CN112710598A

CN112710598A - Flow cytometry sorting instrument and flow cytometry sorting method

Info

Publication number: CN112710598A
Application number: CN202011522686.2A
Authority: CN
Inventors: 潘文强; 蓝科; 于大维
Original assignee: Shanghai Micro Electronics Equipment Co Ltd
Current assignee: Shanghai Micro Electronics Equipment Co Ltd
Priority date: 2020-12-21
Filing date: 2020-12-21
Publication date: 2021-04-27

Abstract

The embodiment of the invention discloses a flow cytometry sorting instrument and a flow cytometry sorting method, wherein the flow cytometry sorting instrument comprises a liquid drop delay time measuring module and a liquid drop shunting module; the liquid drop delay time measuring module is used for measuring liquid drop delay time according to the time of a liquid flow passing through a liquid drop breaking point; the droplet delay time measurement module feeds forward the droplet delay time to the droplet diversion module to control the time at which the droplets are charged. By adopting the technical scheme, the liquid drop delay time is measured by the liquid drop delay time measuring module according to the time of the liquid flow passing through the liquid drop fracture point; the delay time of the liquid drops is fed forward to the liquid drop distribution module, so that the delay time of the liquid drops can be accurately obtained by the liquid drop distribution module, the time for charging the liquid drops is accurately controlled, the charged liquid drops can accurately enter a liquid drop sorting channel, the liquid drop sorting accuracy is high, and the cell sorting accuracy in the liquid drops is high.

Description

Flow cytometry sorting instrument and flow cytometry sorting method

Technical Field

The embodiment of the invention relates to the technical field of cell sorting, in particular to a flow cytometry sorting instrument and a flow cytometry sorting method.

Background

Flow cytometers are typical particle analysis sorting devices that rely on the flow of cells or other particles in a liquid flow stream to determine one or more characteristics of the particle under study. For example, a liquid sample containing cells is passed through a flow cytometer in a rapidly moving liquid stream such that each cell successively passes through a sensing region to obtain characteristic information of the cell, after which cell sorting is achieved by charging a droplet containing the cell, which is deflected under the influence of a subsequent electric field.

The charging time of the liquid drop is determined by the delay time of the liquid drop, the delay time of the liquid drop can not be directly measured in the prior art, whether the delay time of the liquid drop is accurate or not is deduced by processing a large amount of data of information contained in the sorted cells, the consumed time is long, experimental articles such as microspheres and the like are consumed greatly, and the efficiency is low; and meanwhile, the delay time of the liquid drop cannot be acquired in real time.

Disclosure of Invention

In view of this, embodiments of the present invention provide a flow cytometer and a flow cytometry sorting method for directly measuring a delay time of a droplet, accurately controlling a charging time of the droplet, and improving sorting efficiency.

In a first aspect, an embodiment of the present invention provides a flow cytometer, including a droplet delay time measurement module and a droplet splitting module;

the liquid drop delay time measuring module is used for measuring liquid drop delay time according to the time of a liquid flow passing through a liquid drop breaking point;

the droplet delay time measurement module feeds forward the droplet delay time to the droplet diversion module to control the time at which the droplets are charged.

Optionally, the droplet delay time measuring module includes a detection point measuring submodule, a fracture point measuring submodule and a droplet delay time calculating submodule;

the detection point measuring submodule is used for measuring the first time when the liquid flow passes through the liquid flow detection point;

the break-off point measuring submodule is used for measuring a second time for the liquid flow to break off and form a liquid drop;

the droplet delay time calculation submodule is used for calculating the droplet delay time according to the first time and the second time.

Optionally, the detection point measurement submodule is configured to detect a first detection signal of the liquid flow passing through the liquid flow detection point;

the fracture point measuring submodule comprises a fracture position monitoring unit; the break-off position monitoring unit is used for determining the break-off point position of the liquid drop; the break-off point measuring submodule is used for detecting a second detection signal of the liquid drop passing through the break-off point position of the liquid drop;

the droplet delay time calculation submodule is used for recording a first receiving time of the first detection signal and a second receiving time of the second detection signal, and calculating droplet delay time according to the first receiving time and the second receiving time.

Optionally, the first detection signal and the second detection signal are both fluorescence signals excited by the detection particles;

alternatively, the first detection signal and the second detection signal are both scattering signals excited by the detection particles.

Optionally, the fracture position monitoring unit includes a monitoring camera and a stroboscopic light source;

the monitoring camera is used for acquiring a liquid drop moving image;

the stroboscopic light source is used for providing an illumination signal for the monitoring camera.

Optionally, the stroboscopic light source includes a light emitting diode or a semiconductor laser, and an exposure time of the stroboscopic light source is matched with a liquid flow velocity.

Optionally, the exposure time T satisfies T <5 μ s.

Optionally, the detection point measuring sub-module includes a first laser light source and a forward detection signal receiving unit.

Optionally, the fracture point measuring sub-module includes a second laser light source, and an emission angle of the second laser light source is adjustable.

Optionally, the detection point measuring submodule further includes a light shielding diaphragm, and the light shielding diaphragm is configured to shield the laser beam emitted by the first laser light source from directly irradiating the forward detection signal receiving unit.

Optionally, the droplet delay time measuring module further includes a light path reflector set, where the light path reflector set is configured to reflect the first laser light source to the droplet breaking point, so as to excite the detection particles passing through the droplet breaking point;

the posture of the light path reflector set is adjustable.

Optionally, the optical path reflecting mirror group includes a first reflecting mirror and a second reflecting mirror, and the first reflecting mirror is located on the optical path of the first laser light source and is configured to reflect the laser beam emitted by the first laser light source to form a first reflected light beam; the second reflecting mirror is positioned on the light path of the first reflecting beam and used for reflecting the first reflecting beam to form a second reflecting beam and controlling the second reflecting beam to be at least incident to the liquid drop breaking point.

Optionally, the angles of the first mirror and the second mirror are adjustable.

Optionally, the optical path reflecting mirror group further includes a first optical filter;

the first optical filter is positioned on the light path of the first reflected light beam and is used for filtering interference light in the first reflected light beam; or the first optical filter is positioned on the light path of the second reflected light beam and is used for filtering interference light in the second reflected light beam.

Optionally, the first mirror comprises a plane mirror, the second mirror comprises a plane mirror,

or the first reflector comprises a spherical reflector and the second reflector comprises a spherical reflector.

Optionally, the first reflecting mirror includes a plane reflecting mirror, the second reflecting mirror includes a plane reflecting mirror, and the optical path reflecting mirror group further includes an amplifying lens;

the magnifying lens is located on the light path of the first reflected light beam.

Optionally, the forward detection signal receiving unit includes a forward lens, and the first mirror is located at a focal plane position of the forward lens.

Optionally, the first laser light source is configured to emit a first laser beam and a second laser beam;

the first reflecting mirror is used for reflecting the first laser beam to form a third reflected light beam and reflecting the second laser beam to form a fourth reflected light beam;

the second reflecting mirrors are respectively positioned on the light paths of the third reflected light beam and the fourth reflected light beam and are used for reflecting the third reflected light beam to a first position on the liquid flow path and reflecting the fourth reflected light beam to a second position on the liquid flow path, wherein the first position is positioned between the liquid flow detection point and the second position, the second position is positioned on one side of the liquid drop breaking point close to the liquid flow detection point, or the second position is coincided with the liquid drop breaking point.

Optionally, the droplet delay time measuring module further includes a droplet speed measuring sub-module, and the droplet speed measuring sub-module is configured to determine whether the liquid flow reaches a stable and stable state according to the first position, the second position, the time when the third reflected light beam is incident on the first position, and the time when the fourth reflected light beam is incident on the second position.

