CN101915855B - Method and device for detecting flying speeds of particles in cytometer by using secondary light-scattering method - Google Patents
Method and device for detecting flying speeds of particles in cytometer by using secondary light-scattering method Download PDFInfo
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- CN101915855B CN101915855B CN2010102413829A CN201010241382A CN101915855B CN 101915855 B CN101915855 B CN 101915855B CN 2010102413829 A CN2010102413829 A CN 2010102413829A CN 201010241382 A CN201010241382 A CN 201010241382A CN 101915855 B CN101915855 B CN 101915855B
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Abstract
The invention relates to a video-detecting flow cytometer, in particular to a method and an implementation device for detecting the flying speeds of particles in a cytometer by using a secondary light-scattering method so as to accurately measure the speeds in real time. The invention adopts the technical scheme that the method for detecting the flying speeds of particles in the cytometer by using the secondary light-scattering method comprises the following steps of: enabling the same particle to continuously pass through two beams of parallel light to generate twice scattering, wherein the two beams of parallel light are emitted from a cytometer nozzle and have a distance D; focalizing scattered light and receiving by a photosensitive sensor; respectively detecting the two light scatterings by the photosensitive sensor; measuring a time interval T between the two light scatterings through an electronic timing circuit; and calculating the average flying speed of the particles according to a speed formula: V=D/T. The invention also provides a corresponding implementation device. The invention is mainly applied to detecting the flying speeds of the particles in the cytometer.
Description
Technical field
The present invention relates to detect the imaging cytometer appearance.Specifically, relate to method and the device for carrying out said that the secondary light scattering method detects particle flying speed in the cell instrument.
Background technology
Present technique is mainly used in the imaging cytometer appearance, and the imaging cytometer appearance is evolved from traditional flow cytometer.Traditional flow cytometer is a kind ofly to be used for pair cell or particle is analyzed and sorting, thereby reaches the instrument that carries out early stage medical diagnosis on disease (like cancer) or purposes such as pair cell influence such as research drugs, ray from cellular level.This instrument the time needs pair cell or particle to carry out the specific fluorescence mark in work, and computing machine is confirmed the physics and the chemical information of cell or particle through scattered light and fluorescence information, thereby operating personnel can't see the real image of cell or particle.For addressing these problems, the imaging cytometer appearance can realize taking in the short time a large amount of particles, may observe cell or particle true picture and individual cells or particle functions such as " souvenirs of taking pictures " one by one.
Imaging cytometer appearance technology is that blood sample after the dilution or other biological are learned sample; Spray from micropore nozzle with sheath fluid (parcel blood sample); Use high speed photography that the particle in the jet flow is taken pictures one by one, use image processing techniques to realize identification, classification and counting again particle.
The measurement of so-called " particle flying speed " refers to by the velocity survey of the particle in the blood sample of sheath fluid parcel in fact in the present technique, and the speed of particle must be decided by to wrap up its sheath fluid, so adjustment sheath fluid speed is in fact also promptly adjusted particle speed.The measurement effect of particle flying speed has two:
1, in the imaging cytometer appearance, sample and sheath stream need very to stablize from the speed of nozzle ejection, and in order to stablize flow stream velocity, at first will measure flow stream velocity.
2, in the imaging cytometer appearance, camera shutter and flashlamp must get into the proper timing unlatching of photographed region at the particle that is taken.Realize this purpose, at first need detect the arrival of particle, its less important its flying speed of in time measuring is so that the moment that decision camera shutter and flashlamp start.
In the tradition low cytometric analysis, also need stablize the speed that sample goes out from nozzle ejection.For this reason, also need measure nozzle velocity, so that for jetting system provides feedback parameter, again through regulating pressure or the additive method in sample pool or the sheath flow pool, stable ejection speed.
In tradition flow cytometer and the imaging cytometer appearance, existing flow-speed measurement method can be divided into two types.At least two laser instruments of one type of needs; Like in-vivo imaging flow cytometer (In Vivo Imaging Flow Cytometer) is to come computing velocity through measurement count slit and the distance that postpones the inner cell motion of imaging pulse time period; This method need count and the form images laser instrument of two functions; And for example multispectral image cell instrument (Multispectral Imaging Flow Cytometer) with velocity survey as subsystem, an and additional laser instrument in the subsystem; Another kind of speed-measuring method is to infer speed through formula, is not to measure in real time, and for example diffraction imaging flow cytometer (Diffraction Imaging Flow Cytometer) calculates speed according to vertical positions different in the fluid column by formula.
