting type flow cytometer.(5-1) be the fluidic cell box, minute to select the sorting collection systems such as deflecting plate and collection tube (5=2), (5-3) be the fluid monitor video camera, (5-4) be the micro objective of collecting fluorescence and Side direction light scattering, (5-5) be three laser instruments, (5-6) be 8 optical prisms that change optical path direction, (5-7) be the photodiode that receives forward scattering light, (5-8) be the photomultiplier that receives side scattered light, be 8 photomultipliers that receive fluorescence signal to (5-9-8) (5-9-1), (5-10) be remaining spectroscope, optical filter, lens etc.Can notice, the fluorescence signal of different wave length is blended in the same light path out the time from the micro objective (5-4) of collecting fluorescence and Side direction light scattering, the all fluorescence signals that will tell single wavelength, just must use at least one spectroscope, optical filter (sometimes also will with a plurality of) just has totally 10 these class eyeglass, not only complex structures among Fig. 5, light path is difficult transfers, and loss is large.Device of the present invention is only used single minute dispersive optical element, has unrivaled superior in structure, loss and performance.
Summary of the invention
The present invention be I another invention Chinese patent " the coaxial means of illumination of Ku Erte micropore and analytical instrument thereof in the global function blood analysis instrument "
＜7 〉And the U.S. " Coaxial Illumination Of Coulter Aperture In Full Function Hematology Analyzer "
＜8 〉Extension and expansion.The present invention is applied in the flow cytometer the coaxial quadrature of Ku Erte micropore is bright, can with in the existing main flow flow cytometer that accounts for flow cytometer market leading position in the world fluidic cell box and the complicated fluid focus system of requisite costliness cast off once and for all, make to the responsive especially backward scattered light of inside particles constituent and structure survey and the detection first Application of backward fluorescence signal in commercial flow cytometer, thereby improved greatly the distinguishing ability of instrument.Because the application of this unique lighting system, make the sensitivity of instrument improve one more than the order of magnitude, make instrument of the present invention become a high sensitivity, fine resolution, low-cost, simple in structure, the practical flow cytometer of high performance-price ratio easy and simple to handle, not only can break Chinese flow cytometer market by foreign company's monopolization passive situation of decades, and because its low price, present domestic flow cytometer can be mainly used in scientific research and sphere of learning, the clinical examination that is generalized to hospital is used, greatly improve the clinical examination ability of general hospital, and opened up the new prospect in flow cytometer market.
The present invention lighting system, namely to the directly coaxial parallel illumination of the Ku Erte micropore of classics, the meaning of coaxial parallel illumination is exactly that the optical axis of the direction of beam propagation overlaps the illumination of (coaxial) with Ku Erte micropore axle.The employing of this lighting system, can thoroughly abandon in the existing flow cytometer fluidic cell box and the complicated fluid focus system of requisite costliness, make inside particles constituent and the responsive especially backward scattered light of structure are surveyed first Application in commercial flow cytometer, thereby improved greatly the distinguishing ability of instrument.
Fluidic cell instrument apparatus of the present invention, the signal that can survey comprises: Ku Erte dc pulse signal (DC), forward light scattering signal (FLS) and rear light scattering signal (BLS), forward and backward fluorescence signal.For the first time backward scattered light and backward fluorescence signal to be used for business-like practical flow cytometer in the world so far.
To achieve these goals, the present invention is applied to the coaxial parallel means of illumination of Ku Erte micropore in the flow cytometer first, it makes light scattering method and fluorescent penetrant method can directly apply to traditional Ku Erte micropore, it is characterized in that, with the optical axis of the illuminating bundle direction of propagation and the axis coaxle of Ku Erte micropore.
In addition, for the micropore pond (Fig. 1) that the makes Coulter principle Ku Erte micropore (Fig. 1) in (.1-1) (.1-5) can become the part of the light path of excitation laser light beam, scattered light signal, fluorescence signal, the front and back of the relative Ku Erte micropore in micropore pond of particular design have two optical windows.
Two optical windows of the front and back of the relative micropore in further described micropore pond, each described optical window can be an optical flat, a pair of lens, fibre bundle, photoconductive tube (light guide) etc. or other optical element.
The present invention also provides a kind of fluidic cell instrument apparatus without the fluidic cell box, and it comprises lamp optical system (hybrid optical system that comprises multiwavelength laser), micropore pond, forward and backward scattered light signal and fluorescence signal beam splitting system and the detection optical system of logical light window are installed.It is characterized in that the described optical axis of the illuminating bundle direction of propagation and the axis coaxle of Ku Erte micropore of exciting, multiwavelength laser mixed optical unify scattered light signal and fluorescence signal beam splitting system all adopt single optical element.
