CN111948118B - Liquid drop delay calculating device and calculating method thereof - Google Patents
Liquid drop delay calculating device and calculating method thereof Download PDFInfo
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Abstract
The invention relates to a liquid drop delay calculating device and a calculating method thereof, wherein the device comprises at least one laser, a flow chamber, a nozzle, a first detection unit, a second laser, a second detection unit, an image acquisition unit, a charging device and a sorting plate; the first detection unit comprises a second detector, the second detector is used for detecting the scattered light signal and acquiring the time when the particles reach the detection point; the second laser is used for emitting at least one laser, the second detection unit is used for analyzing a second fluorescence signal, and the second detection unit comprises a third detector which is used for detecting the second fluorescence signal and acquiring the time of the particles reaching the fracture point; according to the invention, the first detector, the second detector and the third detector are arranged, so that the delay time of the liquid drops can be directly measured, the delay time of the liquid drops is prevented from being adjusted, the advantages of short time consumption and material consumption saving are achieved, and the sorting efficiency is greatly improved.
Description
Technical Field
The invention relates to the technical field of cell sorting equipment, in particular to a device and a method capable of automatically calculating liquid drop delay.
Background
With the development of socioeconomic and the progress of science and technology, the liquid drops are widely applied in industry, medicine and life. The method comprises the steps of applying periodic disturbance to a liquid flow sprayed by a nozzle to enable the liquid flow to be gradually broken into liquid drops wrapping cells, and when a laser beam just sprays from the nozzle, scattering light generated by one cell wrapping the liquid flow is projected to a photomultiplier tube and converted into an electric signal to be identified. In this process, the time from when a cell is recognized to when a droplet containing the cell leaves the stream becomes the droplet delay.
At present, the conventional device for calculating the liquid drop delay mainly comprises the following devices:
the charged deflected droplets are illuminated by a laser and a detector is used to detect a fluorescent signal in the droplets. If the liquid drop delay is accurate, the fluorescence signal only appears in the sorting channel; if the droplet delay is inaccurate, the fluorescent signal can be detected in non-gated channels as well, at this time. The drop delay needs to be adjusted so that all fluorescence signals are present in the sorting channel.
However, the above device cannot directly measure the delay time of the liquid drop, and needs to judge whether the delay time of the liquid drop is accurate or not by means of energy detection, and because the speed of the liquid drop is high, the detection mode is not real-time detection but statistical analysis, so that the delay time of the liquid drop needs to be continuously adjusted, and whether the charging of the liquid drop is correct or not is judged by performing statistical analysis on a large amount of data, so that the time consumption is long, experimental supplies such as microspheres and the like are consumed greatly, and the efficiency is low.
The droplet delay is automatically obtained by measuring the fluorescent signal of the microspheres in the waste liquid. And measuring the fluorescence signal intensity in the waste liquid corresponding to different liquid drop delays within the specified liquid drop delay range. And combining with subsequent data processing, the liquid drop delay corresponding to the lowest fluorescence signal intensity is the optimal liquid drop delay time of the system.
The device also needs to process and calculate a large amount of data, cannot calculate the liquid drop delay through word connection, consumes long time, consumes large experimental supplies such as microspheres and the like, and is low in efficiency.
By setting different droplet delays, each droplet delay corresponds to 100 droplets, each 100 droplets are charged and then fall on a specified position of a microscope cover glass, and the distribution condition of fluorescent microspheres on the cover glass is observed when 10 positions (10000 droplets) are filled, so that the droplet delay time is obtained.
However, the method for setting the liquid drop delay usually needs to measure for many times to obtain accurate liquid drop delay time, and is long in time consumption, inconvenient to operate and low in efficiency.
Disclosure of Invention
In view of the above-mentioned drawbacks of the prior art, it is an object of the present invention to provide a droplet delay calculating device and a calculating method thereof. Compared with the prior art, the method can directly measure the liquid drop delay, greatly improves the sorting efficiency and saves the consumable materials.
