CN112748057A

CN112748057A - Liquid drop delay time determination method and flow cytometry sorting instrument

Info

Publication number: CN112748057A
Application number: CN202011533789.9A
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: 2020-12-22
Filing date: 2020-12-22
Publication date: 2021-05-04

Abstract

The invention discloses a method for determining delay time of liquid drops and a flow cytometry sorter, wherein the flow cytometry sorter comprises N groups of charged liquid drop receiving units and a group of uncharged liquid drop receiving units, and the N groups of charged liquid drop receiving units correspond to N initial liquid drop delay times; the method for determining the delay time of the liquid drop comprises the following steps: respectively acquiring first detection signal intensity in a charged droplet receiving unit and second detection signal intensity in an uncharged droplet receiving unit corresponding to N initial droplet delay times; determining a jth initial drop delay time T of the N initial drop delay times according to the first and second detection signal intensities_jFor the drop delay time, or for determining the g-1 st initial drop delay time T of the N initial drop delay times_g‑1Delay time T from g +1 th initial drop_g+1A certain time T in between_mIs the drop delay time. In conclusion, the method of multi-channel detection and comparison judgment can be used for rapidly determining the liquid drop delay time, and the liquid drop delay time determining efficiency is high.

Description

Liquid drop delay time determination method and flow cytometry sorting instrument

Technical Field

The embodiment of the invention relates to the technical field of cell sorting, in particular to a method for determining liquid drop delay time and a flow cytometry sorter.

Background

Flow cytometers are typical particle analysis sorting devices that rely on the flow of cells or other particles in a liquid flow to determine one or more characteristics of the particles 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 droplet charging time is determined by the droplet delay time, and in the prior art, the droplet delay time is determined by repeatedly adjusting the droplet delay time through a group of detection channels, so that the accurate delay time of the droplets is determined, and the method is long in time consumption and low in efficiency.

Disclosure of Invention

In view of this, embodiments of the present invention provide a method for determining a droplet delay time and a flow cytometer, which can determine a droplet delay time quickly and with high efficiency.

In a first aspect, an embodiment of the present invention provides a method for determining a droplet delay time, which is applied to a flow cytometer, where the flow cytometer includes a droplet receiving module, where the droplet receiving module includes N sets of charged droplet receiving units and one set of uncharged droplet receiving units; the N groups of charged droplet receiving units correspond to N initial droplet delay times, wherein any one initial droplet delay time T_iSatisfy 0 < T_i＜T_i+1I is more than or equal to 1 and less than N, and i is an integer;

the method for determining the drop delay time comprises the following steps:

respectively acquiring first detection signal intensity in a charged droplet receiving unit and second detection signal intensity in an uncharged droplet receiving unit corresponding to the N initial droplet delay times;

determining a jth initial drop delay time T of the N initial drop delay times based on the first and second detection signal strengths_jFor the drop delay time, or for determining the g-1 st initial drop delay time T of the N initial drop delay times_g-1Delay time T from g +1 th initial drop_g+1A certain time T in between_mDelay time for liquid dropWherein T is_g-1≤T_m≤T_g+1(ii) a J is more than or equal to 1 and less than or equal to N, and j is an integer; g is more than or equal to 2 and less than or equal to N-1, and g is an integer.

Optionally, determining a jth initial droplet delay time T of the N initial droplet delay times according to the first and second detection signal intensities_jFor the drop delay time, or for determining the g-1 st initial drop delay time T of the N initial drop delay times_g-1Delay time T from g +1 th initial drop_g+1A certain time T in between_mIs a drop delay time, comprising:

delay time T when the jth initial drop_jDetermining the jth initial droplet delay time T when the ratio of the corresponding first and second detection signal intensities satisfies a predetermined ratio_jIs the drop delay time;

delay time T when the g-th initial droplet_gWhen the intensity of the corresponding second detection signal is minimum, re-determining N initial droplet delay times corresponding to the N groups of charged droplet receiving units, wherein the jth initial droplet delay time corresponding to the jth group of charged droplet receiving units is T_j', the g-th initial drop delay time corresponding to the g-th group of charged drop receiving units is T_g’，T_g-1≤T_j’≤T_g+1，T_g-1≤T_g’≤T_g+1；

And repeating the steps until the ratio of the first detection signal intensity and the second detection signal intensity corresponding to the mth initial droplet delay time meets a preset ratio, determining the mth initial droplet delay time as the droplet delay time, and enabling m to be larger than or equal to 1 and smaller than or equal to N.

delay time T when the j-1 st initial drop_j-1Corresponding first detection signal intensity, the jth initial drop delay time T_jCorresponding first detection signal intensity and the (j + 1) th initial droplet delay time T_j+1The corresponding first detection signal intensity is the same, and the jth initial droplet delay time T_jDetermining the jth initial droplet delay time T when the ratio of the corresponding first and second detection signal intensities satisfies a predetermined ratio_jIs the drop delay time;

And repeating the steps until the first detection signal intensity corresponding to the m-1 th initial droplet delay time, the first detection signal intensity corresponding to the m-1 th initial droplet delay time and the first detection signal intensity corresponding to the m +1 th initial droplet delay time are the same, and when the ratio of the first detection signal intensity corresponding to the m-1 th initial droplet delay time to the second detection signal intensity meets a preset ratio, determining the m-th initial droplet delay time as the droplet delay time, wherein m is more than or equal to 1 and less than or equal to N.

Optionally, after determining the droplet delay time, the method further includes:

the drop delay time is verified.

Optionally, verifying the droplet delay time comprises:

acquiring the fluorescence signal intensity of the liquid drops in the 1 st group to the Nth group of charged liquid drop receiving units respectively based on the liquid drop delay time;

judging whether the intensities of the droplet fluorescence signals in the 1 st group to the Nth group of charged droplet receiving units are the same or not; and verifying that the droplet delay time is accurate when the intensities of the droplet fluorescence signals in the 1 st to Nth groups of charged droplet receiving units are the same.

Optionally, before obtaining the first detection signal intensity in the charged droplet receiving unit and the second detection signal intensity in the uncharged droplet receiving unit corresponding to the N initial droplet delay times, respectively, the method further includes:

and determining the charging electric quantity corresponding to each group of the charged liquid drop receiving units in the N groups of the charged liquid drop receiving units.

Optionally, T_i-T_i-1＝T_i+1-T_i；T_j’-T_j-1’＝T_j+1’-T_j’。

In a second aspect, an embodiment of the present invention further provides a flow cytometer, which determines a droplet delay time by using the droplet delay time determination method described in the first aspect; the flow cytometry sorter comprises a liquid drop delay time determining module, a liquid drop receiving module and a liquid drop shunting module;

the drop receiving module comprises N groups of charged drop receiving units and one group of uncharged drop receiving units; the N groups of charged droplet receiving units correspond to N initial droplet delay times, wherein any one initial droplet delay time T_iSatisfy 0 < T_i＜T_i+1I is more than or equal to 1 and less than N, and i is an integer;

the droplet delay time determining module is used for respectively acquiring first detection signal intensity in a charged droplet receiving unit and second detection signal intensity in an uncharged droplet receiving unit corresponding to the N initial droplet delay times; determining the delay time of the liquid drop according to the first detection signal intensity and the second detection signal intensity;

the droplet delay time determination module is further used for feeding back the droplet delay time to the droplet shunting module so as to control the time for which the droplets are charged.