Advantage of the present invention and the technique effect that reaches show the following aspects:
1) described in background technology 6, all flow cytometers all are the vertical illumination modes of usefulness in the world now.The present invention is owing to excite the optical axis of the illuminating bundle direction of propagation and the axis coaxle of Ku Erte micropore, it is coaxial parallel lighting system, the zone of light beam and particle interaction is whole Ku Erte micropore just like this, the zone of light beam and particle interaction and time, all improve one more than the order of magnitude than vertical illumination mode.In the vertical illumination mode, see Fig. 6 (6-7), when particle began to enter laser beam and begins to produce scattered light or fluorescence, photodetector began to produce electric pulse, such as Fig. 6 (6-1).When particle was in the laser beam center, scattered light or fluorescence reached maximal value, such as Fig. 6 (6-2).Corresponding electric pulse also reaches peak value, and when particle left laser beam, electric pulse was got back to noise level, such as Fig. 6 (6-3).The time width of whole pulse be particle by the time of laser beam, and laser beam only has 20 μ m～25 μ m at the particle direct of travel.And in parallel illumination, (special optics and photoelectricity design make detector only produce corresponding to the signal in space characteristic zone when particle begins to enter the Ku Erte micropore, see following 2), particle and laser beam effect also begin to produce scattered light or the fluorescence that detector can be responded to, photodetector begins to produce electric pulse, such as Fig. 6 (6-4).Because illuminating bundle light intensity in the Ku Erte micropore is that (special optical design reaches this purpose in evenly distribution, see following 3), in a single day particle enters the Ku Erte micropore, its signal that sends namely reaches maximal value, and be maintained to particle and leave the Ku Erte micropore, the wave mode of photoimpact such as Fig. 6 (6-12).Fig. 6 (6-4), (6-5), (6-6) be respectively particle just entered Ku Erte micropore, particle in the Ku Erte micropore, particle just left the Ku Erte micropore, wave mode such as Fig. 6 (6-12) of corresponding photoimpact.Because the corresponding broadening of the time width of light signal, just the requirement of time response of detector descended accordingly, thereby can use the larger area photodetector, and increase the selectivity of receiving angle scope.Fig. 6 (6-8) is the optical axis of laser in the parallel illumination, also is Ku Erte micropore central shaft.Fig. 6 (6-9) is the laser beam in the parallel illumination.Fig. 6 (6-10) is the cell particle, and Fig. 6 (6-11) is the thickness (300 μ m) of Ku Erte micro pore sheet, also is the length of Ku Erte micropore.The diameter of Ku Erte micropore is 70 μ m among Fig. 6.
2) the present invention has used optical system and the photoelectricity design of particular design, make detector only produce response to the signal in space characteristic zone, the light scattering signal and the fluorescence signal that namely only the particle in the detection sensitizing range (7-3) among Fig. 7 (haemocyte B among the figure) are produced have response, to surveying the particle (haemocyte A, C, D among the figure) outside the sensitizing range, even be in the illuminating bundle, the light scattering signal that it sends and fluorescence signal can not arrive photodetector.As for the optical window from the micropore pool wall, more can't enter photodetector such as the backward background noise of formation of the window glass sheet of Fig. 8 (8-1) reflection.Fig. 8 (8-2) is the Ku Erte micropore, and Fig. 8 (8-3) is the disk that the Ku Erte micropore is installed.The optical system of particular design make instrument of the present invention especially rear to extremely low noise is only arranged, the signal of rear orientation light and backward fluorescence signal to noise than (S/N) even be higher than DC (S/N), as shown in figure 13
3) since the micropore pool wall on optical window from Ku Erte micropore center (laser focusing waist) from
Far away, the laser intensity on the optical window greatly reduces the light intensity that reflects from optical window much smaller than the laser intensity at waist place, thus the background noise when greatly having reduced the back scattering photodetection.This also is based on and surveys unsurmountable technical bottleneck of back scattering light time in the blood analysis instrument of flow cytometer principle and the existing flow cytometer
＜9 〉As shown in Figure 4, surveying the back scattering light time, laser focusing Shu Yaoxian shines the surface (apart from the about 2mm in Ku Erte micropore center) of rectangular cylinder, and then shine the garden cylindrical surface (about 25 microns apart from Ku Erte micropore center) of Ku Erte micropore, because the laser focusing beam intensity here is very large, the light intensity of reflection is corresponding also larger, makes script intensity can't reach the acceptable signal-to-interference ratio requirement with regard to the detection of very weak rear orientation light (than little about 3 orders of magnitude of forward scattering light intensity) too greatly because of the background noise that the wall reflection produces.And means of illumination of the present invention just can overcome the technical bottleneck of this back scattering photodetection.
4) the whole Ku Erte micropore of definition district surveys the sensitizing range and can comprise whole Ku Erte micropore for surveying the sensitizing range, also can be the middle part of micropore, the front portion of micropore, the rear portion of micropore, or the combination of several parts of micropore.More than the advantage of coaxial parallel lighting system, can make the zone of light beam and particle interaction just be whole Ku Erte micropore, the zone of light beam and particle interaction and time, all improve one more than the order of magnitude than the vertical illumination mode order of magnitude, scattered light signal and fluorescence signal obtain the raising of the corresponding order of magnitude.Intensity increases by 500 at least, in conjunction with an above-mentioned acquisition of signal optical system to specific region, space sensitivity, the signal of rear orientation light signal and fluorescence signal is greatly improved than (S/N) noise, thereby greatly improved the sensitivity of instrument and to the distinguishing ability of variety classes cell.