In order to achieve the purpose, the technical scheme of the invention is as follows:
the device comprises at least one laser, a flow chamber, a nozzle, a first detection unit, a second laser, a second detection unit, an image acquisition unit, a charging device and a sorting plate;
the laser is used for emitting at least one laser which irradiates on the particles in the flow chamber;
the flow chamber is used for limiting the flow of the particles, so that the particles are sequentially irradiated by the laser to generate a first optical signal, and the first optical signal comprises at least one scattered optical signal and a first fluorescent signal;
the nozzle is arranged at the outlet of the flow chamber, so that the liquid flow sprayed out from the nozzle is broken into liquid drops wrapping the particles;
the first detection unit is used for analyzing the scattered light signal, and the first detection unit comprises a second detector which is used for detecting the scattered light signal and acquiring the time t when the particle reaches a detection point1；
The second laser is used for emitting at least one laser, the laser irradiates the liquid flow breaking point, and the particles passing through the breaking point are irradiated by the laser to generate a second optical signal, wherein the second optical signal comprises a second fluorescent signal;
the second detection unit is used for analyzing the second fluorescence signal and comprises a third detector which is used for detecting the second fluorescence signal and is used for acquiring the time t when the particles reach the breaking point2；
The image acquisition unit is used for acquiring a shape image of the liquid flow flowing out of the nozzle;
the charging device is used for charging the liquid drops at the breaking points;
the sorting plate is used for placing the particles into a specified position by utilizing the characteristics of the particles.
Optionally, the first detection unit further includes a first detector, and the first detector is configured to detect the scattered light signal or the first fluorescence signal, and is configured to obtain a time when the particle reaches a detection point or obtain fluorescence characteristic information of the particle.
Optionally, the liquid drop delay calculating device further comprises a speed measuring device, and the speed measuring device is used for calculating the flow speed of the particles in the liquid flow; the speed measuring device comprises a third laser, a fourth detector and a fifth detector;
optionally, the third laser is configured to emit laser light and obtain a first irradiation spot at a first point of the liquid flow, and a third fluorescence signal is generated when a particle in the liquid flow passes through the first irradiation spot;
optionally, the fourth laser emits laser light and obtains a second irradiation spot at a second point of the liquid flow, and the liquid optionally generates a fourth fluorescence signal when particles in the flow pass through the second irradiation spot;
optionally, the fifth detector is configured to detect the third fluorescence signal, and is configured to acquire a time t3 when the particle passes through the first irradiation spot;
optionally, the fourth detector is configured to detect the fourth fluorescence signal, and is configured to acquire a time t4 when the particle passes through the second irradiation spot.
The speed measuring device further comprises a reflecting device, and the reflecting device is used for reflecting the laser beam emitted by the first laser to replace the laser emitted by the third laser and the laser emitted by the fourth laser; the reflecting device comprises at least two reflecting mirrors;
optionally, the one mirror is configured to reflect at least one laser light emitted by the first laser to form a first reflected light;
optionally, the other mirror is configured to reflect the first reflected light to form a second reflected light, and reflect the second reflected light at the first point and/or the second point of the liquid flow to obtain the first illumination spot and/or the second illumination spot.
Optionally, the system further comprises an illumination unit, wherein the at least one illumination unit is used for providing illumination for the image acquisition unit, and the exposure time of the illumination unit is less than 5 microseconds.
Correspondingly, the invention also provides a method for carrying out liquid drop delay calculation by using the liquid drop delay calculation device, which comprises the following steps:
a laser emits at least one laser, the laser irradiates the particles at the detection point of the flow chamber, and the particles are irradiated by the laser to generate a first optical signal, wherein the first optical signal comprises at least one scattered light signal and a first fluorescence signal;
detecting the scattered light signal by a second detector for obtaining the time t when the particle reaches the detection point1Or detecting the scattered light signal and the first fluorescence signal by using a first detector to acquire the time when the particles reach a detection pointTime t1And fluorescence signature information of the particles;
the second laser emits at least one laser, the laser irradiates the breaking point liquid drop, and the particles passing through the breaking point liquid drop are irradiated by the laser to generate a second optical signal, wherein the second optical signal comprises a second fluorescence signal;
detecting the second fluorescence signal with a third detector for obtaining the time t for the particle to reach the break point2；
Time Δ t of droplet delay t2-t1。
Optionally, before calculating the time Δ t of the droplet delay, a flow velocity V of the particles in the droplet needs to be detected for determining whether the liquid flow is in a stable state or an unstable state, where the flow velocity V is calculated as follows:
V＝d/△t1
wherein d is the spacing between the first and second illumination spots, Δ t1Is the time interval between the passage of the particle through the first and second illumination spots.