There is obvious drawback in these two kinds of existing methods: first kind of required laser instrument is more, the big and debugging difficulty increase of expense; On every picture that second kind can not guarantee take cell or particle are arranged all, because institute's degree of testing the speed might not be accurate, therefore efficient reduce.
Summary of the invention
For overcoming the deficiency of prior art; Can carry out in real time, carry out accurately velocity survey; The technical scheme that the present invention takes is that the secondary light scattering method detects the method for particle flying speed in the cell instrument, makes the directional light that two bundle distances are known as D that continues to pass through from the ejection of cell instrument nozzle; Produce double scattering; Received by light sensor after the scattered light line focus, the time interval T that light sensor is surveyed twice light scattering respectively and taken place through twice light scattering of electric time-keeping circuit measuring calculates the average velocity that particle flies through speed V=D/T.
The secondary light scattering method detects the device of particle flying speed in the cell instrument, comprising:
C shape bearing 2, near the sheath that flows through the nozzle ejection its center from top to bottom fast flows 1;
The slit A that the incident beam 4 that laser tube 3 sends passes on C shape bearing 2 one sides gets into C shape bearing 2; Shine sheath stream 1 back and get into the slit A ' of a relative side with light incident side on the C shape bearing 2; Penetrate after right-angle prism 5 twice reflection become outgoing beam 6 from slit; Outgoing beam 6 passes with slit A ' and is positioned at C shape bearing 2 homonymies and slit B ' above it, sheath stream 1, and from bearing 2, penetrates with slit A homonymy and slit B above it, and incident beam 4 is parallel with outgoing beam 6; Apart is determined by the distance of slit A on the C shape bearing 2 and B, and is known as D;
Lens are positioned on the C type bearing 2, are used to focus on scattered light, shine after the focusing on the photosensor 7 at lens rear;
Subsequent conditioning circuit is used for according to speed V=D/T, and T is the scattered light signal time interval, calculates the average velocity V of particle flight.
Laser tube is the laser instrument parallel light tube, and the laser beam cross-section of generation is a rectangle or near the ellipse of rectangle.
Described device, during Installation and Debugging, whether have laser penetrate, have only when when slit B observes the laser that reflects if can observe at slit B place, explain that just the whole optical path adjusting is correct.
Its characteristics of the present invention are:
1, only use a laser instrument can produce desired two bundle collimated laser beams.On the one hand, reduce and use the number of laser instrument effectively to cut down cost, because the laser price still accounts for larger specific gravity than whole cell instrument cost at present; On the other hand; The combination of a laser and a right-angle prism is debugged than two laser more easily: during Installation and Debugging, cover slit B with thin paper, start laser instrument; The position of adjustment laser instrument; When having laser facula and facula area maximum on the thin paper at slit B place, show that this moment, laser instrument was in the optimum position, fixing locked laser instrument gets final product.Article two, light is inevitable parallel, if with two laser instruments, then can not guarantee.
2, because can carry out in real time, velocity survey accurately, the image guaranteeing all to have on every photo a particle under the situation of system's operate as normal has improved efficient, has practiced thrift cost.
Description of drawings
Fig. 1 measurement mechanism schematic perspective view, among the figure: the 1st, sheath stream, the 2nd, C shape bearing (support), the 3rd, laser instrument, the 4th, incoming laser beam, the 5th, right-angle prism, the 6th, outgoing laser beam, the 7th, photosensor.
Fig. 2 measurement mechanism front view.
Fig. 3 measurement mechanism vertical view.
Fig. 4: scattered light detects and the processing section functional block diagram.
Fig. 5: actual measurement pulse waveform.
Embodiment
The present invention adopts light scattering principle to realize.When continuing to pass through to produce when two bundle distances are known as the directional light of D, same particle respectively produces primary scattering; Can be received by light sensor after the scattered light line focus; The time interval T that light sensor is surveyed twice light scattering respectively and taken place through twice light scattering of electric time-keeping circuit measuring calculates the average velocity that particle flies through V=D/T.