Optionally, the droplet velocity measurement sub-module is specifically configured to calculate a first average velocity of the droplet between the first position and the second position according to the time when the third reflected light beam is incident on the first position and the time when the fourth reflected light beam is incident on the second position, and determine that the liquid flow reaches a stable motion state when the first average velocity meets a preset requirement.

Optionally, the droplet speed measuring sub-module includes a second optical filter, a first detecting lens, a first field diaphragm, a second field diaphragm, a first detector and a second detector; the second optical filter and the first detection lens are simultaneously positioned on detection paths of the first detector and the second detector; the first field diaphragm is positioned on a detection path of the first detector; the second field stop is located in a detection path of the second detector.

Optionally, the detection point measurement sub-module includes a forward detection signal receiving unit and/or a lateral detection signal receiving unit;

the forward detection signal receiving subunit comprises a forward lens and a forward detector;

the lateral detection signal receiving subunit comprises a lateral lens and a lateral detector.

Optionally, the flow cytometer further comprises a fluid stream storage module, a droplet formation module, and a droplet sorting module;

the liquid flow storage module is used for storing the liquid flow, and the liquid flow flows through the liquid drop forming module and is broken into liquid drops at the positions of the liquid drop breaking points;

the liquid drop sorting module comprises a deflection electrode plate and a liquid drop receiving test tube, wherein the deflection electrode plate is used for controlling the charged liquid drops to deflect, and the liquid drop receiving test tube is used for receiving the deflected liquid drops.

In a second aspect, an embodiment of the present invention further provides a flow cytometer, including a droplet speed measurement module;

the liquid drop speed measurement module comprises a third laser light source, the third laser light source is used for emitting a third laser beam and a fourth laser beam, the third laser beam is incident to a third position on the liquid flow path where the liquid drop speed is measured, and the fourth laser beam is incident to a fourth position on the liquid flow path where the liquid drop speed is measured;

the liquid drop speed measuring module is used for determining whether the liquid flow reaches a stable and stable state according to the third position, the fourth position, the time of the third laser beam entering the third position and the time of the fourth laser beam entering the fourth position.

Optionally, the liquid drop velocity measurement module is specifically configured to calculate a second average velocity of the liquid drop between the third position and the fourth position according to the time when the third laser beam is incident on the third position and the time when the fourth laser beam is incident on the fourth position, and determine that the liquid flow reaches a stable motion state when the second average velocity meets a preset requirement.

Optionally, the liquid drop velocity measurement module further includes a fourth optical filter, a second detection lens, a third field diaphragm, a fourth detector, and a fifth detector; the fourth optical filter and the second detection lens are simultaneously positioned on detection paths of the fourth detector and the fifth detector; the third field diaphragm is positioned on a detection path of the fourth detector; the third field stop is located in a detection path of the fifth detector.

In a third aspect, an embodiment of the present invention further provides a flow cytometry sorting method applied to a flow cytometry sorter, where the flow cytometry sorter includes a detection point measurement module, a droplet delay time measurement module, and a droplet splitting module; the flow cytometric sorting method comprises the following steps:

the detection point measuring module acquires an optical detection signal of a sample when liquid flows through a liquid flow detection point, and acquires sorting target information according to the optical detection signal;

the liquid drop delay time measuring module measures liquid drop delay time according to the time of a liquid flow passing through a liquid drop breaking point; and the liquid drop shunting module receives the sorting target information and the liquid drop delay time, and sorts and charges the sorting target according to the liquid drop delay time.

Optionally, the flow cytometer further includes a liquid flow velocity measurement module, and when the liquid flow velocity measured by the liquid flow velocity measurement module reaches a stable state, the liquid drop breaking point measured by the liquid drop delay time measurement module is determined to be an accurate liquid drop breaking point.

The embodiment of the invention provides a flow cytometry sorter and a flow cytometry sorting method, wherein the flow cytometry sorter comprises a liquid drop delay time measuring module and a liquid drop shunting module; measuring the liquid drop delay time according to the time of the liquid flow passing through the liquid drop breaking point by a liquid drop delay time measuring module; and the delay time of the liquid drops is fed forward to the liquid drop shunting module, so that the delay time of the liquid drops can be accurately known by the liquid drop shunting module, the time for charging the liquid drops is accurately controlled, the charged liquid drops can accurately enter a liquid drop sorting channel, the liquid drop sorting accuracy is high, and the cell sorting accuracy in the liquid drops is high.

Drawings

Other features, objects and advantages of the invention will become more apparent upon reading of the detailed description of non-limiting embodiments made with reference to the following drawings:

FIG. 1 is a schematic diagram of a prior art flow cytometer;

FIG. 2 is a schematic diagram of a flow cytometer in accordance with an embodiment of the present invention;

FIG. 3 is a schematic diagram of another flow cytometer in accordance with an embodiment of the present invention;

FIG. 4 is a schematic structural diagram of a physical apparatus of a flow cytometer provided in an embodiment of the present invention;

FIG. 5 is a schematic illustration of a drop image provided by an embodiment of the present invention;

FIG. 6 is a schematic diagram of a drop delay provided by an embodiment of the present invention;

FIG. 7 is a schematic diagram of an apparatus for performing another flow cytometry sorting apparatus according to an embodiment of the present invention;

FIG. 8 is a schematic diagram of an alternative embodiment of a flow cytometer;

FIG. 9 is a schematic diagram of an alternative embodiment of a flow cytometer;

FIG. 10 is a schematic diagram of an alternative embodiment of a flow cytometer;

FIG. 11 is a schematic illustration of another drop image provided by an embodiment of the present invention;

FIG. 12 is a schematic view of the flow of fluid through a first position and a second position in accordance with an embodiment of the present invention;

FIG. 13 is a schematic diagram of an alternative embodiment of a flow cytometer;

FIG. 14 is a schematic diagram of an alternative embodiment of a flow cytometer;

FIG. 15 is a schematic illustration of another drop image provided by an embodiment of the present invention;

FIG. 16 is a schematic diagram of another flow cytometer in accordance with an embodiment of the present invention;

fig. 17 is a schematic flow chart of a flow cytometric sorting method according to an embodiment of the present invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention clearer, the technical solutions of the present invention will be fully described by the detailed description with reference to the accompanying drawings in the embodiments of the present invention. It is obvious that the described embodiments are a part of the embodiments of the present invention, not all embodiments, and all other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without inventive efforts fall within the scope of the present invention.

Fig. 1 is a schematic structural diagram of a flow cytometer in the prior art, and as shown in fig. 1, the flow cytometer includes a charging unit 1, a deflecting electrode plate 2, a collecting test tube 3, and a waste liquid collecting bin 4, droplets are charged by the charging unit 1 at a droplet breaking point, the charged droplets are deflected under the action of the deflecting electrode plate 2 and finally collected by the collecting test tube 3, and uncharged droplets fall into the waste liquid collecting bin 4. In the cell sorting process, the delay time of the liquid drop needs to be accurately known, and the time for charging the liquid drop is accurately known. If the droplet delay is accurate, the droplet containing the fluorescence signal is only present in the sorting channel; if the droplet delay is inaccurate, a fluorescent signal can also be detected in the non-sorting channel (i.e., waste collection bin). In the prior art, one scheme for acquiring the delay time of the liquid drop is to judge whether the delay time of the liquid drop is correct or not in an energy detection mode, the detection mode is not real-time detection but is analyzed by a statistical method, and the method has the limitation 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 method is long in time consumption, large in consumption of experimental supplies such as detection particles and the like, and low in efficiency. In the other scheme of obtaining the droplet delay time in the prior art, the droplet delay time is automatically obtained by detecting the fluorescent signal of the particles in the waste liquid collecting bin, specifically, the fluorescent signal intensity in the waste liquid corresponding to different droplet delay times is measured in a specified droplet delay range, and the droplet delay time corresponding to the lowest fluorescent signal intensity is the optimal droplet delay time of the system by combining with subsequent data processing. The data needs to process and calculate a large amount of data, the delay of liquid drops cannot be directly measured, the consumed time is long, experimental articles such as particles are detected, the consumption is high, and the efficiency is low.