Optionally, the droplet delay time determination module includes a signal intensity detection sub-module, and the signal intensity detection sub-module includes a first laser light source and a first monitoring camera assembly.

Optionally, the signal intensity detection sub-module further includes a converging lens and a mirror group;

the converging lens is positioned on a transmission path of a laser signal emitted by the first laser light source;

the reflector group comprises N reflectors, and the reflectors are used for reflecting the laser signals to flow paths of droplets corresponding to the charged droplet receiving units and the uncharged droplet receiving units.

Optionally, the ith reflector in the reflector group is located between the ith charged droplet receiving unit and the (i + 1) th charged droplet receiving unit, and is configured to reflect the laser signal to the (i + 1) th charged droplet receiving unit;

or the ith reflector is positioned between the ith charged droplet receiving unit and the uncharged droplet receiving unit and used for reflecting the laser signal to the uncharged droplet receiving unit;

or, the ith reflector is located between the uncharged droplet receiving unit and the (i + 1) th charged droplet receiving unit and is used for reflecting the laser signal to the (i + 1) th charged droplet receiving unit.

Optionally, the mirror comprises a plane mirror and/or a spherical mirror.

Optionally, the first monitoring camera component comprises a wide-angle monitoring camera;

the wide-view monitoring camera is used for respectively acquiring fluorescence signals and/or scattered light signals of laser signals passing through the N groups of charged liquid drop receiving units and uncharged liquid drop receiving units.

Optionally, the first monitoring camera component includes N +1 monitoring cameras;

the first to nth monitoring cameras in the N +1 monitoring cameras are used for respectively acquiring fluorescence signals and/or scattered light signals of laser signals passing through the N groups of charged liquid drop receiving units; and the (N + 1) th monitoring camera in the (N + 1) monitoring cameras is used for acquiring a fluorescence signal and/or a scattered light signal of the laser signal after passing through the uncharged droplet receiving unit.

Optionally, both the first detection signal intensity and the second detection signal intensity are fluorescence signal intensities excited by the detection particles in the droplet excited by the first laser light source;

or both the first detection signal intensity and the second detection signal intensity are the scattering signal intensity excited by the detection particles in the droplet excited by the first laser light source.

Optionally, the flow cytometer further comprises at least one illumination source for providing illumination to the charged drop receiving unit and the uncharged drop receiving unit.

Optionally, the droplet delay time determination module is specifically configured to determine a jth initial droplet delay time T of the N initial droplet delay times_jFor the drop delay time, or for determining the g-1 st initial drop delay time T of the N initial drop delay times_g-1Delay time T from g +1 th initial drop_g+1A certain time T in between_mIs the drop delay time, where T_g-1≤T_m≤T_g+1(ii) a J is more than or equal to 1 and less than or equal to N, and j is an integer; g is more than or equal to 2 and less than or equal to N-1, and g is an integer.

Optionally, the droplet delay time determination module includes a signal intensity detection sub-module and a droplet delay time determination sub-module;

the signal intensity detection submodule is used for respectively acquiring first detection signal intensity in the charged liquid drop receiving unit and second detection signal intensity in the uncharged liquid drop receiving unit corresponding to the N liquid drop delay times;

the drop delay time determination submodule is used for determining the jth drop delay time T in the N drop delay times according to the first detection signal intensity and the second detection signal intensity_jFor drop delay time, or for determining the g-1 drop delay time T of the N drop delay times_g-1Delay time T from g +1 th droplet_g+1A certain time T of_mIs the drop delay time.

Optionally, the flow cytometer further comprises an initial droplet delay time providing module, wherein the initial droplet delay time providing module comprises a detection point recording submodule and a breakpoint calculation submodule;

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

and the break-off point calculation submodule is used for respectively calculating the charging time of the liquid drop at the break-off point according to the first time and the initial liquid drop delay time.

Optionally, the detection point recording sub-module includes a second laser light source, and further includes a forward detection signal receiving unit and/or a lateral detection signal receiving unit.

Optionally, the forward detection signal receiving unit includes a light shielding diaphragm, a forward lens and a forward detector;

the lateral detection signal receiving unit comprises a lateral objective lens and a lateral detector.

Optionally, the detection point recording sub-module further includes an illumination lens, and the illumination lens is located on a transmission path of the laser signal emitted by the second laser light source.

Optionally, the breakpoint calculation sub-module 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 flow cytometer further comprises a deflecting electrode plate.

The embodiment of the invention provides a method for determining the delay time of liquid drops and a flow cytometry sorter, wherein the flow cytometry sorter comprises N groups of charged liquid drop receiving units and a group of uncharged liquid drop receiving units, and the N groups of charged liquid drop receiving units correspond to N initial liquid drop delay times; directly determining the jth initial drop delay in the N initial drop delay times according to the first detection signal intensity in the charged drop receiving unit and the second detection signal intensity in the uncharged drop receiving unit corresponding to the N initial drop delay timesTime T_jFor the drop delay time, or for determining the g-1 st initial drop delay time T of the N initial drop delay times_g-1Delay time T from g +1 th initial drop_g+1A certain time T in between_mIs the drop delay time. Because the flow cytometry sorter comprises a plurality of groups of charged liquid drop receiving units and corresponds to a plurality of liquid drop delay detection channels, the liquid drop delay time can be quickly determined by using a multi-channel detection and comparison and judgment method, and the liquid drop delay time determination efficiency 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 flow chart of a method for determining a drop delay time according to an embodiment of the present invention;

FIG. 4 is a schematic flow chart of another method for determining drop delay according to an embodiment of the present invention;

FIG. 5 is a schematic flow chart of another method for determining drop delay according to an embodiment of the present invention;

FIG. 6 is a schematic diagram of a droplet sorting result provided by an embodiment of the present invention;

FIG. 7 is a schematic diagram of another droplet sorting result provided by an embodiment of the present invention;

FIG. 8 is a schematic diagram of another droplet sorting result provided by an embodiment of the present invention;

FIG. 9 is a schematic diagram of another droplet sorting result provided by an embodiment of the present invention;

FIG. 10 is a block diagram of a flow cytometer in accordance with an embodiment of the present invention;

fig. 11 is a schematic structural diagram of a signal strength detection sub-module according to an embodiment of the present invention;

fig. 12 is a schematic structural diagram of another signal strength detection sub-module 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, a collection test tube is generally arranged in a scheme for acquiring the delay time of the liquid drop, the collection test tube corresponds to the delay time of the liquid drop, and in the process of measuring the delay time of the liquid drop, the accurate delay time of the liquid drop is determined by repeatedly adjusting the delay time of the liquid drop, so that the time consumption is long and the efficiency is low.