Optionally, the particle passes through the time interval Δ t between the first and second illumination spots1The calculation formula of (a) is as follows:
△t1＝t4-t3
wherein t is4Is the time of the particle passing through the second illumination spot, t3Is the time that the particle passes through the first illumination spot.
Optionally, a calculation formula of a distance d between the first illumination spot and the second illumination spot is as follows:
d＝n×u/m
n denotes a pixel of an interval between the first irradiation spot and the second irradiation spot, u denotes a pixel size of the image pickup unit, and m denotes a magnification of a lens in the image pickup unit.
Compared with the prior art, the invention has the following beneficial technical effects:
according to the invention, the first detector, the second detector and the third detector are arranged, so that the delay time of the liquid drops can be directly measured, the delay time of the liquid drops is prevented from being adjusted, the advantages of short time consumption and material consumption saving are achieved, and the sorting efficiency is greatly improved.
The invention can calculate the flow speed of the particles in the liquid drop by arranging the speed measuring device, judges whether the liquid flow is in a stable state or not according to the measuring result, and accurately calculates the liquid drop time when the liquid flow is in the stable state, thereby obtaining the accurate liquid drop delay.
The invention can prevent direct light signals from entering the forward detector by arranging the reflecting device, so that the liquid drop delay is accurate.
Drawings
Fig. 1 shows a schematic structural diagram of a liquid drop delay calculation device according to an embodiment of the present invention.
Fig. 2 is a schematic diagram showing a flow image obtained at a break-off point in a droplet delay calculating device according to an embodiment of the present invention.
Fig. 3 is a schematic diagram showing calculation of a droplet delay time in the droplet delay calculating device according to the embodiment of the present invention.
FIG. 4 is a diagram showing a flow velocity measurement scheme of microspheres in a droplet delay computing device according to an embodiment of the invention.
FIG. 5 is a schematic diagram of a flow image of microspheres obtained during measurement of flow rate of droplets in a droplet delay calculating device according to an embodiment of the present invention.
FIG. 6 is a schematic diagram showing the measurement of the flow velocity of the microspheres in the droplets in the droplet delay calculation device according to the embodiment of the invention.
Fig. 7 is a schematic diagram showing a first embodiment of a reflecting device in a liquid drop delay calculating device according to an embodiment of the present invention.
Fig. 8 is a schematic diagram of a second embodiment of a reflecting device in a droplet delay calculating device according to an embodiment of the present invention.
Fig. 9 is a schematic diagram showing a third embodiment of a reflecting means in a droplet delay calculating device according to an embodiment of the present invention.
In the drawings, the reference numbers: 101. a first laser; 102. a first lens; 103. a flow chamber; 104. a second lens; 105. a first detector; 106. a first diaphragm; 107. a third lens; 108. a second detector; 109. a nozzle; 110. a stroboscopic light source; 111. an image acquisition unit; 112. a second laser; 113. a second diaphragm; 114. detecting points; 115. a third detector; 116. a third diaphragm; 117. a first optical filter; 118. a charging device; 119. a breaking point; 120. a sorting plate; 121. collecting the test tubes; 122. a waste liquid collecting bin;
2. a speed measuring device; 201. a second optical filter; 202. a fourth lens; 203. a fourth diaphragm; 204. a fourth detector; 205. a fifth detector; 206. a third laser; 207. a fourth laser;
3. a reflecting device; 301. a first reflector; 302. a fifth lens; 303. a third optical filter; 304. a second reflector; 305. and a fourth filter.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more clearly understood, the following detailed description is made of a droplet delay calculating device and a calculating method thereof according to the present invention with reference to the accompanying drawings and the detailed description. The advantages and features of the present invention will become more apparent from the following description. It is to be noted that the drawings are in a very simplified form and are all used in a non-precise scale for the purpose of facilitating and distinctly aiding in the description of the embodiments of the present invention. To make the objects, features and advantages of the present invention comprehensible, reference is made to the accompanying drawings. It should be understood that the structures, ratios, sizes, and the like shown in the drawings and described in the specification are only used for matching with the disclosure of the specification, so as to be understood and read by those skilled in the art, and are not used to limit the implementation conditions of the present invention, so that the present invention has no technical significance, and any structural modification, ratio relationship change or size adjustment should still fall within the scope of the present invention without affecting the efficacy and the achievable purpose of the present invention.