Based on this, the embodiment of the invention provides a flow cytometer, which comprises a droplet delay time measuring module and a droplet shunting module; the liquid drop delay time measuring module is used for measuring liquid drop delay time according to the time of the liquid flow passing through the liquid drop breaking point; the droplet delay time measurement module feeds forward the droplet delay time to the droplet diversion module to control the time at which the droplets are charged. According to the flow cytometry sorting instrument provided by the embodiment of the invention, the liquid drop delay time is directly measured through the liquid drop delay time measuring module, and is fed forward to the liquid drop sorting module, so that the liquid drop sorting module can accurately obtain the liquid drop delay time, the liquid drop is accurately charged at a liquid drop fracture point, the liquid drop charging time is accurately controlled, the liquid drop sorting is accurate, and the liquid drop sorting efficiency is high; and the method is different from the existing method for reversely estimating the delay time of the liquid drops according to the sorting condition of the liquid drops, the method does not need to repeatedly operate for multiple times to reversely estimate the delay time of the liquid drops, the measurement of the delay time of the liquid drops is accurate, the measurement of the delay time of the liquid drops is simple, and the consumable material is saved.

The above is the core idea of the present invention, and the technical solution in the embodiment of the present invention will be clearly and completely described below with reference to the drawings in the embodiment of the present invention. All other embodiments obtained by a person of ordinary skill in the art based on the embodiments of the present invention without any creative work belong to the protection scope of the present invention.

Fig. 2 is a schematic structural diagram of a flow cytometer provided in an embodiment of the present invention, and as shown in fig. 2, the flow cytometer provided in an embodiment of the present invention includes a droplet delay time measurement module 10 and a droplet splitting module 20; the liquid drop delay time measuring module 10 is used for measuring the liquid drop delay time according to the time when the liquid flow passes through the liquid drop breaking point; the drop delay time measurement module 10 feeds forward the drop delay time to the drop diversion module 20 to control the time at which the drops are charged.

For example, the droplet break-off point can be understood as the position where the liquid changes from the liquid stream form to the droplet form during the flow, and the droplet delay time measuring module 10 is used to measure the droplet delay time according to the time when the liquid stream passes through the droplet break-off point. Specifically, the time for the liquid to enter the liquid flow storage module can be determined as the starting time of the droplet delay time, the time for the liquid flow to flow through the detection point is determined as the starting time of the droplet delay time, the time for the liquid to change from the liquid flow form to the droplet form is determined as the ending time of the droplet delay time, and the droplet delay time is determined according to the starting time and the ending time; alternatively, the time when the liquid changes from the liquid flow form to the droplet form is determined as the end time of the droplet delay time, and the droplet delay time is determined according to the start time and the end time. The liquid drop delay time measuring module 10 feeds the acquired liquid drop delay time forward to the liquid drop shunting module 20, the liquid drop shunting module 20 accurately grasps the liquid drop delay time, when cell sorting starts, the charged time of the liquid drop is determined according to the liquid drop delay time, the liquid drop is ensured to be charged at the position of a liquid drop fracture point, the charged liquid drop is deflected under the action of a deflection electrode plate and accurately enters a sorting channel, and high liquid drop sorting accuracy is ensured.

It can be understood that, in the flow cytometer provided in the embodiments of the present invention, the droplet delay time measurement module operates in the process before the formal sorting of the cells, and the droplet shunt module operates in the process of the formal sorting of the cells. The droplet delay time is determined by the droplet delay time measuring module before the formal cell sorting starts, and the droplet delay time is fed forward to the droplet shunting module, the droplet shunting module determines the charging time of the droplets according to the droplet delay time in the formal cell sorting process, so that the droplets are accurately charged at the droplet breaking point, the droplet charging time is accurately controlled, the droplet sorting is accurate, and the droplet sorting efficiency is high.

In summary, different from the method for reversely estimating the droplet delay time according to the sorting condition of the droplets in the prior art, the flow cytometer provided in the embodiment of the present invention directly measures the droplet delay time through the droplet delay time measurement module, and feeds the droplet delay time forward to the droplet sorting module, and the droplet sorting module can accurately grasp the droplet delay time, so as to ensure that the droplets are accurately charged at the droplet break point, the droplet charging time is accurately controlled, the droplet sorting is accurate, and the droplet sorting efficiency is high; and the method is different from the existing method for reversely estimating the delay time of the liquid drops according to the sorting condition of the liquid drops, the method does not need to repeatedly operate for multiple times to reversely estimate the delay time of the liquid drops, the measurement of the delay time of the liquid drops is accurate, the measurement of the delay time of the liquid drops is simple, and the consumable material is saved.

Alternatively, fig. 3 is a schematic structural diagram of another flow cytometer provided in the embodiment of the present invention, and as shown in fig. 3, on the basis of the above embodiment, the droplet delay measurement module 10 may include a checkpoint measurement submodule 11, a breakpoint measurement submodule 12, and a droplet delay time calculation submodule 13; the detection point measuring submodule 11 is used for measuring the first time when the liquid flow passes through the liquid flow detection point; the break-off point measuring submodule 12 is used for measuring a second time for the liquid flow to break off and form a liquid drop; the drop delay time calculation submodule 13 is configured to calculate a drop delay time based on the first time and the second time.

Illustratively, the embodiment of the invention determines the time of the liquid flow flowing through the detection point as the starting moment of the liquid drop delay time. Specifically, the detection point measuring submodule 11 is configured to measure a first time when the liquid flow passes through the liquid flow detection point, where the liquid flow detection point may be a detection position in the liquid flow storage module before the droplet break-off point, and record the time when the liquid flow passes through the liquid flow detection point as the first time; the break-off point measuring submodule 12 is used for measuring a second time when the liquid flow breaks to form the liquid drop, and recording the time when the liquid flow passes through the break-off point of the liquid drop as the second time; the droplet delay time calculation submodule 13 is configured to calculate a droplet delay time according to the first time and the second time, and specifically, to calculate a droplet delay time according to a difference between the second time and the first time.

In the above-mentioned technical aspects of the embodiments, fig. 4 is a schematic structural diagram of a physical device of a flow cytometer according to an embodiment of the present invention, fig. 5 is a schematic structural diagram of a droplet image according to an embodiment of the present invention, fig. 6 is a schematic structural diagram of a droplet delay time according to an embodiment of the present invention, and in conjunction with fig. 4-6, the checkpoint measuring submodule 11 is configured to detect a first detection signal when a fluid stream passes through a checkpoint of the fluid stream; the fracture point measuring submodule 12 includes a fracture position monitoring unit 121; the break-off position monitoring unit 121 is used for determining the position of a break-off point of the liquid drop; the break point measuring submodule 12 is used for detecting a second detection signal of the liquid drop passing through the break point position of the liquid drop; the liquid drop delay time calculation submodule is used for recording the first receiving time of the first detection signal and the second receiving time of the second detection signal and calculating the liquid drop delay time according to the first receiving time and the second receiving time.