Based on this, the embodiment of the present invention provides a method for determining a droplet delay time, which is applied to a flow cytometer, where the flow cytometer includes a droplet receiving module, and the droplet receiving module includes N sets of charged droplet receiving units and one set of uncharged droplet receiving units; n groups of charged droplet receiving units correspond to N initial droplet delay times, wherein any initial droplet delay time T_iSatisfy 0 < T_i＜T_i+1，1≤i is less than N, and i is an integer; the method for determining the delay time of the liquid drop comprises the following steps: respectively acquiring first detection signal intensity in a charged droplet receiving unit and second detection signal intensity in an uncharged droplet receiving unit corresponding to N initial droplet delay times; determining a jth initial drop delay time T of the N initial drop delay times according to the first and second detection signal intensities_jFor the drop delay time, or for determining the g-1 st initial drop delay time T of the N initial drop delay times_g-1Delay time T from g +1 th initial drop_g+1A certain time T in between_mIs the drop delay time, where T_g-1≤T_m≤T_g+1(ii) a J is more than or equal to 1 and less than or equal to N, and j is an integer; g is more than or equal to 2 and less than or equal to N-1, and g is an integer. According to the method for determining the delay time of the liquid drop, the flow cytometry sorter is provided with the plurality of charged liquid drop receiving units, and the plurality of charged liquid drop receiving units correspond to the plurality of initial liquid drop delay times, so that the delay time of the plurality of liquid drops can be verified at one time in the process of determining the delay time of the liquid drops, the delay time of the liquid drops can be determined quickly, and the efficiency of determining the delay time of the liquid drops is improved.

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 fig. 3 is a method for determining a droplet delay time provided in an embodiment of the present invention, and as shown in fig. 2 and fig. 3, the flow cytometer provided in an embodiment of the present invention includes N sets of charged droplet receiving units 11 and one set of uncharged droplet receiving units 12; the N groups of charged droplet receiving units 11 correspond to N initial droplet delay times, wherein any one initial droplet delay time T_iSatisfy 0 < T_i＜T_i+1I is more than or equal to 1 and less than N, and i is an integer;

the method for determining the delay time of the liquid drop comprises the following steps:

and S110, respectively acquiring first detection signal intensity in the charged droplet receiving unit and second detection signal intensity in the uncharged droplet receiving unit corresponding to the N initial droplet delay times.

S120, determining the jth initial droplet delay time T in the N initial droplet delay times according to the first detection signal intensity and the second detection signal intensity_jFor the drop delay time, or for determining the g-1 st initial drop delay time T of the N initial drop delay times_g-1Delay time T from g +1 th initial drop_g+1A certain time T in between_mIs the drop delay time, where T_g-1≤T_m≤T_g+1(ii) a J is more than or equal to 1 and less than or equal to N, and j is an integer; g is more than or equal to 2 and less than or equal to N-1, and g is an integer.

Illustratively, as shown in fig. 2, the flow cytometer provided by the embodiment of the present invention includes N sets of charged droplet receiving units 11 and one set of uncharged droplet receiving units 12, where each set of the uncharged droplet receiving units 11 corresponds to one initial droplet delay time, and N sets of the charged droplet receiving units 11 correspond to N initial droplet delay times. In the droplet delay time determining process, the first detection signal intensity in the charged droplet receiving unit 11 and the second detection signal intensity in the uncharged droplet receiving unit 12 corresponding to the N initial droplet delay times are respectively obtained, so that N sets of the first detection signal intensity and the second detection signal intensity can be obtained, and according to the N sets of the first detection signal intensity and the second detection signal intensity, one of the N initial droplet delay times can be directly determined as the droplet delay time, for example, the jth initial droplet delay time T_jFor drop delay time, or for determining one of two adjacent ones of N initial drop delay times as a drop delay time, e.g. the g-1 st initial drop delay time T_g-1Delay time T from g +1 th initial drop_g+1A certain time T in between_mIs the drop delay time, where T_g-1≤ T_m≤T_g+1(ii) a J is more than or equal to 1 and less than or equal to N, and j is an integer; g is more than or equal to 2 and less than or equal to N-1, and g is an integer. Thus, by settingA plurality of charging liquid drop receiving units are arranged in the flow cytometry sorting device, the plurality of charging liquid drop receiving units correspond to a plurality of initial liquid drop delay times, so that the plurality of liquid drop delay times can be verified at one time in the liquid drop delay time determining process, the liquid drop delay time can be determined quickly, and the liquid drop delay time determining efficiency is improved.

Specifically, the jth initial droplet delay time T in the N initial droplet delay times is determined according to the first detection signal intensity and the second detection signal intensity_jFor the drop delay time, or for determining the g-1 st initial drop delay time T of the N initial drop delay times_g-1Delay time T from g +1 th initial drop_g+1A certain time T in between_mThe delay time of the liquid drop can comprise:

delay time T when j initial drop_jWhen the ratio of the corresponding first detection signal intensity and the second detection signal intensity meets a preset ratio, determining the jth initial droplet delay time T_jIs the drop delay time;

And repeating the steps until the ratio of the first detection signal intensity and the second detection signal intensity corresponding to the mth initial droplet delay time meets a preset ratio, and determining the mth initial droplet delay time as the droplet delay time, wherein m is more than or equal to 1 and less than or equal to N.

For example, after acquiring the first detection signal strengths of the charged droplet receiving units 11 corresponding to the N initial droplet delay times, the initial droplet delay time corresponding to the charged droplet receiving unit 11 with the strongest first detection signal strength, for example, T, is determined_j(ii) a All in oneTime of day T_jSecond detected signal strength in the corresponding uncharged drop receiving unit 12 if T_jWhen the ratio of the corresponding first detection signal intensity to the second detection signal intensity satisfies a predetermined ratio, the droplet delay time T is T ═ T_jI.e. determining the jth initial drop delay time T_jIs the drop delay time. Specifically, the jth initial drop delay time T_jThe ratio of the intensity of the first detection signal to the intensity of the second detection signal satisfies a predetermined ratio, which is defined as T_jThe corresponding first detection signal intensity is much greater than the second detection signal intensity, i.e. T_jThe corresponding first detection signal strength is sufficiently large and the second detection signal strength is sufficiently small. Illustratively, the preset ratio a may satisfy a ≧ 9, which is not limited in the embodiment of the present invention.

Or, after acquiring the first detection signal intensities in the charged droplet receiving units 11 corresponding to the N initial droplet delay times, respectively, the initial droplet delay time corresponding to the charged droplet receiving unit 11 with the strongest first detection signal intensity, for example, T, is determined_g(ii) a Simultaneous acquisition of T_gSecond detected signal strength in the corresponding uncharged drop receiving unit 12 if T_gWhen the ratio of the first detection signal intensity to the second detection signal intensity is not greater than the predetermined ratio, and T is greater than T_gThe intensity of the second detection signal in the corresponding uncharged droplet receiving unit 12 is smaller than the intensity of the second detection signal corresponding to the other initial droplet delay time, which indicates that T_gNot the exact drop delay, but at T_gIn the vicinity, the accurate drop delay time can be determined at this time to be between the g-1 st initial drop delay time and the g +1 st initial drop delay time, i.e. the jth initial drop delay time corresponding to the jth group of charged drop receiving units is Tj' and T_g-1≤T_j’≤T_g+1The delay time of the g initial droplet corresponding to the g group of charged droplet receiving units is T_g' satisfy T_g-1≤T_g’≤T_g+1. Thus, the above steps are repeated according to the newly determined initial drop delay time until the mth initial drop delay time pairAnd when the ratio of the first detection signal intensity to the second detection signal intensity meets a preset ratio, determining the mth initial droplet delay time as the droplet delay time, wherein m is more than or equal to 1 and less than or equal to N.