The first embodiment is as follows:
fig. 1 is a schematic structural diagram showing a liquid drop delay calculation device according to an embodiment of the present invention.
Referring to fig. 1, the droplet delay calculating device includes a first laser 101, a flow chamber 103, a nozzle 109, a first detecting unit, a second laser 112, a second detecting unit, an image acquiring unit 111, and a sorting plate 120.
The first laser 101 is used to emit at least one laser beam that is irradiated onto the particles at the detection point 114 in the flow cell 103 (the laser beam is not limited to being irradiated only at the detection point 114, and it may be irradiated at any point in the flow cell 103, and the detection point 114 is used only as described in the present embodiment). In the device for calculating the delay of a droplet provided in this embodiment, the first laser 101 is a single-wavelength laser, and the excitation wavelength of the illumination is selected by replacing the laser with a different wavelength.
In another embodiment of the present invention, the first laser 101 may be a plurality of lasers, and is used as an illumination excitation light source. Or a specific wavelength combination is selected as an illumination excitation light source, or a white light laser is adopted, and a specific wavelength is selected as an illumination excitation light source through a light splitting/filtering device.
In the droplet delay calculating device provided in the first embodiment of the present invention, the droplet delay calculating device further includes a first lens 102, the first lens 102 is disposed on a side of the first laser 101 emitting laser light, and is configured to converge at least one laser light emitted by the first laser 101 to a point, and the first laser 101, the first lens 102, and the flow chamber 103 are on the same horizontal line. Preferably, the first lens 102 may be any one or combination of cylindrical lens, prism or diffractive optical element (e.g., shaping lens).
The flow cell 103 is used for limiting the flow of the particles, so that the particles passing through the detection point 114 are sequentially irradiated by the laser to generate a first optical signal, for example, the flow cell 103 allows only one of the particles to pass through, marks the particles by a fluorescein marking method, a fluorescent dye coloring method, or the like, and sequentially irradiates the marked particles by the laser to generate a first optical signal, wherein the first optical signal includes at least a scattered light signal and a first fluorescent signal. In the droplet delay calculation apparatus provided in this embodiment, the first optical signal is a forward scattered light signal, a side scattered light signal, and a first fluorescence signal.
The particles may be cells, bacteria, etc., including but not limited to biological particulate matter, such as microorganisms including bacteria such as E.coli, viruses such as tobacco mosaic virus, fungi such as yeast, ribosomes, chromosomes, mitochondria, organelles, etc., and biologically-relevant polymers such as nucleic acids, proteins, and complexes thereof; the particles may also be artificial particles such as latex particles, gel particles, industrial particles, and the like, including but not limited to particles formed of organic polymeric materials including polystyrene and the like, inorganic materials including glass, silica, magnetic materials, and the like, and metallic materials including metal colloids and the like, and the like. Although the particulate matter is generally spherical in shape, the particles may have a non-spherical shape. Further, the size, mass, etc. of the particles are also not limited. For example, the particles are encapsulated by the sample fluid and can flow in the flow chamber. Preferably, the diameter of the particles involved in the liquid drop delay calculation device of the embodiment of the present invention may be selected from 1um, 3um, 5um or other sizes.
A nozzle 109 is mounted at the outlet of the flow chamber 103 to break up the stream of liquid ejected through the nozzle 109 into droplets of encapsulated particles. The nozzle 109 has a conventional structure, and an ultrahigh frequency piezoelectric crystal is arranged on the nozzle 109, and vibrates after charging to break the discharged liquid flow into uniform droplets.
The following describes a specific structure of a first detection unit, which is used for analyzing the forward scattered light signal in the first optical signal, and the first detection unit includes a second detector 108, and the second detector 108 is used for detecting the forward scattered light signal to obtain the time t when the particle reaches the detection point 1141. Preferably, in the droplet delay calculating device provided in this embodiment, the second detector 108 is any one of a PDA and a PMT, where the PDA refers to a photodiode detector, the PMT refers to a photomultiplier tube, and both the PDA and the PMT are used to detect light energy and are selected according to the intensity of the energy.
In the droplet delay calculating device provided in the first embodiment of the present invention, the first detecting unit further includes a first diaphragm 106 and a third lens 107, and the forward scattered light is received by the second detector 108 after passing through the first diaphragm 106 and the third lens 107. Preferably, the first diaphragm 106 is a light blocking plate or a mirror, the third lens 107 may be a converging lens, and the first diaphragm 106, the third lens 107 and the second detector 108 are located at the same horizontal position of the first laser 101 and the first lens 102.