The following (see figure 1) of technical scheme:
From the liquid of nozzle ejection is the blood sample by sheath stream 1 parcel.Blood sample is through repeatedly dilution, and concentration is less, thereby each particle or iuntercellular distance is bigger in the blood, thereby allows this device that the flight time of each sample is calculated from each particle of nozzle ejection or the spacing of cell.Sheath stream 1 is with ring shape parcel blood sample, and in the present technique measurement range, the sheath fluid that flows still can be considered cylinder, thereby sheath fluid speed can be considered particle speed, and this condition makes regulates particle rapidity and become rational method through regulating air pressure.
The sheath stream 1 of nozzle ejection flows through near C shape bearing 2 centers of measurement mechanism from top to bottom fast.The slit A that the incident beam 4 that laser tube 3 sends passes on C shape bearing 2 one sides gets into C shape bearing 2; Shine sheath stream 1 back and get into the slit A ' of a relative side with light incident side on the C shape bearing 2; Penetrate after right-angle prism 5 twice reflection become outgoing beam 6 from slit; Outgoing beam 6 passes with slit A ' and is positioned at C shape bearing 2 homonymies and slit B ' above it, sheath stream 1, and from bearing 2, penetrates with slit A homonymy and slit B above it.Incident beam 4 is parallel with outgoing beam 6, and apart is determined by the distance of slit A on the C shape bearing 2 and B, and is known as D.
Particle in the sheath stream 1 ejects from nozzle with sheath stream 1, gets into C shape support and along its downward injection.Saidly know that by preceding sheath stream 1 passes light beam with particle with twice: pass the outgoing beam 6 that is positioned at the top for the first time, pass the incident beam 4 that is positioned at the below for the second time.When particle passes outgoing beam 6, produce a light scattering.Scattered light focuses on through the lens (being positioned on the C type bearing) that are positioned at the adjacent side of slit of two opposite sides, shines on the photosensor 7 at lens rear, and photosensor 7 converts this scattered light signal to electric signal, i.e. electric pulse.Equally, when particle 7 moves downward when passing incident beam 4, also produce a light scattering, photosensor 9 is exported an electric impulse signal too.
Can measure recurrent interval T after two electric pulses are handled by follow-up electronic circuit, thereby according to V=D/T, subsequent conditioning circuit can calculate the average velocity V of particle flight, the motion of particle here can be similar to regards linear uniform motion as.
The xsect of the laser beam 4 that laser instrument 3 sends is rectangle or approximate rectangle; Behind the enough 5 liang of secondary reflections of right-angle prism of width; The xsect of emergent light 6 also is rectangle basically or is similar to rectangle; Such design can reduce incident light and reflected light aiming difficulty greatly, makes no matter the particle of what position all can scattering laser in sheath fluid.
To sum up, the present invention:
1, only use a laser instrument can produce desired two bundle collimated laser beams.On the one hand, reduce and use the number of laser instrument effectively to cut down cost, because the laser price still accounts for larger specific gravity than whole cell instrument cost at present; On the other hand; The combination of a laser and a right-angle prism is debugged than two laser more easily: during Installation and Debugging, cover slit B with thin paper, start laser instrument; The position of adjustment laser instrument; When having laser facula and facula area maximum on the thin paper at slit B place, show that this moment, laser instrument was in the optimum position, fixing locked laser instrument gets final product.Article two, light is inevitable parallel, if with two laser instruments, then can not guarantee.
2, because can carry out in real time, velocity survey accurately, the image guaranteeing all to have on every photo a particle under the situation of system's operate as normal has improved efficient, has practiced thrift cost.