5) the present invention has used the directly coaxial parallel illumination of Ku Erte micropore, thereby thoroughly abandoned in the existing streaming haemocyte instrument fluidic cell box and the complicated fluid focus system of requisite costliness, so have " simple structure, low fault, low cost, easy to operate " etc. series of advantages.
Description of drawings:
Fig. 1: Coulter principle
1-1 micropore pond
The 1-1 microporous pipe
1-3 haemocyte suspending liquid
The 1-4 haemocyte
1-5 Ku Erte micropore
The 1-6 external electrode
Electrode in the 1-7
1-8 micropore electric current
The 1-9 vacuum (6 " Hg)
Fig. 2: the constituent of blood
2-1 Xue Pulp
The visible component of 2-2 blood
The 2-3 blood platelet
The 2-4 red blood cell
The 2-5 white blood cell
The 2-6 neutrophil leucocyte
The 2-7 eosinophilic granulocyte
The 2-8 basophil
The 2-9 lymphocyte
The 2-10 monocyte
Fig. 3: based on the ultimate principle of the light scattering haemanalysis instrument of flow cytometer
3-1 fluidic cell box
3-2 Ku Erte micropore
The 3-3 electrode
3-4 micropore electric current
3-5 sheath cover a fluid stream
3-6 sample (particle) a fluid stream
The 3-7 LASER Light Source
The 3-8 condenser lens
90 ° of light scattering of 3-9 or fluorescent are surveyed
3-10 forward light scattering and axial light loss
Fig. 4: fluidic cell box
The 4-1 laser beam
4-2 sheath cover a fluid stream
4-3 garden cone
4-4 microchannel (Orifice)
4-5 sample (particle) a fluid stream
Fig. 5: BD FACSVatage SE flow cytometer index path
5-1 fluidic cell box
5-2 divides sorting collection systems such as selecting deflecting plate and collection tube
5-3 fluid monitor video camera
5-4 collects the micro objective of fluorescence and Side direction light scattering
Three laser instruments of 5-5
8 optical prisms that change optical path direction of 5-6
5-7 receives the photodiode of forward scattering light
5-8 receives the photomultiplier of side scattered light
5-9 (5-9-1) to (5-9-8) is 8 photomultipliers that receive fluorescence signal
(totally 10 these class eyeglasses) such as remaining spectroscope of 5-10, optical filter, lens
Fig. 6: the interaction of particle and laser in BD vertical illumination and the parallel illumination
Photodetector began to produce electric pulse when particle began to enter laser beam in the 6-1 vertical illumination
When particle was in the laser beam center in the 6-2 vertical illumination, corresponding electric pulse reached peak value
When particle left laser beam in the 6-3 vertical illumination, electric pulse was got back to noise level
Particle has just entered the Ku Erte micropore in the parallel illumination of 6-4
Particle is in the Ku Erte micropore in the parallel illumination of 6-5
Particle has just left the Ku Erte micropore in the parallel illumination of 6-6
6-7 vertical illumination mode
The optical axis of laser in the parallel illumination of 6-8 also is Ku Erte micropore central shaft
Laser beam in the parallel illumination of 6-9
6-10 cell particle
The thickness of 6-11 Ku Erte micro pore sheet (300 μ m)
Electric pulse waveform when particle is by the Ku Erte micropore in the parallel illumination of 6-12
Fig. 7: light scattering and fluorescence signal sensitizing range
The Ku Erte micropore that the 7-1 diameter is 50 microns
The 7-2 laser beam
The 7-3 sensitizing range
Rear portion, 7-4 sensitizing range
Middle part, 7-4 sensitizing range
The 7-6 sensitizing range is anterior
7-7 cell A, B, C, D
7-8 laser beam optical axis and Ku Erte micropore axle (coaxial)
7-9 forward light scattering and forward direction fluorescence signal
7-10 backward scattered light and backward fluorescence signal
The special microwell plate of 7-11 300 micron thickness garden valuts thats
Fig. 8: with the micropore pond of optical window
The 8-1 optical window
8-2 Ku Erte micro pore sheet
8-3 installs the disk of Ku Erte micropore
Fig. 9: the waist of laser focusing bundle is full of the Ku Erte micropore in the coaxial parallel illumination of Ku Erte micropore
The waist of 9-1 laser focusing bundle
9-2 Ku Erte micropore
Figure 10: one of specific implementation method of coaxial parallel illumination
10-1 micropore electric current
The 10-2 electrode
10-3 Ku Erte micropore
The 10-4 haemocyte
10-5 haemocyte suspending liquid
The 10-6 optical window
10-7 laser focusing bundle
The 10-8 lamp optical system
The Real-Time Monitoring optical system of 10-9 laser illuminator intensity
10-10 back scattering and backward fluorescence reception optical system
The 10-11 beam-splitter
10-12 optical fiber
The 10-13 laser instrument
10-14 forward scattering and forward direction fluorescence reception optical system
Figure 11: two of the specific implementation method of coaxial parallel illumination
11-1 micropore electric current
The 11-2 electrode
11-3 Ku Erte micropore
The 11-4 haemocyte
11-5 haemocyte suspending liquid
The 11-6 optical window
11-7 laser focusing bundle
The 11-8 lamp optical system
The Real-Time Monitoring optical system of 11-9 laser illuminator intensity
11-10 back scattering and backward fluorescence reception optical system
The 11-11 beam-splitter
11-12 optical fiber
The 11-13 laser instrument
11-14 forward scattering and forward direction fluorescence reception optical system
Figure 12: the forward scattering light of standard particle and the oscillograph recording of dc pulse signal
Green track (descending) is DC pulse (DC) signal, red track (on) be forward scattering light (FLS) signal.920 millivolts of the direct current backgrounds of forward direction.