In the device for calculating the liquid drop delay provided in the first embodiment of the present invention, the first detecting unit further includes a first detector 105, and the first detector 105 is configured to detect a side scattered light signal and a first fluorescence signal to obtain a time t when a particle reaches the detecting point 1141Or to obtain fluorescence signature information for the particle.
For example, the particles are labeled with different fluorescein or fluorochromes, and the characteristics contained in different particles differ, which may be different cytoplasms, such as antigens, DNA, RNA, etc. The particles containing different features will have different fluorescence signature information when labeled. The fluorescence signature information includes one or more of the following characteristics of the particle: a fluorescence wavelength of the particle, a fluorescence energy of the particle, a fluorescein content of the particle, a feature of the particle, and a quantity of each feature of the particle.
Optionally, a second lens 104 may be further disposed before the first detector 105, and the second lens 104 may be a microscope objective.
The device for calculating the delay of the liquid drop provided by the first embodiment of the invention further comprises a second laser 112, wherein the second laser 112 is used for emitting at least one laser, the laser is irradiated to the breaking point 119 of the liquid flow, so that the particles passing through the breaking point 119 are irradiated by the laser to generate a second optical signal, and the second optical signal comprises a second fluorescence signal. Preferably, the second laser 112 may be emitted by a laser.
The device for calculating the liquid drop delay further comprises a second point detection unit for analyzing a second fluorescence signalThe second spot detection unit comprises a third detector 115, the third detector 115 being adapted for detecting a second fluorescence signal for obtaining the time t at which the particle reaches the break point 1192。
In the liquid drop delay calculating device provided in the first embodiment of the present invention, the second point detecting unit further includes a second diaphragm 113, a third diaphragm 116, and a first optical filter 117, the third diaphragm 116 is located before the receiving end of the third detector 115 that receives the second fluorescent signal, and the first optical filter 117 is located before the third diaphragm 116, preferably, the second diaphragm 113 is a light-shielding diaphragm, the light-shielding diaphragm can be automatically switched to an open state and a closed state, and the image obtaining unit 11 can obtain the image of the bright spot at the center of the breaking point 119 only when the light-shielding diaphragm is opened. The third aperture 116 is a field stop for blocking signals other than the illumination spot, and the first filter 117 allows only the excited second fluorescence signal to pass through.
The liquid drop delay calculation device provided by the first embodiment of the invention further comprises a graph acquisition unit 111 and an illumination unit, wherein the graph acquisition unit 111 is used for acquiring a topography image of the liquid stream flowing out of the nozzle. Preferably, the pattern obtaining unit 111 in the droplet delay calculating device provided in the first embodiment of the present invention is a monitoring camera, the illuminating unit in the droplet delay calculating device provided in the first embodiment of the present invention is a stroboscopic light source 110, the stroboscopic light source 110 may be any one of an LED, an LD, or a pulse laser, an exposure time of the light source is less than 5 microseconds, and the stroboscopic light source 110 is configured to couple laser light emitted by the second laser 112 to irradiate particles at the breaking point 119, so that energy of a signal received by the third detector 115 is more, detection efficiency can be improved, and a more accurate detection effect can be obtained.
The device for calculating the time delay of the liquid drop further comprises a sorting plate 120 and a charging device 118, wherein the counter-sorting plate 120 is used for placing the particles into the designated positions by utilizing the characteristics of the particles, and the charging device 118 is used for charging the liquid drop containing the particles at the breaking point, so that the liquid drop containing the particles is charged after being separated from the flow chamber 103. Preferably, the sorting plate is a deflecting electrode plate, and the deflecting electrode is used for attracting or repelling the charged particle-containing droplets so that the charged particle-containing droplets are deflected, or the uncharged particle-containing droplets are not deflected, so that each particle-containing droplet falls into a designated position, respectively, in the first embodiment, the charged particle-containing droplets fall into the collecting tube 121, and the uncharged particle-containing droplets fall into the waste liquid collecting bin 122.