Claims (4)
1. a secondary light scattering method detects the method for particle flying speed in the cell instrument; Make the sheath stream that two bundle distances are known as the directional light of D that continues to pass through, produce double scattering, received by photosensor (7) after the scattered light line focus from the ejection of cell instrument nozzle; The time interval T that photosensor (7) is surveyed twice light scattering respectively and taken place through twice light scattering of electric time-keeping circuit measuring; Calculate the average velocity that particle flies through speed V=D/T, it is characterized in that the concrete steps of said method are:
The sheath stream (1) of nozzle ejection is flow through near C shape bearing (2) center from top to bottom fast;
The slit A that the incident beam (4) that laser tube (3) is sent passes on C shape bearing (2) one sides gets into C shape bearing (2); Shine sheath stream (1) back and get into the slit A ' that C shape bearing (2) is gone up a relative side with light incident side; Penetrate after right-angle prism (5) twice reflection becoming outgoing beams (6) from slit A '; Outgoing beam (6) passes with slit A ' and is positioned at C shape bearing (2) homonymy and slit B ' above it, sheath stream (1), and goes up and slit A homonymy and the ejaculation of slit B above it from C shape bearing (2), and incident beam (4) is parallel with outgoing beam (6); Apart is determined by the distance of slit A on the C shape bearing (2) and B, and is known as D;
Employing is positioned at the lens focus scattered light on the C type bearing (2), shines after the focusing on the photosensor (7) at lens rear;
Adopt photosensor (7) to convert scattered light signal to electric signal;
Adopt subsequent conditioning circuit according to speed V=D/T, T is the scattered light signal time interval, calculates the average velocity V of particle flight.
2. a kind of secondary light scattering method according to claim 1 detects the device for carrying out said of the method for particle flying speed in the cell instrument, it is characterized in that, comprising:
C shape bearing (2), near the sheath that flows through the nozzle ejection its center from top to bottom fast flows (1);
The slit A that the incident beam (4) that laser tube (3) sends passes on C shape bearing (2) one sides gets into C shape bearing (2); Shine sheath stream (1) back and get into the slit A ' that C shape bearing (2) is gone up a relative side with light incident side; Penetrate after right-angle prism (5) twice reflection becoming outgoing beams (6) from slit A '; Outgoing beam (6) passes with slit A ' and is positioned at C shape bearing (2) homonymy and slit B ' above it, sheath stream (1), and goes up and slit A homonymy and the ejaculation of slit B above it from C shape bearing (2), and incident beam (4) is parallel with outgoing beam (6); Apart is determined by the distance of slit A on the C shape bearing (2) and B, and is known as D;
Lens are positioned on the C type bearing (2), are used to focus on scattered light, shine after the focusing on the photosensor (7) at lens rear;
Photosensor (7) converts scattered light signal to electric signal;
Subsequent conditioning circuit is used for according to speed V=D/T, and T is the scattered light signal time interval, calculates the average velocity V of particle flight.
3. a kind of secondary light scattering method according to claim 2 detects the device for carrying out said of the method for particle flying speed in the cell instrument, it is characterized in that laser tube is the laser instrument parallel light tube, and the laser beam cross-section of generation is a rectangle or near the ellipse of rectangle.
4. a kind of secondary light scattering method according to claim 2 detects the device for carrying out said of the method for particle flying speed in the cell instrument; It is characterized in that; Described device during Installation and Debugging, observes at slit B place whether the laser ejaculation is arranged; Have only when when slit B observes the laser that reflects, explain that just the whole optical path adjusting is correct.
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| CN2010102413829A CN101915855B (en) | 2010-07-30 | 2010-07-30 | Method and device for detecting flying speeds of particles in cytometer by using secondary light-scattering method |
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| CN2010102413829A CN101915855B (en) | 2010-07-30 | 2010-07-30 | Method and device for detecting flying speeds of particles in cytometer by using secondary light-scattering method |
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| CN103926421B (en) * | 2014-05-07 | 2017-02-08 | 河北工业大学 | Autocorrelation speed detection device for cell screening |
| KR101605638B1 (en) * | 2014-12-22 | 2016-03-22 | 고려대학교 산학협력단 | Apparatus for measuring fluid velocity |
| EP3239721B1 (en) * | 2014-12-22 | 2021-01-13 | Absology Co., Ltd. | Apparatus for measuring fluid velocity |
| CN107727882A (en) * | 2017-09-30 | 2018-02-23 | 中国科学院合肥物质科学研究院 | A kind of velocity-measuring system of mm-scale rapid flight frost bullet |
| CN111351956A (en) * | 2020-03-30 | 2020-06-30 | 中科前衍(深圳)工程科学发展有限公司 | High-speed airflow speed measuring device and method for ore pulverization |
| CN114184527A (en) * | 2021-12-15 | 2022-03-15 | 中国日用化学研究院有限公司 | Application of nanoparticle tracking analyzer in characterization of surfactant aggregate |
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| CN2812008Y (en) * | 2005-06-13 | 2006-08-30 | 中国科学院空间科学与应用研究中心 | forward scattering laser speed measurer |
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