Figure 13: the rear orientation light of standard particle and the oscillograph recording of dc pulse signal
Red track (descending) is DC pulse (DC) signal, green track (on) be rear orientation light (BLS) signal.9.60 millivolts of backward direct current backgrounds.
Figure 14: the rear orientation light of Quality Control blood sample and the oscillograph recording of dc pulse signal
Red track (descending) is DC pulse (DC) signal, green track (on) be rear orientation light (BLS) signal.Blue track (in) be that rear orientation light (BLS) is through filtered signal.After
To 24 millivolts of direct current backgrounds.
Figure 15: without the three-dimensional of the part optical system of fluidic cell box desktop flow cytometer experimental prototype
Specific implementation method
Below in conjunction with accompanying drawing and embodiment, the present invention is described in detail.
Enforcement of the present invention mainly is to substitute the fluidic cell box that has now in the flow cytometer with the micropore pond that logical light window is installed, and lamp optical system, forward and backward scattered light signal and fluorescence signal detection optical system etc. are directly involved in core subsystem of the present invention, suitably reasonably integrate by the Instrument Design requirement, thereby become one without the desktop flow cytometer of fluidic cell box.Distinguishing characteristics of the present invention is that the described illuminating bundle direction of propagation is consistent with the axis of Ku Erte micropore in the micropore pond.
Referring to Fig. 9, in the present invention, the optical axis of the direction of propagation of laser illuminator light beam (9-5) is coaxial with the axis (9-1-1) of Ku Erte little (9-1), and it makes light scattering method and fluorescence detection can directly apply to classical Ku Erte micropore.Make a call to the through hole (9-1) of 50～80 micron diameters at the center of several mm dias of a slice, thick 300 microns ruby disk (9-2), just become the Ku Erte micropore of classics.The laser (9-5) of propagating along Ku Erte micropore axle (9-1-1) is focused on the center of micropore (9-1).This lighting system still belongs to pioneering on the world's blood analysis instrument history in more than 60 year so far of Coulter principle invention.Its superiority will be set forth fully at the remainder of this instructions.Haemocyte (9-4) can pass through micropore (9-1).Certainly, the diameter of the material of above-mentioned disk (9-2) and size, micropore can be set according to specific requirement.Because the optical axis of the direction of propagation of illumination light (9-5) is coaxial with the axis (9-1-1) of Ku Erte micropore (9-1), so the optical axis that figure axis (9-3) is illuminating bundle (9-5) relays direction also is the central shaft of cylinder of the Ku Erte micropore (9-1) of open circles column type.
The lighting source that said method uses can be that (gas laser is such as the He-Ne laser instrument for laser instrument, the Ar ion laser, semiconductor laser, the optical fiber lotus root is closed semiconductor laser, solid state laser, the optical fiber lotus root is closed solid state laser, ld pumping solid state laser, tunable laser, fiber laser etc.), also can be light emitting diode (LED), glow discharge spot lamp, the light source etc. that burns certainly.
The wavelength coverage of the light source of above-mentioned use, according to different application, can be from ultraviolet, can see one or more the infrared band.
When illuminating bundle is propagated along Ku Erte micropore axle, should use suitable optical system to focus optical beams to and be full of equably whole Ku Erte micropore.
When the light source that uses during as laser instrument, should make the laser focusing bundle be slightly less than the diameter of Ku Erte micropore in the waist size at focus place, its rayleigh range (Rayleigh Range) is then greater than the length of several Ku Erte micropores.