Referring to fig. 2, the position of the break-off point 119 of the droplet delay calculating device according to the first embodiment of the present invention is determined by the image obtaining unit 111. Specifically, the graph obtaining unit 111 obtains a stable liquid flow image, the liquid flow image includes an image before droplet break and an image after droplet break, and the break point 119 is a droplet to be broken. The attitude of the second laser 112 is adjusted so that the irradiation spot is located at the center of the droplet in the break-off point 119, and when the laser of the second laser 112 irradiates the center of the droplet, an irradiation spot appears, and when a particle passes through the irradiation spot, a second fluorescence signal is excited and received by the third detector 115.
Referring to fig. 4, a liquid drop delay calculating device provided by an embodiment of the present invention includes a speed measuring device 2, where the speed measuring device 2 is used to calculate a flow velocity of a particle in a liquid flow. The speed measuring device 2 comprises a third laser 206, a fourth laser 207, a fourth detector 204 and a fifth detector 205.
The third laser 206 is configured to emit laser light and obtain a first illumination spot at a first point of the fluid stream, wherein a particle in the fluid stream passes through the first illumination spot to generate a third fluorescence signal. The fourth laser 207 emits laser light and acquires a second illumination spot at a second point in the fluid stream, and a fourth fluorescence signal is generated when a particle in the fluid stream passes through the second illumination spot.
The fifth detector 205 is configured to detect the third fluorescence signal, and is configured to obtain a time t3 when the particle passes through the first irradiation spot; the fourth detector 204 is arranged to detect a fourth fluorescence signal for acquiring the time t4 at which the particle passes the second illumination spot. Preferably, the fourth detector 204 and the fifth detector 205 are any one of photodiodes, avalanche diodes, or photomultipliers, which have the advantage of high sensitivity, can detect extremely weak fluorescence signals, and have extremely high response speed in nanoseconds (ns).
In the speed measuring device 2, the first irradiation light spot and the second irradiation light spot can be displayed in a droplet image as shown in fig. 4, the droplet image is captured by the pattern obtaining unit 111, and the distance d between the first irradiation light spot and the second irradiation light spot can be calculated by using the droplet image.
In the embodiment of the present invention, the speed measuring device 2 further includes a second optical filter 201, a fourth lens 202, and a fourth diaphragm 203, and preferably, the second optical filter 201 is a long-pass optical filter, and the long-pass optical filter is configured to prevent wavelengths of laser beams emitted by the third laser 206 and the fourth laser 207 from passing through, so that only the third fluorescence signal and the fourth fluorescence signal pass through. The fourth diaphragm 203 is used for eliminating stray light except for the first irradiation light spot and the second irradiation light spot, and ensuring that the received signal is completely a third fluorescence signal excited by the first irradiation light spot irradiating the particles or a fourth fluorescence signal excited by the second irradiation light spot irradiating the particles.
Referring to fig. 7, in another embodiment of the present invention, the speed measuring device further includes a reflection device 3, and the reflection device 3 is used for reflecting the laser beam emitted by the first laser 101 to replace the laser beams emitted by the third laser 206 and the fourth laser 207.
Specifically, the reflecting device includes at least two reflecting mirrors, the droplet delay calculating device provided in this embodiment is a first reflecting mirror 301 and a second reflecting mirror 304, the first reflecting mirror 301 is configured to reflect the laser beam emitted by the first laser to form a first reflected light, the second reflecting mirror 304 is configured to reflect the first reflected light to form a second reflected light, and the second reflected light is reflected at the first point and/or the second point of the liquid flow, so as to obtain the first irradiation spot and/or the second irradiation spot.
Optionally, a fifth lens 302 may be disposed between the first mirror 301 and the second mirror 304, and the fifth lens 302 is an enlarging lens for enlarging the droplet. The first mirror 301 may prevent a direct optical signal from entering the second detector 108, the first mirror 301 and the second mirror 304 may be any one of a plane mirror and a spherical mirror, and when the first mirror 301 and the second mirror 304 are both spherical mirrors, the fifth lens 302 may not be required. In the droplet delay calculating apparatus provided in the first embodiment of the present invention, the first reflecting mirror 301, the second reflecting mirror 304, and the fifth lens 302 are located at the same position in the vertical direction.
Referring to fig. 8 and 9, optionally, in another embodiment of the present invention, the first reflector 301 may be disposed before the third lens 107 or after the third lens 107 is disposed.