The method for determining the liquid drop delay time can ensure accurate measurement of the liquid drop delay time, meanwhile, because the plurality of charged liquid drop receiving units correspond to the plurality of initial liquid drop delay times, the plurality of initial liquid drop delay times can be evaluated and calculated at the same time, specific range intervals of the liquid drop delay time are determined, the liquid drop delay time can be ensured to be determined quickly in the determined time range, and the liquid drop delay time determination efficiency is improved.

Further, determining the jth initial drop delay time T in the N initial drop delay times according to the first detection signal intensity and the second detection signal intensity_jFor the drop delay time, or for determining the g-1 st initial drop delay time T of the N initial drop delay times_g-1Delay time T from g +1 th initial drop_g+1A certain time T in between_mThe delay time of the liquid drop can comprise:

delay time T when j-1 th initial drop_j-1Corresponding first detection signal intensity, jth initial droplet delay time T_jCorresponding first detection signal intensity and j +1 th initial droplet delay time T_j+1The corresponding first detection signals have the same intensity, and the jth initial droplet delay time T_jWhen the ratio of the corresponding first detection signal intensity and the second detection signal intensity meets a preset ratio, determining the jth initial droplet delay time T_jIs the drop delay time;

Illustratively, when determining the jth initial drop delay time T_jWhen the delay time of the liquid drop is the j-1 st initial liquid drop delay time T_j-1Corresponding first detection signal intensity, jth initial droplet delay time T_jCorresponding first detection signal intensity and j +1 th initial droplet delay time T_j+1Whether the corresponding first detection signal intensity is the same or not is judged, and the delay time T of the j-1 th initial drop is obtained_j-1Corresponding first detection signal intensity, jth initial droplet delay time T_jCorresponding first detection signal intensity and j +1 th initial droplet delay time T_j+1Verifying the jth initial droplet delay time T when the corresponding first detection signal strength is the same_jThe accuracy of judging the delay time of the liquid drops is increased for the accuracy of the delay time of the liquid drops. When determining the mth initial drop delay time T_mWhen the delay time of the droplet is m-1 th initial droplet delay time T_m1Corresponding first detection signal intensity, mth initial droplet delay time T_mCorresponding first detection signal intensity and m +1 th initial droplet delay time T_m+1Whether the corresponding first detection signal intensity is the same or not is judged, and the delay time T is within the m-1 th initial drop_m-1Corresponding first detection signal intensity, mth initial droplet delay time T_mCorresponding first detection signal intensity and m +1 th initial droplet delay time T_m+1Verifying the mth initial droplet delay time T when the corresponding first detection signal intensities are the same_mThe accuracy of judging the delay time of the liquid drops is increased for the accuracy of the delay time of the liquid drops.

Further, in the embodiments of the present invention, the interval between the extension times of any two adjacent initial droplets is the same, i.e. T_i-T_i-1＝T_i+1-T_i；T_j’-T_j-1’＝T_j+1’-T_j' so, the time range interval between any two adjacent initial droplet delay times can be ensured to be the same, and the method for determining the droplet delay time is ensured to be accurate and efficient.

On the basis of the foregoing embodiment, fig. 4 is a schematic flowchart of another method for determining a droplet delay time according to an embodiment of the present invention, and as shown in fig. 4, the method for determining a droplet delay time according to an embodiment of the present invention may include:

s210, respectively acquiring first detection signal intensity in the charged droplet receiving unit and second detection signal intensity in the uncharged droplet receiving unit corresponding to the N initial droplet delay times.

S220, determining the jth initial droplet delay time T in the N initial droplet delay times according to the first detection signal intensity and the second detection signal intensity_jFor the drop delay time, or for determining the g-1 st initial drop delay time T of the N initial drop delay times_g-1Delay time T from g +1 th initial drop_g+1A certain time T in between_mIs the drop delay time, where T_g-1≤T_m≤T_g+1(ii) a J is more than or equal to 1 and less than or equal to N, and j is an integer; g is more than or equal to 2 and less than or equal to N-1, and g is an integer;

and S230, verifying the drop delay time.

Illustratively, after the droplet delay time is determined by the methods of S210 and S220, the droplet delay time needs to be verified to ensure that the droplet delay time is accurate and that the time when the droplet is charged is accurate. Specifically, the procedure for verifying the droplet delay time may be as follows:

acquiring the fluorescent signal intensity of the liquid drops in the 1 st group to the Nth group of charged liquid drop receiving units respectively based on the liquid drop delay time;

judging whether the intensities of the droplet fluorescence signals in the 1 st group to the Nth group of charged droplet receiving units are the same or not; and verifying that the droplet delay time is accurate when the intensities of the droplet fluorescence signals in the 1 st group to the Nth group of the charged droplet receiving units are the same.

It can be understood that, when the delay time of the liquid drop is accurate, the time for charging the liquid drop is accurate, all the microspheres contained in the liquid drop are charged, and the charged microspheres enter the charged liquid drop receiving unit under the action of the deflection electrode, so that the fluorescent signal intensities of the liquid drops in the 1 st group to the Nth group of the charged liquid drop receiving units are the same. Therefore, it is only necessary to verify whether the intensities of the droplet fluorescence signals in the 1 st to nth groups of charged droplet receiving units are the same, and to verify that the droplet delay times are accurate when the intensities of the droplet fluorescence signals in the 1 st to nth groups of charged droplet receiving units are the same.

By verifying the delay time of the liquid drops, the accuracy of the delay time of the liquid drops is further ensured, the accuracy of the charged time of the liquid drops is ensured, the accuracy of liquid drop sorting is ensured, and the precision of the liquid drop sorting is improved.

On the basis of the foregoing embodiment, fig. 5 is a schematic flowchart of another method for determining a droplet delay time according to an embodiment of the present invention, and as shown in fig. 5, the method for determining a droplet delay time according to an embodiment of the present invention may include:

s310, determining the charging electric quantity corresponding to each group of charging droplet receiving units in the N groups of charging droplet receiving units.

And S320, respectively acquiring first detection signal intensity in the charged droplet receiving unit and second detection signal intensity in the uncharged droplet receiving unit corresponding to the N initial droplet delay times.

S330, determining the jth initial droplet delay time T in the N initial droplet delay times according to the first detection signal intensity and the second detection signal intensity_jFor the drop delay time, or for determining the g-1 st initial drop delay time T of the N initial drop delay times_g-1Delay time T from g +1 th initial drop_g+1A certain time T in between_mIs the drop delay time, where T_g-1≤T_m≤T_g+1(ii) a J is more than or equal to 1 and less than or equal to N, j is wholeCounting; g is more than or equal to 2 and less than or equal to N-1, and g is an integer.

For example, in order to ensure that the charged droplets can be received in the charged droplet receiving units, the charging amount corresponding to each group of charged droplet receiving units needs to be determined, and by reasonably setting the charging amount corresponding to each group of charged droplet receiving units, it is ensured that the charged droplets are deflected into the charged droplet receiving units under the action of the deflection electrodes, and accurate detection of the strength of the subsequent first detection signal is ensured.