Figure 10 is the micropore pond energy according to Coulter principle (referring to Fig. 1), and its forms the passage of light beam with illumination Ku Erte micro-pore wall and receiving scattered light and fluorescence signal.The microporous pipe (1-2) of Fig. 1 is changed system into a micropore pond (11-1) be divided into two-part microwell plate (10-2), and relatively open two optical windows (10-7) in the front and back of micropore (10-5) at micropore pond (10-1), as shown in figure 10, window (10-7) can be a pair of optical flat (10-7) as shown in figure 10, also can be as shown in figure 11 a pair of lens or other optical element (11-7).This optical element can be with the beam condenser of light source in micropore (10-5), (11-5).This shows that micropore of the present invention pond (10-1), (11-1) can improve a little on Coulter principle micropore pond commonly used.In Figure 10, Figure 11, (10-3),, (11-3) expression haemocyte suspending liquid, (10-4), (11-4) represent haemocyte, (10-6), (11-6) represent internal and external electrode, (10-8), (11-8) be the micropore electric current that produces.The vacuum (10-9) of micropore pond right part, (11-9) (for example, vacuum tightness can be 6 " Hg), vacuum can make particle enter micropore pond right part from micropore pond left part by micropore.
This shows, because the optical window (10-7) on the above-mentioned micropore pool wall, (11-7) are distant from the center (waist of laser focusing bundle) of Ku Erte micropore (10-5), (11-5), laser intensity on optical window (10-7), (11-7) is much smaller than the laser intensity at waist place, make from the light intensity of optical window (10-7), (11-7) reflection and greatly reduce, thus the background noise when greatly having reduced rear orientation light and the detection of backward fluorescence signal.This also is based on and surveys unsurmountable technical bottleneck of back scattering light time in the existing blood analysis instrument of flow cytometer principle and the existing flow cytometer
＜8 〉Again as shown in Figure 4, situation in prior art, after surveying rear orientation light during to fluorescence signal, laser focusing Shu Yaoxian shines the surface (apart from the about 2mm in Ku Erte micropore center) of rectangular cylinder, and then shine the garden cylindrical surface (about 25 microns apart from Ku Erte micropore center) of Ku Erte micropore, because the laser focusing beam intensity here is very large, the light intensity of reflection is corresponding also larger, makes script intensity can't reach the acceptable signal-to-interference ratio requirement with regard to the detection of very weak rear orientation light (than little about 3 orders of magnitude of forward scattering light intensity) too greatly because of the background noise that the wall reflection produces.And the design of means of illumination of the present invention and micropore pond and optical window just can overcome technical bottleneck and the technological deficiency of this back scattering photodetection.Greatly reduced the background noise when the backward fluorescence signal of rear orientation light is surveyed.
As shown in Figure 7, whole Ku Erte micropore of the present invention district can be defined as and survey sensitizing range (7-3), can comprise whole Ku Erte micropore, also can be the middle part (7-5) of micropore, the front portion (7-6) of micropore, the rear portion (7-4) of micropore, or the combination of several parts of micropore.Its light source can adopt laser beam (7-2).Thereby producible forward light scattering signal (7-, the backward fluorescence signal of rear light scattering signal (7-10).The optical axis of laser beam (7-2) is coaxial (7-8) with the axle of Ku Erte micropore.Certainly, so-called " coaxial " or " coaxial " are actually and show consistent on the direction herein, all be in lower of the precision of certain processing and adjustment can the actual limit that reaches, can not be go up for how much, theoretic 100% coaxial or coaxial.In the present embodiment, disk (7-11) is to adopt the special microwell plate of 300 micron thickness garden valuts thats.Certainly, the diameter of the material of above-mentioned disk and size, micropore can be set according to specific requirement.Because the optical axis of the direction of propagation of laser beam and the axis coaxle of Ku Erte micropore, so figure axis (7-8) is the optical axis of laser beam relay direction, also be the central shaft of cylinder of the Ku Erte micropore of open circles column type.
Signal detection system of the present invention only has the characteristics to certain certain spatial areas sensitivity, the light scattering signal and the fluorescence signal that namely only the particle in the detection sensitizing range among Fig. 7 are produced have response, to surveying the particle outside the sensitizing range, even be in the illuminating bundle, the light scattering signal that it sends and fluorescence signal can not arrive photodetector.
Of the present invention the signal detection system to certain certain spatial areas sensitivity comprises, but be not limited to, optics, machinery, electronics, light-electricity, light-machine, as to reach above several combinations method, or other can this be realized to any method to certain certain spatial areas sensitivity.
The optical detection receiving system that the present invention is used has specific region, space selectivity is accepted the characteristics of scattered light signal and fluorescence signal, thereby has realized the successful detection of rear light scattering signal and fluorescence signal.
To the detection of forward, backward scattered light signal signal and fluorescence signal, can be interval to different angles, or the detection of the combination in several different angles interval.
The light signal of forward direction comprises the signals such as forward scattering light, axial light loss (Axial Light Loss is called for short ALL) and fluorescence signal.
To the detection of forward, backward scattered light signal and fluorescence signal, can be the detection of single wavelength, also can be the light splitting of multi-wavelength light spectrometer is surveyed.
Also can carry out in forward, backward the fluorescence signal detection of monochrome or polychrome, or the inelastic optical scattering signal, such as the acquisition of signal of Raman scattering, anti-Stokes Raman scattering etc.