Referring to fig. 9, when the first reflector 301 is disposed behind the third lens 107, the reflecting device 3 may further include a fourth filter 305, the fourth filter 305 is disposed on one side of the second reflector 304, and the fourth filter 305 is a band-pass filter or a short-pass filter, which only allows two paths of direct light to irradiate onto the liquid stream.
The liquid drop calculating method realized based on the liquid drop delay calculating device comprises the following steps:
referring to fig. 1, 2 and 3, the first laser 101 is controlled to emit a laser beam, and the laser beam irradiates a particle at a detection point 114 in the flow chamber 103, so that the particle is irradiated by the laser beam to generate a first optical signal, wherein the first optical signal is a forward scattered light signal, a side scattered light signal and a first fluorescent signal.
Detecting the forward scattered light signal with a second detector 108 for obtaining the time t at which the particle reaches a detection point1。
The side scatter light signal and the first fluorescence signal are then detected by a first detector 105 to obtain the time t at which the particle reaches the detection point1And fluorescence characteristic information of the particles.
The second laser 112 emits a laser beam, the laser is irradiated to the break-off point 119 liquid drop, so that the particles passing through the break-off point 119 liquid drop are irradiated by the laser to generate a second optical signal, and the second optical signal comprises a second fluorescence signal;
detecting the second fluorescence with a third detector 115Signal for obtaining the time t for the particle to reach the breaking point2；
Referring to fig. 3, the time Δ t of the droplet delay is t2-t1。
Before calculating the time Δ t of the liquid drop delay, the flow velocity V of the particles in the liquid drop needs to be detected for judging whether the liquid flow is in a stable state or an unstable state, and the calculation formula of the flow velocity V is as follows:
V＝d/△t1
wherein d is the spacing between the first and second illumination spots, Δ t1Is the time interval between the passage of the particle through the first and second illumination spots.
Referring to fig. 5 and 6, the particles pass through the time interval Δ t between the first irradiation spot and the second irradiation spot1The calculation formula of (a) is as follows:
△t1＝t4-t3
wherein t is4Is the time of the particle passing through the second illumination spot, t3Is the time that the particle passes through the first illumination spot.
Referring to fig. 5, d can be captured by the pattern obtaining unit 111 to obtain a droplet image, and the droplet image can be used to calculate the distance d between the first illumination spot and the second illumination spot, which is as follows:
d＝n×u/m
n denotes a pixel of an interval between the first irradiation spot and the second irradiation spot, u denotes a pixel size of the image pickup unit, and m denotes a magnification of a lens in the image pickup unit.
The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Claims (8)
1. A droplet delay calculating device, characterized in that: the device comprises at least one first laser (101), a flow chamber (103), a nozzle (109), a first detection unit, a second laser (112), a second detection unit, a speed measuring device (2), an image acquisition unit (111), a charging device (118) and a sorting plate (120);
the first laser (101) is used for emitting at least one laser, and the laser irradiates on the particles in the flow chamber;
the flow chamber (103) is used for limiting the flow of the particles, so that the particles are sequentially irradiated by the laser to generate a first optical signal, and the first optical signal comprises at least one scattered optical signal and a first fluorescent signal;
the nozzle (109) is arranged at the outlet of the flow chamber (103) and breaks the liquid flow sprayed out from the nozzle (109) into liquid drops wrapped with particles;
the first detection unit is used for analyzing the scattered light signal, the first detection unit comprises a second detector (108), the second detector (108) is used for detecting the scattered light signal, and the time t1 when the particle reaches a detection point is obtained;
the second laser (112) is used for emitting at least one laser, the laser irradiates to the liquid flow breaking point, the particles passing through the breaking point are irradiated by the laser to generate a second light signal, and the second light signal comprises a second fluorescence signal;
the second detection unit is used for analyzing the second fluorescence signal, the second detection unit comprises a third detector (115), the third detector (115) is used for detecting the second fluorescence signal, and the time t of the particles reaching the breaking point is obtained2；
The speed measuring device (2) comprises a third laser (206), a fourth laser (207), a fourth detector (204) and a fifth detector (205); wherein
The third laser (206) is used for emitting laser and acquiring a first irradiation spot on a first point of the liquid flow, and a third fluorescence signal is generated when a particle in the liquid flow passes through the first irradiation spot;
the fourth laser (207) emits laser light and acquires a second irradiation spot on a second point of the liquid flow, and a fourth fluorescence signal is generated when particles in the liquid flow pass through the second irradiation spot;
the fifth detector (205) is arranged for detecting the third fluorescence signal for obtaining a time t at which the particle passes the first illumination spot3；
The fourth detector (204) is used for detecting the fourth fluorescence signal and acquiring the time t when the particles pass through the second irradiation spot4；
The image acquisition unit (111) is used for acquiring a shape image of the liquid flow flowing out of the nozzle;
the charging device (118) is used for charging the liquid drops at the breaking points;
the sorting plate (120) is used for placing the particles into a designated position by utilizing the characteristics of the particles.