For example, as shown in fig. 4, the detection point measuring submodule 11 may include a first laser light source 111 and a forward detection signal receiving unit 112, where the first laser light source 111 is configured to emit a first laser signal to the fluid flow detection point a1, the first laser signal excites the detection particles in the fluid flow to generate a first fluorescence signal, and the forward detection signal receiving unit 112 is configured to receive the first fluorescence signal, where the first fluorescence signal is the first detection signal. The first laser light source 111 may be 1 single-wavelength laser, and the illumination excitation wavelength is selected by replacing lasers with different wavelengths; or a plurality of lasers can be used as the illumination and excitation light source at the same time, or a specific wavelength combination is selected as the illumination and excitation light source; or may be a white light laser, and a specific wavelength is selected as the illumination excitation light source through the light splitting/filtering device, which is not limited in the embodiment of the present invention. The probe particles may be standard size microspheres with a diameter of 1 μm, 3 μm, 5 μm, or other sizes, which is not limited by the embodiments of the present invention.

The fracture point measuring sub-module 12 may include a fracture position monitoring unit 121, a second laser light source 122, and a second detection signal receiving unit 123; the break-off position monitoring unit 121 is used to determine the position of the drop break-off point a 2. The second laser light source 122 is configured to emit a second laser signal to the droplet breaking point a2, the second laser signal excites the detection particle in the droplet to generate a second fluorescence signal, and the second detection signal receiving unit 123 is configured to receive the second fluorescence signal, where the second fluorescence signal is the second detection signal.

A droplet delay time calculation submodule (not shown in the figure) is communicatively connected to the forward detection signal receiving unit 112 and the second detection signal receiving unit 123, respectively, for recording a first receiving time T1 of the first detection signal and a second receiving time T2 of the second detection signal, and determining a difference Δ T between the first receiving time T1 and the second receiving time T2 as a droplet delay time, as shown in fig. 6. Further, the droplet delay time calculation submodule is further configured to feed forward the droplet delay time Δ T to the droplet splitting module 20, when the cell sorting formally starts, the droplet splitting module 20 may prestore a first receiving time T1 of the first detection signal, and when the droplet delay time Δ T arrives, the droplet is charged at the droplet breaking point, and the droplet charging time is accurately controlled.

Optionally, as mentioned above, the first detection signal and the second detection signal may both be fluorescence signals excited by the detection particles, or the first detection signal and the second detection signal may also be scattering signals excited by the detection particles, and the scattering signals may include polarization information, for example, the first detection signal may also be a first forward scattering signal excited by the detection particles at the liquid stream detection point a1, and the second detection signal may also be a second forward scattering signal excited by the detection particles at the liquid droplet break-off point a 2. The specific types of the first detection signal and the second detection signal are not limited in the embodiment of the present invention, as long as it is ensured that the forward detection signal receiving unit 112 can receive the excitation signal of the detection particle based on the first laser signal at the liquid flow detection point a1, and the second detection signal receiving unit 123 can receive the excitation signal of the detection particle based on the second laser signal at the droplet breaking point a 2.

Alternatively, as shown with continued reference to fig. 4, the fracture position detection unit 121 may include a monitoring camera 1211 and a stroboscopic light source 1212; among other things, the monitoring camera 1211 may be used to acquire a droplet motion image; the strobe light source 1212 is used to provide an illumination signal for the monitoring camera 1211.

For example, in order to accurately determine the position of the droplet breaking point a2, the flow of the liquid stream may be monitored in the vicinity of the droplet breaking point by using the monitoring camera 1211, a droplet moving image is acquired, and the position of the droplet breaking point is clearly determined according to the droplet moving image. The droplet moving image comprises two parts of a droplet before breaking motion and a droplet after breaking motion, wherein the breaking point is the droplet just to be broken. Further, the emitting angle of the second laser source 122 is adjustable, and the emitting angle of the second laser source 122 is adjusted, so that the irradiation spot of the second laser signal is exactly located at the exact center of the droplet at the position of the droplet breaking point a2, as shown in fig. 5. When the second laser signal is applied to the droplet, the scattered light from the droplet is received by the monitoring camera 1211, so that a bright spot appears at the center of the droplet fracture point a 2. When the detection particles flow through the irradiation spot, the excited fluorescence signal is received by the second detection signal receiving unit 123.

Further, in the monitoring process of the monitoring camera 1211, in order to acquire a clear droplet moving image, the fracture position detection unit 121 may further include a strobe light source 1212, where the strobe light source 1212 is configured to provide an illumination signal to the monitoring camera 1211, so as to ensure that the monitoring area of the monitoring camera 1211 is in a bright field state, and the droplet moving image is clear. Optionally, the strobe light source 1212 may include a light emitting diode or a semiconductor laser, and the exposure time of the strobe light source 1212 is matched to the liquid flow velocity, ensuring that the monitoring camera 1211 can acquire a clear droplet moving image during the exposure time of the strobe light source 1212. Further, the exposure time T may satisfy T <5 μ s.

Optionally, with continuing reference to fig. 4, the detection point measuring submodule 11 provided in the embodiment of the present invention may further include a first light shielding diaphragm 113, where the first light shielding diaphragm 113 is configured to shield a laser beam emitted by the first laser light source 111 from directly irradiating the forward detection signal receiving unit 112, so as to ensure that the first detection signal received by the forward detection signal receiving unit 112 is only a fluorescent signal or a forward scattered light signal obtained by the detection particle based on the excitation of the first laser signal, avoid that the laser beam emitted by the first laser light source 111 directly irradiates the forward detection signal receiving unit 112, so as to cause interference on the first detection signal, and ensure that the first receiving time T1 is accurate.

Optionally, as shown in fig. 4, the detection point measurement sub-module 11 provided in the embodiment of the present invention may further include an illumination lens 114, where the illumination lens 114 is configured to adjust the first laser signal, for example, adjust a focusing power and a divergence angle of the first laser signal, so as to ensure that the first laser signal is focused well. Further, the illumination lens 114 may include a cylindrical mirror, a prism, or a diffractive optical element, and the embodiment of the present invention does not limit the specific type of the illumination lens 114.

Optionally, with continuing reference to fig. 4, the fracture point measurement sub-module 12 provided in the embodiment of the present invention may further include a second light shielding diaphragm 124, where the second light shielding diaphragm 124 is located on an exit path of the second laser signal, and the second light shielding diaphragm 124 may be automatically switched to an open state and a closed state. When the second light blocking diaphragm 124 is in the open state, a bright spot appears at the center of the droplet break-off point a2 by adjusting the emitting angle of the second laser source 122, and the monitor camera 1211 can obtain the pattern of the bright spot at the center of the droplet break-off point a 2.

Optionally, fig. 7 is a schematic structural diagram of another physical device of a flow cytometer according to an embodiment of the present invention, as shown in fig. 7, the droplet delay time measurement module 10 according to an embodiment of the present invention may further include an optical path mirror set 115, where the optical path mirror set 115 is configured to reflect the first laser light source 111 to the droplet breaking point a2 to excite the detection particle passing through the droplet breaking point a 2; the attitude of the optical path mirror assembly 115 is adjustable.

For example, as shown in fig. 7, in addition to the first light shielding diaphragm may be arranged to prevent the first laser signal emitted by the first laser source 111 from being directly incident on the front line detection signal receiving unit 112, a light path reflector set 115 may be arranged on a propagation path of the first laser signal, the light path reflector set 115 reflects the first laser signal to the droplet breaking point a2, and the laser detection particles generate the second detection signal at the droplet breaking point a2, so that not only the second receiving time may be obtained, but also the first laser source 111 is multiplexed into the second laser source 122, thereby ensuring that a set of laser devices is saved, simplifying the structure of the flow cytometer, and reducing the cost of the flow cytometer.