All flow cytometers all are the vertical illumination modes of usefulness in the world now.The present invention is owing to excite the optical axis of the illuminating bundle direction of propagation and the axis coaxle of Ku Erte micropore, it is coaxial parallel lighting system, the zone of light beam and particle interaction is whole Ku Erte micropore just like this, the zone of light beam and particle interaction and time, all improve one more than the order of magnitude than vertical illumination mode.In the vertical illumination mode, see Fig. 6 (6-7), when particle began to enter laser beam and begins to produce scattered light or fluorescence, photodetector began to produce electric pulse, such as Fig. 6 (6-1).When particle was in the laser beam center, scattered light or fluorescence reached maximal value, such as Fig. 6 (6-2).Corresponding electric pulse also reaches peak value, and when particle left laser beam, electric pulse was got back to noise level, such as Fig. 6 (6-3).The time width of whole pulse be particle by the time of laser beam, and laser beam only has 20 μ m～25 μ m at the particle direct of travel.And in parallel illumination, (special optics and photoelectricity design make detector only produce corresponding to the signal in space characteristic zone when particle begins to enter the Ku Erte micropore,), particle and laser beam effect also begin to produce scattered light or the fluorescence that detector can be responded to, photodetector begins to produce electric pulse, such as Fig. 6 (6-4).Because illuminating bundle light intensity in the Ku Erte micropore is evenly distribute (special optical design reaches this purpose), in a single day particle enters the Ku Erte micropore, its signal that sends namely reaches maximal value, and be maintained to particle and leave the Ku Erte micropore, the wave mode of photoimpact such as Fig. 6 (6-12).Fig. 6 (6-4), (6-5), (6-6) are respectively particle and have just entered Ku Erte micropore, particle and just left the Ku Erte micropore, wave mode such as Fig. 6 (6-12) of corresponding photoimpact at, particle.Because the corresponding broadening of the time width of light signal, just the requirement of time response of detector descended accordingly, thereby can use the larger area photodetector, and increase the selectivity of receiving angle scope.Fig. 6 (6-8) is the optical axis that swashs Ku Erte micropore center light in the parallel illumination, also is Ku Erte micropore central shaft.Fig. 6 (6-9) is the laser beam in the parallel illumination.Fig. 6 (6-10) is the cell particle, and Fig. 6 (6-11) is the thickness (300 μ m) of Ku Erte micro pore sheet, also is the length of Ku Erte micropore.The diameter of Ku Erte micropore is 70 μ m among Fig. 6.
Among Fig. 9, (9-1) laser focusing bundle waist in the coaxial parallel lighting system of expression.Its rayleigh range (Rayleigh Range) is greater than the length of several Ku Erte micropores, and laser focusing bundle waist is full of whole Ku Erte micropore (9-2).
Figure 10, Figure 11 are the synoptic diagram of two specific embodiments provided by the invention, and they are on the basis in the micropore pond of Fig. 3 or Fig. 4, adopt the synoptic diagram of embodiment of the detection system of coaxial lighting system of the present invention.This embodiment only illustrates, but practical application can be not limited to these embodiment.Specific as follows:
Embodiment 1):
Referring to Figure 10, laser instrument (10-13), optical fiber coupled lasers for example, output enter lamp optical system (10-8) through the light of optical fiber (10-12) output through beam-splitter (10-11), focused beam (10-7) focuses on the haemocyte (10-4) at micropore center through micropore pond optical window (10-6) (the right among the figure).The interact forward scattering light (FLS) that produces or axial light of irradiation light and haemocyte lost (ALL) and forward direction fluorescence signal and entered Forward receiving optical system (10-14) through micropore pond optical window (10-6) (left side among the figure).The interact rear orientation light (BLS) that produces and backward fluorescence signal of irradiation light and haemocyte (10-4) returns through micropore pond window (10-6) (left side among the figure) and enters backward light-receiving optical system (comprise lamp optical system (10-8), beam-splitter (10-11) and backward light-receiving optical system (10-10), the Real-Time Monitoring optical system (10-9) of laser illuminator intensity is for the Strength Changes of Real-Time Monitoring laser illuminator.So, when each haemocyte passes through micropore, just can obtain simultaneously the signal (DC, FLS, BLS, ALL, and forward and backward fluorescence signal) of a plurality of relevant haemocyte information, these signals namely are used for five classification of haemocyte and the measurement of other multiple parameters with its fine resolution.Among the figure, (10-5) be haemocyte suspending liquid.(10-1) be the micropore electric current that the micropore pond produces.Among the figure, A, B are two parts that Ku Erte micropore pond is separated into by the Ku Erte microwell plate.