Based on the above description of the method for determining the drop delay time, the following describes the operation of the method for determining the drop delay time according to an embodiment of the present invention in a specific embodiment, wherein N groups of charged drop receiving units may correspond to eight groups of charged drop receiving units:

firstly, adjusting the droplet charging parameters corresponding to the N charged droplet receiving units, so that the charged droplets pass through the deflection electrode and then are positioned at the center of the deflection channel corresponding to the charged droplet receiving units. After the adjustment of the droplet charging parameters is completed, a set of initial droplet delay times T1: T9 are set, with the time intervals being evenly distributed, such as 0.5ms, 0.65ms, 0.8ms, 0.95ms, 0ms, 1.1ms, 1.25ms, 1.4ms, 1.55ms, where T5 is 0, because the deflection channel CH5 corresponds to the uncharged receiving unit 12 and no deflection operation is performed. The charging delay time corresponding to the deflection channel CH1 corresponding to the first group of charged droplet receiving units 11 is 0.5ms, M detected probe particles are charged into the deflection channel CH1, the corresponding charging amount is C (1), the number M (1) of probe particles in the deflection channel CH1 at this time is recorded, and the number W (1) of probe particles in the deflection channel CH5 corresponding to the uncharged receiving unit 12, wherein the probe particles may be standard-sized microspheres, and the diameter of the microspheres may be 1 μ M, 3 μ M, 5 μ M or other sizes, which is not limited by the embodiment of the present invention. The second group of charged droplet receiving units 11 has a charging delay of 0.65ms for the deflection channel CH2, and also charges the detected M detection particles to the deflection channel CH2 by a corresponding charge amount of C (2), and records the number M (2) of detection particles in the deflection channel CH2 at that time, and the number W (2) of detection particles in the deflection channel CH5 corresponding to the uncharged receiving unit 12. And so on until the detected particle number M (9) in the deflection channel CH9 corresponding to the eighth group of charged receiving units 11 and the detected particle number W (9) in the deflection channel CH5 corresponding to the uncharged receiving unit 12 are obtained, as shown in table one. The number of channels corresponding to the N groups of charged droplet receiving units may be greater than 8 or less than 8, which is not limited in the embodiment of the present invention, and only eight groups of charged droplet receiving units are taken as an example for description.

Table a channel condition corresponding to different droplet receiving units

After the number of the detection particles in the deflection channel is obtained, the channel with the largest number of the detection particles, such as N (3), is taken, whether N (3)/W (3) meets a preset ratio or not is judged, and if N (3)/W (3) meets the preset ratio, the charging delay time T is T3 and is 0.8 ms. Alternatively, as shown in fig. 6, if more microspheres in the deflection channel CH3 are charged correctly, but some microspheres in the waste channel CH5 are not charged, and N (3)/W (3) does not satisfy the predetermined ratio, a set of charging delay times T1' is selected again: t9'. Wherein T1 'is ≧ T2, T9' is ≦ T4, for example, 0.7ms, 0.73ms, 0.76ms, 0.79ms, 0ms, 0.82ms, 0.85ms, 0.88ms, 0.91 ms. The above steps are repeated, in which the number of detected particles in the deflecting passage CH7 is the largest, but N (7)/W (7) is not more than the preset ratio, as shown in fig. 7. At this time, a set of charging delay times T1 ″ is again taken: t9 ' in the same way, T1 ' is not less than T6 ', and T9 ' is not less than T8 '. For example, 0.83ms, 0.835ms, 0.84ms, 0.845ms, 0ms, 0.85ms, 0.855ms, 0.86ms, 0.865 ms. The above steps are repeated, when the number of the detected particles in the deflecting channel CH4 is the largest and N (4)/W (4) is full of the preset ratio, as shown in fig. 8, the charging delay time T is T4 ═ 0.845 ms.

Further, as shown in fig. 6, more microspheres in the deflecting channel CH3 are charged correctly, but some microspheres in the waste liquid channel CH5 are still not charged, at this time, N (3)/W (3) does not satisfy the preset ratio, N (2) is 0 (4), and N (2) is N (3) is N (4), so that it can be stated that the droplet delay time is not T3, and a set of charging delay times T1' needs to be taken again: t9'. Wherein T1 'is ≧ T2, T9' is ≦ T4, for example, 0.7ms, 0.73ms, 0.76ms, 0.79ms, 0ms, 0.82ms, 0.85ms, 0.88ms, 0.91 ms. The above steps are repeated, in which the number of detected particles in the deflecting passage CH7 is the largest, but N (7)/W (7) is not more than the predetermined ratio, and N (6), N (8) are less than N (7), as shown in fig. 7. At this time, it can be shown that the drop delay time is not T7', and a set of charging delay times T1 ″ needs to be fetched again: t9 ' in the same way, T1 ' is not less than T6 ', and T9 ' is not less than T8 '. For example, 0.83ms, 0.835ms, 0.84ms, 0.845ms, 0ms, 0.85ms, 0.855ms, 0.86ms, 0.865 ms. The above steps are repeated, when the number of detected particles in the deflecting channel CH4 is the largest, and N (4)/W (4) is equal to the preset ratio, and N (3) ═ N (4) ═ N (5), as shown in fig. 8, the charging delay time T ═ T4 ═ 0.845ms is illustrated.

Further, the charging delay is set to 0.845ms, the detection particles are sequentially deflected to the deflection channels of CH1: CH9, each deflection channel can obtain the same number of detection particles, and the ratio of the number of detection particles in each deflection channel to the number of detection particles in the waste liquid channel satisfies a preset ratio, that is, the ratio of N (1) -N (9) to W (5) satisfies a preset ratio, as shown in fig. 9, under the condition of the present embodiment, the preset ratio is 9.

In summary, according to the technical solution of the embodiments of the present invention, the flow cytometer is provided with a plurality of charged droplet receiving units, and the plurality of charged droplet receiving units correspond to a plurality of initial droplet delay times, so that the plurality of droplet delay times can be verified at one time in the droplet delay time determination process, thereby ensuring that the droplet delay time can be determined quickly, and improving the droplet delay time determination efficiency.

Based on the same inventive concept, the embodiment of the invention also provides a flow cytometry sorter, and the flow cytometry sorter determines the droplet delay time by adopting the droplet delay time determination method described in the embodiment. Specifically, fig. 10 is a schematic structural diagram of a module of a flow cytometer provided in an embodiment of the present invention, and referring to fig. 2 and fig. 10 in combination, the flow cytometer provided in an embodiment of the present invention includes a droplet delay time determination module 41, a droplet receiving module 42, and a droplet splitting module 43;

the droplet receiving module 42 includes N sets of charged droplet receiving units 11 and one set of uncharged droplet receiving units 12; the N groups of charged droplet receiving units 11 correspond to N initial droplet delay times, wherein any one initial droplet delay time T_iSatisfy 0 < T_i＜T_i+1I is more than or equal to 1 and less than N, and i is an integer;

the droplet delay time determining module 41 is configured to obtain first detection signal intensities in the charged droplet receiving units and second detection signal intensities in the uncharged droplet receiving units corresponding to the N initial droplet delay times, respectively; determining the delay time of the liquid drop according to the first detection signal intensity and the second detection signal intensity;

the drop delay determination module 41 is also used to feed back the drop delay to the drop diversion module 43 to control the time at which the drops are charged.

In particular, the drop delay determination module 41 is specifically configured to determine the jth initial drop delay T of the N initial drop delays_jFor the drop delay time, or for determining the g-1 st initial drop delay time T of the N initial drop delay times_g-1Delay time T from g +1 th initial drop_g+1A certain time T in between_mIs the drop delay time, where T_g-1≤T_m≤T_g+1(ii) a J is more than or equal to 1 and less than or equal to N, and j is an integer; g is more than or equal to 2 and less than or equal to N-1, and g is an integer.