Further, the posture of the optical path mirror group 115 is adjustable, and by adjusting the posture of the optical path mirror group 115, the irradiation spot of the first laser signal passing through the optical path mirror group 115 is exactly located at the right center of the droplet at the position of the droplet break-off point a2, so that a bright spot appears at the center of the droplet break-off point a2, and it is ensured that the monitoring camera 1211 can obtain the pattern of the bright spot at the center of the droplet break-off point a 2; and the excited fluorescence signal is received by the second detection signal receiving unit 123 when the detection particles flow through the irradiation spot.

Specifically, as shown in fig. 7, the optical path mirror set 115 may include a first mirror 1151 and a second mirror 1152, wherein the first mirror 1151 is located on the optical path of the first laser light source and is configured to reflect the laser light beam emitted by the first laser light source 111 to form a first reflected light beam; the second mirror 1152 is positioned on the optical path of the first reflected beam to reflect the first reflected beam to form a second reflected beam and to control the second reflected beam to be incident on at least the drop break point a 2.

For example, the optical path mirror set 115 may include at least two mirrors to reflect the laser beam emitted from the first laser light source 111 to the droplet breaking point, and fig. 7 illustrates that the optical path mirror set 115 includes a first mirror 1151 and a second mirror 1152. As shown in fig. 7, the optical path reflecting mirror set 115 may include a first reflecting mirror 1151 and a second reflecting mirror 1152, the first reflecting mirror 1151 is configured to form the laser beam emitted from the first laser light source 111 into a first reflected beam and control the first reflected beam to be incident on the second reflecting mirror 1152, and the second reflecting mirror 1152 is configured to reflect the first reflected beam into a second reflected beam and stop the second reflected beam to be incident on the droplet breaking point a 2.

Further, the angles of the first mirror 1151 and the second mirror 1152 are adjustable, and by adjusting the angles of the first mirror 1151 and the second mirror 1152, the irradiation spot of the second reflected light beam is ensured to be exactly located at the exact center of the droplet at the position of the droplet breaking point a2, and the monitoring camera 1211 is ensured to obtain the pattern of the bright spot at the center of the droplet breaking point a 2; and the excited fluorescence signal is received by the second detection signal receiving unit 123 when the detection particles flow through the irradiation spot.

Optionally, as shown with continued reference to fig. 7, the optical path mirror assembly 115 may further include a first optical filter 1153; the first filter 1153 may be located on the optical path of the first reflected light beam, and is configured to filter interference light in the first reflected light beam; or the first optical filter 1153 may also be located on the optical path of the second reflected light beam for filtering out the interference light in the second reflected light beam, and fig. 7 only takes the example that the first optical filter 1153 is located on the optical path of the first reflected light beam as an example for explanation. By arranging the first optical filter 1153, it is ensured that the interference light is filtered, it is ensured that the second detection signals received by the second detection signal receiving unit 123 are excitation signals of detection particles based on the second reflected light beam, and it is ensured that the second receiving time T2 based on the second detection signals is accurately received. Optionally, the first filter 1153 may be a band pass filter or a short pass filter, which is not limited in the embodiment of the present invention, and only needs to filter interference light, such as fluorescence or scattered light excited by the detection particles, to ensure that the light incident to the droplet break point a2 only includes the second reflected light beam, and ensure that the second receiving time T2 is accurate.

Optionally, in the optical path mirror group provided in the embodiment of the present invention, the first mirror 1151 may include a plane mirror, and the second mirror 1152 may include a plane mirror, as shown in fig. 7; alternatively, the first mirror 1151 may comprise a spherical mirror and the second mirror 1152 may comprise a spherical mirror, as shown in FIG. 8. The first reflector 1151 and the second reflector 1152 are not limited in the embodiments of the present invention, for example, the first reflector 1151 and the second reflector 1152 may also be reflective prisms, and it is only necessary to ensure that the laser beam emitted from the first laser light source 111 can be reflected to the droplet breaking point a 2.

Further, since the spherical mirror itself has a magnifying function, when the first mirror 1151 comprises a plane mirror and the second mirror 1152 comprises a plane mirror, the optical path mirror group 115 may further comprise a magnifying lens 1154; the magnifying lens 1154 is located on the optical path of the first reflected light beam, and the second mirror 1152 may be located at the focal plane position of the magnifying lens 1154. The first reflected light beam is converged by the additional magnifying lens 1154, so that the light intensity received by the second reflecting mirror 1152 is ensured to be larger, and the intensity of the second reflected light beam is larger, so that the brightness of the irradiation light spot positioned at the droplet breaking point A2 is ensured to be larger, and the monitoring camera 1211 is facilitated to obtain the position of the droplet breaking point A2; meanwhile, the second reflected light beam with larger intensity excites the detection particles, so that the intensity of the second detection signal is ensured to be larger, and the accuracy of the second receiving time T2 based on the second detection signal is ensured to be higher.

Fig. 9 is a schematic structural diagram of another physical device of a flow cytometer according to an embodiment of the present invention, and as shown in fig. 9, the forward detection signal receiving unit 112 may include a forward lens 1121, and the first mirror 1151 is located at a focal plane position of the forward lens 1121.

Exemplarily, since the laser beam emitted by the first laser light source 111 is a parallel beam, the parallel beam converges at the focal plane of the forward lens 1121 after passing through the forward lens 1121, and the first reflector 1151 is disposed at the focal plane of the forward lens 1121, so as to ensure that the first reflector 1151 can completely reflect the direct beam emitted by the first laser light source 111, and thus, the first detection signal received by the forward detection signal receiving unit 112 is a fluorescent signal or a forward scattering signal of the detection particle based on the laser beam emitted by the first laser light source 111, and it is ensured that the first receiving time based on the first detection signal is not interfered by the direct light, and the first receiving time is accurate. Further, since the detection particles are changed based on the forward scattered light or the fluorescence of the laser beam emitted by the first laser light source 111 based on the propagation direction of the direct laser beam, the forward scattered light or the fluorescence is not converged at the focal plane position of the forward lens 1121, and the first reflector 1151 has a large influence on the forward scattered light or the fluorescence, and does not influence the forward detection signal receiving unit 112 to receive the detection signal.

It can be understood that, before the monitoring camera 1211 determines the droplet breaking point a2 according to the droplet moving image, the monitoring camera 1211 needs to first obtain a stable flow image, and the determined droplet breaking point a2 is the correct droplet breaking point only when the flow is determined to be in a stable state according to the flow image, otherwise, there may be a problem that the droplet is accidentally interrupted due to accidental factors to form the droplet breaking point, which may cause a misjudgment of the droplet breaking point. Next, description will be made of a case where the monitoring camera 1211 is determined to obtain a stable liquid flow image.

Fig. 10 is a schematic diagram of a physical device structure of another flow cytometer provided in an embodiment of the present invention, fig. 11 is a schematic diagram of another droplet image provided in an embodiment of the present invention, fig. 12 is a schematic diagram of a time when a fluid stream flows through a first position and a second position, as shown in fig. 10, 11 and 12, a first laser light source 111 is used for emitting a first laser beam and a second laser beam; the first reflecting mirror 1151 is used for reflecting the first laser beam to form a third reflected beam and reflecting the second laser beam to form a fourth reflected beam; the second mirror 1152 is located on the optical paths of the third reflected light beam and the fourth reflected light beam, respectively, and is configured to reflect the third reflected light beam to a first position A3 on the liquid flow path and reflect the fourth reflected light beam to a second position a4 on the liquid flow path, where the first position A3 is located between the liquid flow detection point a1 and the second position a4, the second position a4 is located on the side of the droplet breaking point a2 near the liquid flow detection point a1, or the second position a4 coincides with the droplet breaking point a 2. Further, the droplet delay time measuring module 11 further includes a droplet speed measuring sub-module 116, and the droplet speed measuring sub-module 116 is configured to determine whether the liquid flow reaches a steady state according to the first position A3, the second position a4, the time T3 when the third reflected light beam is incident to the first position A3, and the time T4 when the fourth reflected light beam is incident to the second position a 4.