Embodiment 2):
Referring to Figure 11, this implementation method and specific implementation method 1) the different illumination optical system backward light-receiving optical systems of unifying that are do not have the part that shares, the design in micropore pond also is different from above-mentioned implementation method 1).The output of optical fiber coupled lasers (11-13) is introduced into lamp optical system (11-8) through optical fiber (11-12), then focuses on the haemocyte (11-4) at micropore center through the optical window (11-6) in micropore pond (the right among the figure) through becoming focused beam (11-7) behind the beam-splitter (11-11).The interact forward scattering light (FLS) that produces or axial light of irradiation light and haemocyte lost (ALL) and forward direction fluorescence signal and entered forward light receiving optics (11-14) through micropore pond optical window (11-6) (left side among the figure).The interact rear orientation light (BLS) that produces and backward fluorescence signal of irradiation light and haemocyte (11-4) returns through micropore pond window (11-6) (the right among the figure), reflected by beam-splitter (11-11) to enter backward light-receiving optical system (11-10).The Real-Time Monitoring optical system (11-9) of laser illuminator intensity is used for the Strength Changes of Real-Time Monitoring laser illuminator.So, when each haemocyte passes through micropore, just can obtain simultaneously the signal (DC, FLS, BLS, ALL, and forward and backward fluorescence signal) of a plurality of relevant haemocyte information, these signals namely are used for five classification of haemocyte and the measurement of other multiple parameters with its fine resolution.
The part of test results example:
1) the Ku Erte dc pulse signal (DC, lower track) of the standard particle of 7 microns of diameters shows (experimental result) with the oscillograph recording of forward-scattering signal (FLS, upper track), sees Figure 12.The direct current background of noting the forward light scattering signal is 920 millivolts.
2) the Ku Erte dc pulse signal (DC, lower track) of the standard particle of 7 microns of diameters shows (experimental result) with the oscillograph recording of back scattering light signal (BLS, upper track), referring to Figure 13.
3) oscillograph recording of the Ku Erte dc pulse signal of Quality Control blood sample (DC, lower track) and back scattering light signal (BLS, lower track, through filtered BLS, middle track) shows (experimental result), referring to Figure 14.
Attention: the direct current background of rear light scattering signal only is 24 millivolts.920 millivolts of the direct current backgrounds of contrast forward light scattering signal have reduced by 34 times.The signal noise ratio of rear light scattering signal (S/N) even also higher than (S/N) of DC pulse, this proves absolutely the being unsurpassed in excellence property of the design of lighting system of the present invention and backward optical detection system.
Figure 15 is the three-dimensional design figure without fluidic cell box desktop flow cytometer experimental prototype middle part spectroscopy system.Above-mentioned oscilloscope signal figure namely on this experimental prototype obtain.
Pass through above-described embodiment, can find out, the present invention is to the direct illumination of Ku Erte micropore, thereby thoroughly abandoned existing flow cytometer and based in the blood cell analysis instrument of flow cytometer principle the fluidic cell box of requisite costliness and complicated fluid focus system, so series of advantages such as " simple structure, low fault, low costs, easy to operate " is arranged.
Claims (19)
1. in flow cytometer, use is to the coaxial parallel means of illumination of Ku Erte micropore, thereby the fluidic cell box of the requisite costliness of institute in the existing main flow flow cytometer in the world and the fluid focus system of complexity have thoroughly been abandoned, it makes the detection of light scattering and fluorescence can directly apply to classical Ku Erte micropore, it is characterized in that, with the optical axis of the illuminating bundle direction of propagation and the axis coaxle of Ku Erte micropore.
2. the coaxial parallel means of illumination of Ku Erte micropore in the flow cytometer according to claim 1, the lighting source of described illuminating bundle, can be following any or several: laser instrument, light emitting diode (LED), glow discharge spot lamp, the light source that burns certainly; Described laser instrument can be following any or several: gas laser, such as the He-Ne laser instrument, the Ar ion laser, semiconductor laser, the optical fiber lotus root is closed semiconductor laser, solid state laser, the optical fiber lotus root is closed solid state laser, ld pumping solid state laser, tunable laser, fiber laser.
3. the coaxial parallel means of illumination of Ku Erte micropore in the flow cytometer according to claim 1, it is characterized in that, the wavelength coverage of the lighting source of described illuminating bundle, according to different application, can be from ultraviolet, can see one or more the infrared band.
4. the coaxial parallel means of illumination of Ku Erte micropore in 3 arbitrary described flow cytometers according to claim 1 is characterized in that when illuminating bundle was propagated along Ku Erte micropore axle, light beam focused on and is full of equably whole Ku Erte micropore.
5. the coaxial parallel means of illumination of Ku Erte micropore in 3 arbitrary described flow cytometers according to claim 1, it is characterized in that, when the light source that uses during as laser instrument, the laser focusing bundle is slightly less than the diameter of Ku Erte micropore in the waist size at focus place, and its rayleigh range (Rayleigh Range) is then greater than the length of several Ku Erte micropores.
6. the coaxial parallel means of illumination of Ku Erte micropore in the flow cytometer according to claim 1, it is characterized in that, can form the passage of light beam for the micropore pond that makes Coulter principle with illumination Ku Erte micropore and detection light signal, microporous pipe is transformed into a micropore pond and is divided into two-part microwell plate, and opens two optical windows in the front and back of the relative micropore in micropore pond.