Illustratively, the drop receiving module 42 includes N groups of charged drop receiving cells 11 and one group of uncharged drop receiving cells 12, each group of the uncharged drop receiving cells 11 corresponding to one initial drop delay time, and N groups of the charged drop receiving cells 11 corresponding to N initial drop delay times. In the droplet delay time determination process, the first detection signal intensity in the charged droplet receiving unit 11 and the second detection signal intensity in the uncharged droplet receiving unit 12 corresponding to the N initial droplet delay times are acquired, respectively, so that N sets of the first detection signal intensity and the second detection signal intensity can be acquired, and the first detection signal intensity and the second detection signal intensity are calculated based on the N sets of the first detection signal intensity and the second detection signal intensityThe signal intensity can be directly determined as the drop delay time, for example, the jth initial drop delay time T_jFor drop delay time, or for determining one of two adjacent ones of N initial drop delay times as a drop delay time, e.g. the g-1 st initial drop delay time T_g-1Delay time T from g +1 th initial drop_g+1A certain time T in between_mIs the drop delay time. So, set up a plurality of charging liquid drop receiving unit in setting up flow cytometer, a plurality of charging liquid drop receiving unit correspond a plurality of initial liquid drop delay time, so can once verify a plurality of liquid drop delay time at liquid drop delay time determination in-process, guarantee can confirm liquid drop delay time fast, promote liquid drop delay time and confirm efficiency.

It should be noted that, in the flow cytometer provided in the embodiment of the present invention, the droplet delay time determining module operates in a process before formal cell sorting, determines the droplet delay time before formal cell sorting starts through the droplet delay time determining module, and feeds back the droplet delay time to the droplet shunting module, and the droplet shunting module determines the time for charging the droplet according to the droplet delay time in the formal cell sorting process, so as to ensure that the droplet is accurately charged at the droplet breaking point, and the droplet charging time is accurately controlled, so that accurate droplet sorting is ensured, and the droplet sorting efficiency is high.

In summary, the flow cytometer provided in the embodiment of the present invention includes a plurality of sets of charged droplet receiving units in the flow cytometer, and corresponds to a plurality of droplet delay detection channels, and a method of multi-channel detection and comparison and judgment is used to quickly determine droplet delay time, so that the droplet delay time determination efficiency is high, and meanwhile, the droplet charging time control is accurate, thereby ensuring accurate droplet sorting and high droplet sorting efficiency.

Further, the droplet delay time determination module 41 may include a signal intensity detection sub-module and a droplet delay time determination sub-module; the signal intensity detection submodule is used for respectively acquiring N liquid drop delay time correspondencesA first detection signal strength in the charged droplet receiving unit and a second detection signal strength in the uncharged droplet receiving unit of (a); the drop delay time determination submodule is used for determining the jth drop delay time T in the N drop delay times according to the first detection signal intensity and the second detection signal intensity_jFor drop delay time, or for determining the g-1 drop delay time T of the N drop delay times_g-1Delay time T from g +1 th droplet_g+1A certain time T of_mIs the drop delay time.

For example, after acquiring the first detection signal strengths of the charged droplet receiving units 11 corresponding to the N initial droplet delay times, the initial droplet delay time corresponding to the charged droplet receiving unit 11 with the strongest first detection signal strength, for example, T, is determined_j(ii) a Simultaneous acquisition of T_jSecond detected signal strength in the corresponding uncharged drop receiving unit 12 if T_jIf the ratio of the first detection signal intensity to the second detection signal intensity satisfies a predetermined ratio, the droplet delay time T is T ═ T_jI.e. determining the jth initial drop delay time T_jIs the drop delay time. Or, after acquiring the first detection signal intensities in the charged droplet receiving units 11 corresponding to the N initial droplet delay times, respectively, the initial droplet delay time corresponding to the charged droplet receiving unit 11 with the strongest first detection signal intensity, for example, T, is determined_g(ii) a Simultaneous acquisition of T_gSecond detected signal strength in the corresponding uncharged drop receiving unit 12 if T_gThe ratio of the intensity of the corresponding first detection signal to the intensity of the corresponding second detection signal is not more than a preset ratio, and T_gThe intensity of the second detection signal in the corresponding uncharged droplet receiving unit 12 is smaller than the intensity of the second detection signal corresponding to the other initial droplet delay time, which indicates that T_gNot the exact drop delay, but at T_gIn the vicinity, the accurate drop delay time can be determined at the moment so as to be between the g-1 st initial drop delay time and the g +1 st initial drop delay time, that is, the jth initial drop delay time corresponding to the jth group of charged drop receiving units is T_j’， T_g-1≤T_j’≤T_g+1The delay time of the g initial droplet corresponding to the g group of charged droplet receiving units is T_g' satisfy T_g-1≤T_g’≤T_g+1. And repeating the steps according to the redetermined initial droplet delay time until the ratio of the first detection signal intensity and the second detection signal intensity corresponding to the mth initial droplet delay time meets a preset ratio, and determining the mth initial droplet delay time as the droplet delay time, wherein m is more than or equal to 1 and less than or equal to N. Therefore, accurate and efficient measurement of the delay time of the liquid drop can be ensured.

Optionally, with continued reference to fig. 2, in the flow cytometer provided in the embodiment of the present invention, the droplet delay time determination module 41 may include a signal intensity detection sub-module 411, and the signal intensity detection sub-module 411 may include a first laser light source 4111 and a first monitoring camera assembly 4112.

Specifically, the first laser light source 4111 is configured to detect whether the charged droplet contains a detection particle, the surface of the detection particle contains fluorescein, and the laser light emitted by the first laser light source 4111 irradiates the detection particle and excites a specific fluorescence.

The first monitoring camera component 4112 is used to acquire images of scattered light from the droplets and to detect fluorescence from the particles. Scattered light image of the droplet as shown in fig. 7, the channel boundaries are automatically generated by the first monitoring camera component 4112 and the scattered light signal of the droplet excitation is located right in the center of the deflection channel.

Optionally, the intensity of the first detection signal and the intensity of the second detection signal may both be the intensity of a fluorescence signal excited by the first laser light source 4111 exciting the detection particle in the droplet; or both the first detection signal intensity and the second detection signal intensity are the scattering signal intensity excited by the detection particles in the droplet excited by the first laser light source. 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 first monitoring camera component 4112 can receive the excitation signal of the detection particle based on the laser emitted by the first laser light source 4111.

Further, fig. 11 is a schematic structural diagram of a signal strength detection sub-module according to an embodiment of the present invention, and as shown in fig. 11, the signal strength detection sub-module 411 may further include a converging lens 4113 and a mirror group 4114;

the converging lens 4113 is located on a transmission path of the laser signal emitted by the first laser light source 4111;

the mirror group 4114 includes N mirrors 4115, and the mirrors 4115 are used for reflecting laser signals to flow paths of droplets corresponding to the charged droplet receiving unit 11 and the uncharged droplet receiving unit 12.

Illustratively, parallel laser light emitted by the first laser light source 4111 passes through the converging lens 4113 and then converges in the charged droplet receiving unit 11, and then converges in another charged droplet receiving unit 11 or an uncharged droplet receiving unit 12 through the reflector 4115. The reflector 4115 is used to divert the focused laser spot to the deflection channel corresponding to the charged droplet receiving unit 11 or the uncharged droplet receiving unit 12, so that each deflection channel is illuminated by the same size of illumination spot. When the same microsphere passes through the deflection channels, the intensities of the excited fluorescence signals are equal, the intensity of the first detection signal or the intensity of the second detection signal is ensured to be the same, and the accurate measurement of the delay time is ensured.