2. The drop delay calculation device of claim 1, wherein: the first detection unit further comprises a first detector (105), the first detector (105) is used for detecting the scattered light signal or the first fluorescence signal, and is used for acquiring the time when the particle reaches a detection point or acquiring the fluorescence characteristic information of the particle.
3. The drop delay calculation device of claim 1, wherein: the speed measuring device further comprises a reflecting device (3), wherein the reflecting device (3) is used for reflecting the laser beam emitted by the first laser (101) to replace the laser emitted by the third laser (206) and the fourth laser (207); the reflecting device (3) comprises at least two reflecting mirrors;
one of the mirrors is used for reflecting at least one laser light emitted by the first laser (101) to form a first reflected light;
and the other reflector is used for reflecting the first reflected light to form second reflected light, and reflecting the second reflected light at the first point and/or the second point of the liquid flow to obtain a first illumination spot and/or a second illumination spot.
4. The drop delay calculation device of claim 1, wherein: the device also comprises an illumination unit, wherein the illumination unit is used for providing illumination for the image acquisition unit, and the exposure time of the illumination unit is less than 5 microseconds.
5. A method for calculating the delay of a liquid drop by using the liquid drop delay calculating device of any one of claims 1 to 4, which is characterized by comprising the following steps:
a laser emits at least one laser, the laser irradiates the particles at the detection point of the flow chamber, and the particles are irradiated by the laser to generate a first optical signal, wherein the first optical signal comprises at least one scattered light signal and a first fluorescence signal;
detecting the scattered light signal by a second detector for obtaining the time t when the particle reaches the detection point1Or detecting the scattered light signal and the first fluorescence signal by using a first detector to obtain the time t when the particles reach the detection point1And fluorescence signature information of the particles;
the second laser emits at least one laser, the laser irradiates the breaking point liquid drop, and the particles passing through the breaking point liquid drop are irradiated by the laser to generate a second optical signal, wherein the second optical signal comprises a second fluorescence signal;
detecting the second fluorescence signal with a third detector for obtaining the time t for the particle to reach the break point2；
Time of delay of liquid drop t2-t1。
6. A method of performing a droplet delay calculation in a droplet delay calculation apparatus according to claim 5, wherein: before calculating the time delta t of the liquid drop delay, the flow velocity V of the particles in the liquid drop needs to be detected for judging whether the liquid flow is in a stable state or an unstable state, and the calculation formula of the flow velocity V is as follows:
V＝d/Δt1
wherein d is the separation between the first and second illumination spots, Δ t1Is the time interval between the passage of the particle through the first and second illumination spots.
7. A method of performing a droplet delay calculation in a droplet delay calculation apparatus according to claim 6, wherein: the calculation formula of the time interval for the particle to pass through the first irradiation spot and the second irradiation spot is as follows:
Δt1＝t4-t3
wherein t is4Is the time of the particle passing through the second illumination spot, t3Is the time that the particle passes through the first illumination spot.
8. A method of performing a droplet delay calculation in a droplet delay calculation apparatus according to claim 6, wherein: the distance d between the first illumination spot and the second illumination spot is calculated according to the following formula:
d＝n×u/m
n denotes a pixel of an interval between the first irradiation spot and the second irradiation spot, u denotes a pixel size of the image pickup unit, and m denotes a magnification of a lens in the image pickup unit.
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| CN112903569A (en) * | 2021-01-20 | 2021-06-04 | 贝克曼库尔特生物科技（苏州）有限公司 | System and method for calculating drop delay time and sorting device |
| US20220236164A1 (en) * | 2021-01-25 | 2022-07-28 | Becton, Dickinson And Company | Method and systems for determing drop delay using scatter signals across spatially separated lasers |
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