Fig. 10 illustrates an example in which the first position A3 is located between the liquid flow detection point a1 and the second position a4, and the second position a4 is located on the side of the droplet break-off point a2 near the liquid flow detection point a 1. As shown in fig. 10, the first laser light source 111 emits a first laser beam and a second laser beam, where the first laser beam and the second laser beam may be directly emitted from the first laser light source 111, and at this time, the first laser light source 111 may include two or more lasers, which ensures that two laser beams can be emitted simultaneously; alternatively, the first laser beam and the second laser beam may be obtained by splitting a single laser beam emitted from the first laser light source 111, for example, a splitting device is disposed on a path of the single laser beam emitted from the first laser light source 111 to split the single laser beam into the first laser beam and the second laser beam. The embodiment of the present invention does not describe how the first laser light source 111 obtains the first laser beam and the second laser beam. With continued reference to fig. 10, the first laser beam and the second laser beam are reflected by the first mirror 1151 and the second mirror 1152, respectively, and then incident on the liquid stream flow path at a first position A3 and a second position a4, respectively, wherein the first position and the second position are located between the liquid stream detection point a1 and the liquid drop breaking point a2, and the second position a4 is located at a side of the first position A3 close to the liquid drop breaking point a 2. The droplet speed measurement sub-module 116 is used to determine whether the liquid flow reaches a stable steady state according to the first position A3, the second position a4, the time T3 when the third reflected light beam is incident on the first position A3, and the time T4 when the fourth reflected light beam is incident on the second position a 4.

Specifically, the droplet velocity measuring sub-module 116 is specifically configured to calculate a first average velocity of the droplet between the first position A3 and the second position a4 according to the time T3 when the third reflected light beam is incident to the first position A3 and the time T4 when the fourth reflected light beam is incident to the second position a4, and determine that the liquid flow reaches the steady motion state when the first average velocity meets a preset requirement.

Illustratively, as shown in fig. 11, the first position A3 corresponds to a first detection position A3 'on the surveillance camera 1211, and the second position a4 corresponds to a second detection position a 4' on the surveillance camera 1211, wherein a distance d between the detection position A3 'and the second detection position a 4' satisfies d-n u/M; a first average velocity v of the droplet between the first position A3 and the second position a4 satisfies v ═ d/(T4-T3). Wherein n represents the number of pixels between the first detected position A3 'and the second detected position a 4'; u denotes the size of a single pixel in the monitoring camera 1211; m denotes the magnification of the monitoring camera. And when the first average speed is determined to meet the preset requirement according to the first average speed obtained by the calculation, the liquid flow can be determined to reach a stable motion state. Where the first average speed meets the predetermined requirement may be that the first average speed is maintained within a range of values, for example 10 m/s; or the difference between any two first average speeds v1 and v2 is maintained within a range of values, such as 1 m/s; or the percentage difference between any two of the first average speeds v1 and v2 is maintained within a range of values, such as (v1-v2)/v2 ≦ 10%.

In summary, the above embodiments describe how to determine that the liquid flow is in a steady state, and when the liquid flow is determined to be in a steady state, the droplet break-off point a2 determined by the monitoring camera 1211 is the accurate droplet break-off point.

Optionally, with continued reference to fig. 10, the droplet speed measuring sub-module 116 may include a second optical filter 1161, a first detecting lens 1162, a first field stop 1163, a second field stop 1164, a first detector 1165, and a second detector 1166; the second optical filter 1161 and the first detection lens 1162 are located on detection paths of the first detector 1165 and the second detector 1166 at the same time; the first field stop 1163 is located on the detection path of the first detector 1165; the second field stop 1164 is located in the detection path of the second detector 1166.

Alternatively, the second filter 1161 may be a band pass filter or a long pass filter, and functions not to pass the third reflected light beam and the fourth reflected light beam, but to pass only the fluorescence signals excited by the detection particles based on the third reflected light beam and the fourth reflected light beam. The first and second field stops 1163, 1164 are used to eliminate interfering light other than the fluorescent signal, thereby ensuring that the signals received by the first and

second detectors

1165, 1166 are completely fluorescent signals excited by the detected particles based on the third and fourth reflected light beams. The first detector 1165 and the second detector 1166 may be photodiodes, avalanche diodes, or photomultiplier tubes, and are characterized by high sensitivity, capability of detecting weak fluorescence signals, fast response speed, and response speed in ns order.

Optionally, with continuing reference to fig. 4, 7, 8 and 9, the second detection signal receiving unit 123 may include a third optical filter 1231, a third field diaphragm 1232 and a third detector 1233, where the third optical filter 123 and the third field diaphragm 1232 are sequentially located on a detection path of the third detector 1233, the third optical filter 126 may be a band-pass optical filter or a long-pass optical filter, and functions to allow only the second detection signal to pass through, and the third field diaphragm 125 is used to block other optical signals before the second detection signal, so as to ensure that the second receiving time T2 based on the second detection signal is accurately received. The third detector 1233 may be a photodiode, an avalanche diode, or a photomultiplier, and is characterized by high sensitivity, capability of detecting weak fluorescence signals, fast response speed, and ns-order response speed.

Further, as shown in fig. 4, 7, 8, 9 and 10, when the third reflected beam or the fourth reflected beam is reflected by the second mirror 1152 to the droplet break-off point a2, i.e. the second position a4 coincides with the droplet break-off point a2, for example, the fourth reflected beam is reflected by the second mirror 1152 to the droplet break-off point a2, at this time, the second filter 1161 can be multiplexed as the third filter 1231, the second field stop 1164 can be multiplexed as the third field stop 1232, and the second detector 1166 can be multiplexed as the third detector 1233, which ensures the flow cytometer has a simple structure.

Optionally, the checkpoint measurement submodule 11 provided in the embodiment of the present invention may include a forward detection signal receiving unit 112 and/or a lateral detection signal receiving unit 117, fig. 4, fig. 7, fig. 8, fig. 9, and fig. 10 all describe that the checkpoint measurement submodule 11 includes the forward detection signal receiving unit 112, and fig. 13 describes that the checkpoint measurement submodule 11 includes the lateral detection signal receiving unit 11. As shown in fig. 4, 7, 8, 9 and 10, the forward detection signal receiving unit 112 may include a forward lens 1121 and a forward detector 1122, the forward lens 1121 is configured to focus a fluorescent signal or a forward scattered light signal excited by the detection particle based on the first laser signal, so as to ensure that the fluorescent signal or the forward scattered light signal can enter the forward detection signal receiving unit 112 more, and ensure that the first receiving time T1 based on the first detection signal is received accurately; the forward detector 1122 is a photodetector or a photomultiplier tube, and the embodiment of the present invention does not limit the forward detection signal receiving unit 112. As shown in fig. 13, the lateral detection signal receiving unit 117 may include a lateral lens 1171 and a lateral detector 1172, wherein the lateral lens 1171 is used for focusing the fluorescence signal or the laterally scattered light signal excited by the detection particle based on the first laser signal, so as to ensure that the fluorescence signal or the laterally scattered light signal can enter the lateral detector 1172 more, and ensure that the first receiving time T1 based on the first detection signal is received accurately; optionally, the side lens 1171 may be a microscope objective with a numerical aperture NA satisfying NA >0.6 and a field of view greater than 0.5 mm. . The side detector 1172 is a photodetector or photomultiplier, and the embodiment of the present invention does not limit the side detector 1172.