Further, as shown in fig. 11, the ith mirror 4115 in the mirror group 4114 is located between the ith charged droplet receiving unit 12 and the (i + 1) th charged droplet receiving unit 11, and is used for reflecting the laser signal to the (i + 1) th charged droplet receiving unit 11; alternatively, the ith mirror 4115 is located between the ith charged droplet receiving unit 11 and the uncharged droplet receiving unit 12, and is used for reflecting the laser signal to the uncharged droplet receiving unit 12; or, the ith reflector 4115 is located between the uncharged droplet receiving unit 12 and the i +1 th charged droplet receiving unit 11, and is configured to reflect the laser signal to the i +1 th charged droplet receiving unit 11, so as to ensure that each of the charged droplet receiving unit 11 and the uncharged droplet receiving unit 12 can receive the laser signal emitted by the first laser light source 4111, the first monitoring camera component 4112 can obtain the first detection signal intensity of all the charged droplet receiving units 11 and the second detection signal intensity of the uncharged droplet receiving unit 12, and the droplet extension time can be determined based on the first detection signal intensity and the second detection signal intensity.

Optionally, the reflector 4115 may include a plane reflector and/or a spherical reflector, which is not limited in this embodiment of the present invention, and fig. 11 only illustrates that the reflector is a spherical reflector.

Optionally, as shown in fig. 11, the first monitoring camera component 4112 provided in this embodiment of the present invention may be a wide-angle monitoring camera, so that the wide-angle monitoring camera may respectively obtain fluorescence signals and/or scattered light signals of laser signals passing through N sets of charged droplet receiving units and uncharged droplet receiving units, and one wide-angle monitoring camera may obtain all the intensities of the first detection signal and the second detection signal, thereby ensuring that the first monitoring camera component 4112 is simple in configuration.

Optionally, the first monitoring camera component 4112 provided in the embodiment of the present invention may include a plurality of non-wide-viewing-angle monitoring cameras, as shown in fig. 12, the first monitoring camera component 4112 may include N +1 monitoring cameras 4116; wherein, the first to nth monitoring cameras 4116 in the N +1 monitoring cameras 4116 are configured to respectively acquire fluorescence signals and/or scattered light signals of the laser signals after passing through the N sets of charged droplet receiving units 11; the (N + 1) th monitoring camera 4116 of the (N + 1) monitoring cameras 4116 is used to acquire a fluorescence signal and/or a scattered light signal of the laser signal after passing through the uncharged droplet receiving unit 12. By setting N +1 non-wide view angle monitoring cameras to acquire detection signal intensities in the N charged droplet receiving units 11 and the uncharged droplet receiving units 12, respectively, the droplet extension time can be determined based on the detection signal intensities.

Optionally, as shown in fig. 2, fig. 11 and fig. 12, the flow cytometer provided by the embodiment of the present invention may further include at least one illumination light source 44, where the illumination light source 44 is used to provide illumination for the charged droplet receiving unit 11 and the uncharged droplet receiving unit 12. Alternatively, the illumination light source 44 may be an LED light source, which is not limited in this embodiment of the present invention.

Optionally, with continued reference to fig. 2, the flow cytometer provided in the embodiment of the present invention may further include an initial droplet delay time providing module 45, where the initial droplet delay time providing module 45 may include a checkpoint recording sub-module 451 and a breakpoint calculation sub-module 452; the detection point recording sub-module 451 may be configured to record a first time that the fluid stream passes a fluid stream detection point; the break-off point calculation submodule 452 may be configured to calculate a charging time of the droplet at the break-off point based on the first time and the initial droplet delay time, respectively.

For example, the liquid stream detection point may be a detection position located before the liquid drop breaking point, and the liquid drop breaking point is a position where the liquid stream breaks to form a liquid drop. The detection point recording submodule 451 may be configured to record a first time when the liquid stream passes through the detection point of the liquid stream, and the break-off point calculating submodule 452 may be configured to calculate a charging time of the liquid drop at the break-off point according to the first time and a preset initial drop delay time.

Further, the detection point recording sub-module 451 may include a second laser light source 4511, and may further include a forward detection signal receiving unit 4512 and/or a lateral detection signal receiving unit 4513, and fig. 2 illustrates that the detection point recording sub-module 451 includes both the forward detection signal receiving unit 4512 and the lateral detection signal receiving unit 4513.

Illustratively, the second laser source 4511 is configured to emit a laser signal to the liquid stream detection point a1, the laser signal excites the detection particles in the liquid stream to generate a fluorescence signal, and the forward detection signal receiving unit 4512 is configured to receive the fluorescence 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.

Further, as shown with continued reference to fig. 2, the forward detection signal receiving unit 4512 may include a first light blocking diaphragm 4513, a forward lens 4514, and a forward detector 4515. The first light shielding diaphragm 4514 is configured to shield a laser beam emitted by the second laser light source 4511 from directly irradiating the forward detection signal receiving unit 4512, and it is ensured that a detection signal received by the forward detection signal receiving unit 4512 is only a fluorescent signal or a forward scattering light signal obtained by a detection particle based on excitation of laser emitted by the second laser light source 4511, and it is avoided that the laser beam emitted by the second laser light source 4511 directly irradiates the forward detection signal receiving unit 4512, which causes interference on the detection signal, and it is ensured that the first receiving time is accurate. The forward lens 4515 is used to focus the fluorescence signal or forward scattered light signal of the detected particle, so as to ensure that the fluorescence signal or forward scattered light signal can enter the forward detection signal receiving unit 4512 more, and ensure that the first receiving time is received accurately. The forward detector 4516 is a photodetector or a photomultiplier tube, and the embodiment of the present invention does not limit the specific type of the forward detector 4516.

Further, as shown in fig. 2, the side detection signal receiving unit 4513 may include a side lens 4517 and a side detector 4518, and the side lens 4517 is configured to focus a fluorescence signal or a side scattered light signal excited by the detection particle based on the second laser signal, so as to ensure that the fluorescence signal or the side scattered light signal can enter the side detector 4518 more, and ensure that the first receiving time based on the detection signal is received accurately. The side lens 4517 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 4518 may be a photoelectric detector or a photomultiplier tube, and the embodiment of the present invention does not limit the side detector 4518 specifically.

Optionally, with continued reference to fig. 2, the sub detection point recording module 451 provided in the embodiment of the present invention may further include an illumination lens 4519, where the illumination lens 4519 is located on a transmission path of a laser signal emitted by the second laser light source 4511.

Illustratively, the illumination lens 4519 is used to adjust the laser signal emitted from the second laser light source 4511, for example, adjust the focusing power and the divergence angle of the laser signal, so as to ensure that the focusing effect of the laser signal is good. Further, the illumination lens 4519 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 4519.

Optionally, with continued reference to fig. 2, the breakpoint calculation sub-module 452 may include a monitoring camera 4521 and a frequency flash light source 4522; the monitoring camera 4521 is used to acquire droplet motion images; a strobe light source 4522 is used to provide an illumination signal for the monitoring camera.