7. the coaxial parallel means of illumination of Ku Erte micropore in the flow cytometer according to claim 6, it is characterized in that, two optical windows of the front and back of the relative micropore in described micropore pond, every a pair of described optical window can be a pair of optical flat, a pair of lens or other optical element.
8. according to claim 1,4 with 5 described flow cytometers in the coaxial parallel means of illumination of Ku Erte micropore, it is characterized in that, whole Ku Erte micropore district is for surveying the sensitizing range, survey the sensitizing range and can comprise whole Ku Erte micropore, also can be the middle part of micropore, the front portion of micropore, the rear portion of micropore, or the combination of several parts of micropore.
According to claim 1 with 8 described flow cytometers in the coaxial parallel means of illumination of Ku Erte micropore, it is characterized in that, signal detection system has only responsive to certain certain spatial areas, the light scattering signal that namely only the particle of surveying in the sensitizing range is produced has response, to surveying the particle outside the sensitizing range, even be in the illuminating bundle, the light scattering that it sends and fluorescence signal can not arrive photodetector.
10. the coaxial parallel means of illumination of Ku Erte micropore in according to claim 9 the flow cytometer, it is characterized in that, only to the signal detection system of certain certain spatial areas sensitivity can be optics, machinery, electronics, light-electricity, light-machine, and more than the method for several combination, or other any method that can reach certain certain spatial areas sensitivity is realized.
11. the coaxial parallel means of illumination of Ku Erte micropore in the flow cytometer according to claim 9, it is characterized in that, have specific region, space selectivity is accepted the characteristics of scattered light and fluorescence signal, thereby realized the successful detection of rear light scattering signal and backward fluorescence signal.
12. according to claim 9 with 11 flow cytometer in the coaxial parallel means of illumination of Ku Erte micropore, it is characterized in that, to the detection of forward, backward scattered light signal and fluorescence signal, can be interval to different angles, or the detection of the combination in several different angles interval.
13. according to claim 9, the coaxial parallel means of illumination of Ku Erte micropore in 11,12 the flow cytometer, it is characterized in that, the light signal of forward direction comprises the signals such as forward scattering light, axial light loss (Axial Light Loss is called for short ALL) and forward direction fluorescence.
14. according to claim 9, the coaxial parallel means of illumination of Ku Erte micropore in 11,12,13 the flow cytometer, it is characterized in that, to the detection of forward, backward scattered light and fluorescence signal, can be the detection of single wavelength, also can be the light splitting of multi-wavelength light spectrometer is surveyed.
15. according to claim 9, the coaxial parallel means of illumination of Ku Erte micropore in 11,12,13 the flow cytometer, it is characterized in that, also can carry out in forward, backward the fluorescence signal detection of monochrome or polychrome, or the inelastic optical scattering signal, such as the acquisition of signal of Raman scattering, anti-Stokes Raman scattering etc.
16. the coaxial parallel illumination flow cytometer of Yi Zhong Ku Erte micropore, it comprises lamp optical system, the micropore pond of logical light window, forward and backward scattered light and fluorescence signal detection optical system is arranged, it is characterized in that, consistent with the axis of Ku Erte micropore at the optical axis of the illuminating bundle direction of propagation described in the micropore pond.
17. the application of the coaxial parallel illumination of Yi Zhong Ku Erte micropore, the optical axis of the described illuminating bundle direction of propagation and the axis coaxle of Ku Erte micropore, it not only can be used for flow cytometer, also can be used for other cells, animal blood cell, other fine particles, and have granularity and inside particles composition that each industrial circle of requirement is arranged to material, such as the purity that can be used to detect fuel in the detection of food crystalline substance, pharmacy, chemical industry, petrochemical industry and the aerospace industry etc.
18. according to claim 1, the coaxial parallel means of illumination of Ku Erte micropore in 3,13,14,15 the flow cytometer,
The beam combiner (beam combiner) of the excitation source of multi-wavelength (such as the laser of a plurality of different wave lengths), adopted single dispersion element, comprise, but be not limited to, plane and concave reflection grating, plane and concave surface transmission grating, multimode fiber-optic bundle, the wavelength mixer of the single elements such as light pipe (Lightpipr), optical waveguide (Lightguide).
19. according to claim 1, the coaxial parallel means of illumination of Ku Erte micropore in 3,13,14,15 the flow cytometer,
The wavelength separated of the fluorescence signal of multi-wavelength, changed the complex combination of a plurality of elements of spectroscope commonly used in the existing flow cytometer, optical filter, dichroscope etc., adopted single dispersion element, comprise, but be not limited to the dispersion element of the single elements such as plane and concave reflection grating, plane and concave surface transmission grating.
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| CN103983556A (en) * | 2013-11-03 | 2014-08-13 | 龚维燕 | Multi-wavelength light beam combination device based on optical grating reverse dispersion and applications thereof |
| CN106680186A (en) * | 2016-11-02 | 2017-05-17 | 北京信息科技大学 | Multi-type scattered light detection system of flow cytometer |
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