Optionally, with continued reference to fig. 4, the flow cytometer provided in the embodiment of the present invention may further include a fluid stream storage module 13, a droplet formation module 14, and a droplet sorting module 15; the liquid flow storage module 13 is used for storing liquid flow, and the liquid flow flows through the liquid drop forming module 14 and is broken at the position of a liquid drop breaking point A2 to form a liquid drop; the droplet sorting module 15 includes a deflecting electrode plate 151 and a droplet receiving tube 152, the deflecting electrode plate 151 is used for controlling the charged droplets to deflect, and the droplet receiving tube 152 is used for receiving the deflected droplets, so as to complete the cell sorting operation.

In summary, the flow cytometer provided in the embodiment of the present invention directly measures the droplet delay time through the droplet delay time measurement module, and feeds the droplet delay time forward to the droplet sorting module, and the droplet sorting module can accurately grasp the droplet delay time, ensure that the droplet is accurately charged at the droplet break point, accurately control the droplet charging time, ensure that the droplet is accurately sorted, and have high droplet sorting efficiency; the liquid drop delay time is accurately measured, the liquid drop delay time is simply measured, and consumables are saved; furthermore, the flow cytometry sorter is simple in structure and high in integration level through reasonably arranging devices in the flow cytometry sorter and arrangement positions of the devices.

Fig. 14 is a schematic structural diagram of an entity apparatus of another flow cytometer provided in the embodiment of the present invention, and as shown in fig. 14, the flow cytometer provided in the embodiment of the present invention may include a droplet speed measurement module 30, where the droplet speed measurement module 30 includes a third laser light source 301, the third laser light source 301 is configured to emit a third laser light beam and a fourth laser light beam, the third laser light beam is incident on a third position a5 where a speed of a droplet on the flow path of the liquid stream is measured, and the fourth laser light beam is incident on a fourth position a6 where a speed of a droplet on the flow path of the liquid stream is measured; the liquid drop velocity measurement module 30 is configured to determine whether the liquid flow reaches a stable and stable state according to the third position a5, the fourth position a6, the time when the third laser beam is incident on the third position, and the time when the fourth laser beam is incident on the fourth position.

For example, the third laser light source 301 emits a third laser beam and a fourth laser beam, where the third laser beam and the fourth laser beam may be directly emitted from the third laser light source 301, and at this time, the third laser light source 301 may include two or more lasers, so as to ensure that two laser beams can be emitted simultaneously; alternatively, the third laser beam and the fourth laser beam may be obtained by splitting a single laser beam emitted from the third laser light source 301, for example, a splitting device is disposed on a path of the single laser beam emitted from the third laser light source 301 to split the single laser beam into the third laser beam and the fourth laser beam. The embodiment of the present invention does not describe how the third laser light source 301 obtains the third laser beam and the fourth laser beam. With continued reference to fig. 14, the third laser beam is incident on a third location a5 on the velocity measurement of the drop in the fluid flow path and the fourth laser beam is incident on a fourth location a6 on the velocity measurement of the drop in the fluid flow path. The liquid drop velocity measurement module 30 is configured to determine whether the liquid flow reaches a stable and stable state according to the third position a5, the fourth position a6, the time T5 when the third laser beam is incident to the third position a5, and the time T5 when the fourth laser beam is incident to the fourth position a 6.

Specifically, the liquid drop velocity measurement module 30 is specifically configured to calculate an average velocity second average velocity of the liquid drop between the third position a5 and the fourth position a6 according to a time T5 when the third laser beam is incident to the third position a5 and a time T6 when the fourth laser beam is incident to the fourth position a6, and determine that the liquid flow reaches a stable motion state when the second average velocity meets a preset requirement.

For example, fig. 15 is a schematic diagram of another droplet image provided by the embodiment of the present invention, as shown in fig. 15, the third position a5 corresponds to a third detection position a5 'on the monitoring camera, and the fourth position a6 corresponds to a fourth detection position a 6' on the monitoring camera 1211, where a distance d 'between the third detection position a 5' and the fourth detection position a6 'satisfies d' ═ n u/M; the average velocity of the droplet between the third position a5 and the fourth position a6 the second average velocity v ' satisfies v ' ═ d '/(T6-T5). Wherein n represents the number of pixels between the third detected position a5 'and the fourth detected position a 6'; u represents the size of a single pixel in the surveillance camera; m denotes the magnification of the monitoring camera. And when the second average speed is determined to meet the preset requirement according to the second average speed obtained by the calculation, the liquid flow can be determined to reach a stable motion state. Where the second average speed meets the predetermined requirement may be that the second average speed is maintained within a range of values, for example 10 m/s; or the difference between any two of the second average speeds v1 'and v 2' is maintained within a range of values, such as 1 m/s; or any two of the average velocities, the percent difference between the second average velocities v1 ' and v2 ', is maintained within a range of values, such as (v1 ' -v2 ')/v 2 ' ≦ 10%.

Optionally, with continued reference to fig. 14, the droplet speed measuring module 30 may include a fourth optical filter 302, a second detection lens 303, a third field diaphragm 304, a fourth field diaphragm 305, a fourth detector 306, and a fifth detector 307; the fourth filter 302 and the second detection lens 303 are simultaneously located on the detection paths of the fourth detector 306 and the fifth detector 307; the third field stop 304 is located in the detection path of the fourth detector 306; a fourth field stop 305 is located in the detection path of the fifth detector 307.

Optionally, the fourth filter 302 may be a band-pass filter or a long-pass filter, and functions to not pass the third laser beam and the fourth laser beam, but only detect the fluorescent signal excited by the particles based on the third laser beam and the fourth laser beam. The third field stop 304 and the fourth field stop 305 are used to eliminate interfering light other than the fluorescence signal, thereby ensuring that the signals received by the fourth detector 306 and the fifth detector 307 are completely fluorescence signals excited by the detected particles based on the third laser beam and the fourth laser beam. The fourth detector 306 and the fifth detector 307 may be photodiodes, avalanche diodes, or photomultiplier tubes, and are characterized by high sensitivity, capability of detecting weak fluorescence signals, fast response speed, and response speed in ns order.

In summary, the cell sorter provided by the embodiment of the invention can accurately measure the movement speed of the liquid drop, and has the advantages of simple structure and strong practicability.

Optionally, fig. 16 is a schematic structural diagram of another flow cytometer provided in an embodiment of the present invention, and fig. 17 is a schematic flow chart of a flow cell sorting method provided in an embodiment of the present invention, and on the basis of the above embodiment, an embodiment of the present invention further provides a flow cell sorting method applied to the flow cytometer shown in fig. 16, as shown in fig. 16 and fig. 17, the flow cytometer provided in an embodiment of the present invention may include a checkpoint measurement module 40, a droplet delay time measurement module 41, and a droplet diversion module 42; the flow cytometric sorting method comprises the following steps:

s110, the detection point measuring module obtains an optical detection signal of the sample when the liquid flow passes through the liquid flow detection point, and obtains the sorting target information according to the optical detection signal.

And S120, the liquid drop delay time measuring module measures the liquid drop delay time according to the time when the liquid flow passes through the liquid drop breaking point.

S130, the liquid drop shunting module receives the sorting target information and the liquid drop delay time, and sorting and charging are carried out on the sorting target according to the liquid drop delay time.