For example, in order to accurately determine the position of the droplet breaking point a2, the monitoring camera 4521 may be used to monitor the flow of the liquid stream in the vicinity of the droplet breaking point, acquire a droplet moving image, and clearly determine the position of the droplet breaking point 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. The stroboscopic light source 4522 is used for providing an illumination signal for the monitoring camera 4521, so that a monitoring area of the monitoring camera 4521 is ensured to be in a bright field state, and a moving image of liquid drops is ensured to be clear. Optionally, the strobe light source 4522 may include a light emitting diode or a semiconductor laser, and the exposure time of the strobe light source 4522 is matched with the liquid flow velocity, ensuring that the monitoring camera 4521 can acquire a clear droplet moving image during the exposure time of the strobe light source 4522. Further, the exposure time T may satisfy T <5 μ s.

Optionally, with continuing reference to fig. 2, the flow cytometer provided in the embodiment of the present invention may further include a deflecting electrode plate 46, where the deflecting electrode plate 46 is configured to control the charged droplets to deflect, and the charged droplet receiving unit 11 is configured to receive the deflected droplets, so as to complete the cell sorting operation.

In summary, in the flow cytometer provided in the embodiments of the present invention, multiple groups of charged droplet receiving units and multiple charged droplet receiving units are arranged to correspond to multiple initial droplet delay times, so that multiple droplet delay times can be verified at one time in a droplet delay time determination process, it is ensured that the droplet delay times can be determined quickly, and the droplet delay time determination efficiency is improved; meanwhile, the liquid drop sorting module can accurately master the liquid drop delay time, guarantee that the liquid drops are accurately charged at the liquid drop fracture point, accurately control the liquid drop charging time, guarantee that the liquid drops are accurately sorted, and have high liquid drop sorting efficiency; 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.

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 method for determining the delay time of liquid drops is applied to a flow cytometry sorter, wherein the flow cytometry sorter comprises a liquid drop receiving module, and the liquid drop receiving module comprises N groups of charged liquid drop receiving units and one group of uncharged liquid drop receiving units; the N groups of charged droplet receiving units correspond to N initial droplet delay times, wherein any one initial droplet delay time T_iSatisfy 0 < T_i＜T_i+1I is more than or equal to 1 and less than N, and i is an integer;

the method for determining the drop delay time comprises the following steps:

determining a jth initial drop delay time T of the N initial drop delay times based on the first and second detection signal strengths_jFor the drop delay time, or for determining the g-1 st initial drop delay time T of the N initial drop delay times_g-1Delay time T from g +1 th initial drop_g+1A certain time T in between_mIs the drop delay time, where T_g-1≤T_m≤T_g+1(ii) a J is more than or equal to 1 and less than or equal to N, and j is an integer; g is more than or equal to 2 and less than or equal to N-1, and g is an integer.

2. The method of determining a drop delay time of claim 1, wherein the jth initial drop delay time T of the N initial drop delay times is determined based on the first and second detection signal strengths_jFor the drop delay time, or for determining the g-1 st initial drop delay time T of the N initial drop delay times_g-1Delay time T from g +1 th initial drop_g+1A certain time T in between_mIs a drop delay time, comprising:

3. The method of claim 2, wherein the first detection signal strength and the second detection signal strength are based onIntensity, determining the jth initial drop delay time T of the N initial drop delay times_jFor the drop delay time, or for determining the g-1 st initial drop delay time T of the N initial drop delay times_g-1Delay time T from g +1 th initial drop_g+1A certain time T in between_mIs a drop delay time, comprising:

4. The method of determining a drop delay time of claim 1, wherein after determining the drop delay time, further comprising:

the drop delay time is verified.

5. The method of determining a drop delay time of claim 4, wherein verifying the drop delay time comprises:

6. The method of determining drop delay time according to claim 1, wherein before acquiring the first detection signal strength in the charged drop receiving unit and the second detection signal strength in the uncharged drop receiving unit corresponding to the N initial drop delay times, respectively, further comprises:

7. The method of determining drop delay of claim 2, wherein T is_i-T_i-1＝T_i+1-T_i；T_j’-T_j-1’＝T_j+1’-T_j’。

8. A flow cytometer for determining a droplet delay time by using the droplet delay time determination method according to any one of claims 1 to 7, wherein the flow cytometer comprises a droplet delay time determination module, a droplet receiving module, and a droplet splitting module;

the drop receiving module comprises N groups of charged drop receiving units and one group of uncharged drop receiving units; the N groups of charged droplet receiving unit pairsN initial drop delay times, wherein any initial drop delay time T_iSatisfy 0 < T_i＜T_i+1I is more than or equal to 1 and less than N, and i is an integer;

9. The flow cytometer of claim 8 wherein the drop delay time determination module comprises a signal intensity detection sub-module comprising a first laser light source and a first monitoring camera assembly.

10. The flow cytometer of claim 9 wherein the signal strength detection sub-module further comprises a collection lens and a set of mirrors;

11. A flow cytometer as described in claim 10 wherein the ith mirror in said mirror group is located between the ith charged drop receiving unit and the (i + 1) th charged drop receiving unit for reflecting said laser signal to said (i + 1) th charged drop receiving unit;

12. A flow cytometer as described in claim 10 wherein said mirror comprises a planar mirror and/or a spherical mirror.

13. The flow cytometer of claim 9 wherein the first monitoring camera assembly comprises a wide view monitoring camera;

14. The flow cytometer of claim 9 wherein the first monitoring camera assembly comprises N +1 monitoring cameras;

15. The flow cytometer of claim 9, wherein the first detection signal intensity and the second detection signal intensity are both fluorescence signal intensities excited by the detection particles in the droplet excited by the first laser light source;

16. The flow cytometric analyzer of claim 8, further comprising at least one illumination light source for providing illumination to the charged drop receiving unit and the uncharged drop receiving unit.

17. A flow cytometer as described in claim 8 wherein said drop delay time determination module is specifically configured to determine a jth initial drop delay time T of N initial drop delay times_jFor the drop delay time, or for determining the g-1 st initial drop delay time T of the N initial drop delay times_g-1Delay time T from g +1 th initial drop_g+1A certain time T in between_mIs the drop delay time, where T_g-1≤T_m≤T_g+1(ii) a J is more than or equal to 1 and less than or equal to N, and j is an integer; g is more than or equal to 2 and less than or equal to N-1, and g is an integer.

18. The flow cytometer of claim 8 wherein the drop delay time determination module comprises a signal strength detection sub-module and a drop delay time determination sub-module;

19. The flow cytometer of claim 8 further comprising an initial drop delay time providing module comprising a checkpoint recording sub-module and a breakpoint calculation sub-module;

20. A flow cytometer as described in claim 19 wherein said checkpoint recording sub-module comprises a second laser light source and further comprises a forward detection signal receiving unit and/or a lateral detection signal receiving unit.

21. The flow cytometer of claim 20 wherein the forward detection signal receiving unit comprises an optical stop, a forward lens and a forward detector;

22. The flow cytometer of claim 20 wherein the detection point recording sub-module further comprises an illumination lens, wherein the illumination lens is positioned on the transmission path of the laser signal emitted from the second laser light source.

23. The flow cytometer of claim 19 wherein the breakpoint calculation sub-module comprises a monitoring camera and a stroboscopic light source;

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

24. The flow cytometric classifier of claim 8, further comprising a deflecting electrode plate.