Illustratively, the detection point measurement module obtains an optical detection signal of the sample when the fluid stream flows through the detection point, and obtains the sorting target information according to the analysis of the optical detection signal. The droplet delay time measurement module obtains an accurate delay time as the fluid stream passes through the break-off point. The liquid drop shunting module is in communication connection with the detection point measuring module and the liquid drop delay time measuring module, receives the information of the sorting target and the liquid drop delay time, and needs to charge the sorting target after the liquid drop delay time is reached so as to control the sorting target to deflect under the action of the deflecting electric field and finish sorting.

In summary, the flow cytometry sorting method provided by the embodiment of the invention has the advantages that the allowable target and the droplet delay time are determined accurately, and the sorting result is ensured to be accurate.

Optionally, on the basis of the above embodiment, the flow cytometer provided in the embodiment of the present invention may further include a liquid flow velocity measurement module, and when the liquid flow velocity measured by the liquid flow velocity measurement module reaches a steady state, the droplet break-off point measured by the droplet delay time measurement module is determined to be an accurate droplet break-off point.

Illustratively, the flow cytometer provided in the embodiment of the present invention may further include a flow velocity measurement module, and the above embodiment describes how to determine that the flow is in a steady state. Optionally, the flow cytometry sorting method provided in the embodiment of the present invention may further include that when it is determined that the liquid flow is in a stable state, the droplet break-off point determined by the droplet delay time measurement module is an accurate droplet break-off point, and the droplet splitting module performs sorting charging on the sorting target at the droplet break-off point according to the droplet delay time, so as to ensure accurate cell sorting.

It is to be noted that the foregoing is only illustrative of the preferred embodiments of the present invention and the technical principles employed. Those skilled in the art will appreciate that the present invention is not limited to the specific embodiments described herein, and that the features of the various embodiments of the invention may be partially or fully coupled to each other or combined and may be capable of cooperating with each other in various ways and of being technically driven. Numerous variations, rearrangements, combinations, and substitutions will now become apparent to those skilled in the art without departing from the scope of the invention. Therefore, although the present invention has been described in greater detail by the above embodiments, the present invention is not limited to the above embodiments, and may include other equivalent embodiments without departing from the spirit of the present invention, and the scope of the present invention is determined by the scope of the appended claims.

Claims

1. A flow cytometer comprises a droplet delay time measuring module and a droplet shunting module;

2. The flow cytometer of claim 1 wherein the droplet delay time measurement module comprises a checkpoint measurement sub-module, a breakpoint measurement sub-module, and a droplet delay time calculation sub-module;

3. The flow cytometer of claim 2 wherein the detection point measurement sub-module is configured to detect a first detection signal of the flow stream passing the flow stream detection point;

4. A flow cytometer as described in claim 3 wherein said first detection signal and said second detection signal are both fluorescent signals excited by the detection particle;

5. The flow cytometer of claim 3 wherein the break location monitoring unit comprises a monitoring camera and a stroboscopic light source;

the monitoring camera is used for acquiring a liquid drop moving image;

6. The flow cytometer of claim 5 wherein the strobed light source comprises a light emitting diode or a semiconductor laser and the exposure time of the strobed light source is matched to the fluid flow velocity.

7. A flow cytometer as described in claim 6 wherein said exposure time T satisfies T <5 μ s.

8. The flow cytometer of claim 3 wherein the detection point measurement sub-module comprises a first laser light source and a forward detection signal receiving unit.

9. The flow cytometer of claim 8 wherein the break point measurement sub-module comprises a second laser light source, the second laser light source having an adjustable exit angle.

10. The flow cytometer of claim 9, wherein the detection point measurement sub-module further comprises a first light blocking diaphragm, and the first light blocking diaphragm is configured to block the laser beam emitted by the first laser light source from being directly incident on the forward detection signal receiving unit.

11. The flow cytometer of claim 8 wherein the droplet delay time measurement module further comprises a set of optical path reflectors for reflecting the first laser light source to the droplet break-off point to excite a detection particle passing through the droplet break-off point;

the posture of the light path reflector set is adjustable.

12. The flow cytometer of claim 11, wherein the optical path mirror set comprises a first mirror and a second mirror, the first mirror is located on the optical path of the first laser light source for reflecting the laser beam emitted from the first laser light source to form a first reflected beam; the second reflecting mirror is positioned on the light path of the first reflecting beam and used for reflecting the first reflecting beam to form a second reflecting beam and controlling the second reflecting beam to be at least incident to the liquid drop breaking point.

13. A flow cytometer as described in claim 12 wherein the angles of said first and second mirrors are adjustable.

14. A flow cytometer as described in claim 12 wherein said optical path mirror assembly further comprises a first optical filter;

15. A flow cytometer as described in claim 12 wherein said first mirror comprises a plane mirror and said second mirror comprises a plane mirror,

16. A flow cytometer as described in claim 15 wherein said first mirror comprises a plane mirror, said second mirror comprises a plane mirror, said optical path mirror group further comprises an amplification lens;

17. The flow cytometer of claim 12 wherein the forward detection signal receiving unit comprises a forward lens, the first mirror being located at a focal plane location of the forward lens.

18. A flow cytometer as described in claim 12 wherein said first laser light source is configured to emit a first laser beam and a second laser beam;

19. The flow cytometer of claim 18 wherein the drop delay time measurement module further comprises a drop velocimeter sub-module for determining whether the flow stream has reached a steady state based on the first location, the second location, the time at which the third reflected beam is incident at the first location, and the time at which the fourth reflected beam is incident at the second location.

20. The flow cytometer of claim 19, wherein the drop velocity measurement sub-module is specifically configured to calculate a first average velocity of the drop between the first position and the second position according to the time of the third reflected beam incident on the first position and the time of the fourth reflected beam incident on the second position, and determine that the flow reaches a steady motion state when the first average velocity meets a preset requirement.

21. The flow cytometer of claim 19 wherein the drop velocimetry sub-module comprises a second optical filter, a first detection lens, a first field stop, a second field stop, a first detector and a second detector; the second optical filter and the first detection lens are simultaneously positioned on detection paths of the first detector and the second detector; the first field diaphragm is positioned on a detection path of the first detector; the second field stop is located in a detection path of the second detector.

22. The flow cytometer of claim 2 wherein the detection point measurement sub-module comprises a forward detection signal receiving unit and/or a lateral detection signal receiving unit;

23. The flow cytometric sorter of claim 1 further comprising a fluid stream storage module, a droplet formation module, and a droplet sorting module;

24. A flow cytometry sorter is characterized by comprising a liquid drop speed measurement module;

25. The flow cytometer of claim 24, wherein the droplet velocity measurement module is specifically configured to calculate a second average velocity of the droplet between the third position and the fourth position according to the time of the third laser beam incident on the third position and the time of the fourth laser beam incident on the fourth position, and determine that the liquid stream reaches a steady motion state when the second average velocity meets a preset requirement.

26. The flow cytometer of claim 24 wherein the drop velocimetry module further comprises a fourth optical filter, a second detection lens, a third field stop, a fourth detector and a fifth detector; the fourth optical filter and the second detection lens are simultaneously positioned on detection paths of the fourth detector and the fifth detector; the third field diaphragm is positioned on a detection path of the fourth detector; the third field stop is located in a detection path of the fifth detector.

27. The flow cytometry sorting method is characterized by being applied to a flow cytometry sorter, wherein the flow cytometry sorter comprises a detection point measuring module, a liquid drop delay time measuring module and a liquid drop shunting module; the flow cytometric sorting method comprises the following steps:

28. A flow cytometric sorting method according to claim 27, characterized in that the flow cytometric sorting instrument further comprises a liquid flow velocity measuring module, when the liquid flow velocity measured by the liquid flow velocity measuring module reaches a steady state, the liquid drop breaking point measured by the liquid drop delay time measuring module is determined to be an accurate liquid drop breaking point.