CN208399340U - A kind of flow cytometry device - Google Patents
A kind of flow cytometry device Download PDFInfo
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- CN208399340U CN208399340U CN201821202366.7U CN201821202366U CN208399340U CN 208399340 U CN208399340 U CN 208399340U CN 201821202366 U CN201821202366 U CN 201821202366U CN 208399340 U CN208399340 U CN 208399340U
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Abstract
The utility model relates to a kind of flow cytometry devices, including flow chamber to rectify room, and impedance rectifies room, impedance aperture, impedance positive electrode, specimen needle, flow chamber, laser light source module and scattering light collection module.Impedance method, scattered light method in different time, are associated by the device by two signals that the same cell generates.Optical detection center mean flow rate v is flow to from gem hole center according to cell；The time difference Δ t=L/v that same cell passes through two sensors is calculated to optical detection centre distance L in gem hole center.Further according to the expanded window of two time point delineation impedance channels of Ts_ex- Δ t and Te_ex- Δ t, unique corresponding impedance method signal is found in impedance signal expanded window, carries out signal alignment.The utility model is clear in structure, and assembling is simple, and optics is integrated with impedance detection, and test sample is high-efficient, secondary sheath stream protect sample will not contaminant sensor, synchronous signal is high-quality.
Description
Technical field
The utility model relates to medical instruments field more particularly to a kind of flow cytometry devices.
Background technique
Light scattering principle or impedance method (Coulter principle) are generally used in blood cell/field of cell analysis, to solution
The cell of middle suspension counted, volume (size), Morphology observation.
When detecting cell using light scattering principle, flow cytometry is core technology difficult point, and basic skills is using sheath
Sample cell is constrained to unicellular stream by liquid, by sensor, by determining that the laser irradiation cell detection of spot size scatters light
Variation, generates the pulse of different amplitudes and quantity, to carry out the analysis of the information such as counting and size to cell.Use impedance method
When (Coulter principle) detects cell, classical method is sample by (tens microns) of a micropore tiny isolation, while small
Hole both ends are equipped with positive and negative electrode, are placed in aperture in certain electric field；Negative pressure absorbing sample liquid, sample are passed through in the other end of aperture
State is laminar when originally passing through aperture, cell position determines, when each cell is through small holes, resistivity rises in instantaneous aperture,
Pulse, recording impulse quantity, amplitude, to analyze cell will be generated on the electrode.
Existing stream type cell device has the following problems: (1) in a flow path while cannot carry out two kinds of impedance, optics sides
Science of law detection, therefore counting can not be verified in real time as a result, can only detect again if result mistake.(2) two set of modules need two
Mating fluid path structural support function is covered, structure is complicated, high failure rate, and use cost is high.
Utility model content
The purpose of this utility model is that in place of solving the above deficiencies in the existing technologies.
To achieve the above object, in a first aspect, the utility model provides a kind of flow cytometry device, including flow chamber
Room is rectified, impedance rectifies room, and impedance aperture, impedance positive electrode, specimen needle, flow chamber, laser light source module and scattering light are collected
Module；Specimen needle is located at impedance rectification chamber interior, and impedance aperture is located at the rear end of impedance rectification room, and the outlet end of impedance aperture connects
Impedance positive electrode is connect, specimen needle connects cathode；The rear end of impedance rectification room is located at the inside of flow chamber rectification room, and flow chamber is located at
The rear end of impedance aperture.
Preferably, flow chamber include side length be 200 be micron square fluid channel.
Preferably, flow cytometry device further includes laser light source module and scattering light collection module, laser light source mould
Block and scattering light collection module are located at the opposite two sides of flow chamber.
Preferably, flow chamber include side length be 200 be micron square fluid channel.
Second aspect, the utility model provide a kind of compensation method of flow cytometry device, are applied to first aspect
Flow cytometry device, comprising the following steps:
Step 1: impedance signal is cached using memory, wherein caching depth is D=(L/v) * S；
Step 2: detection optical channel rising edge obtains optical channel rising edge triggering moment TsRise with optical channel
Moment T is trembled along disappearingse；
Step 3: detection optical channel failing edge and peak value obtain optical channel failing edge triggering moment TesIt is logical with optics
Road failing edge, which disappears, trembles moment Te；
Step 4: expanded window before and after setting optical channel pulse: according to optical channel rising edge triggering moment TsExpanded
Open up the first moment of window Ts_ex, disappeared according to optical channel failing edge and tremble moment TeBe expanded the second moment of window Te_ex；
Step 5: setting impedance effective impulse section: being T at the beginning of impedance effective impulse sections_ex+ D, at the end of
Carving is Te_ex+D；
Step 6: detection impedance channel rising edge obtains impedance channel rising edge triggering moment Ts_iRise with impedance channel
Moment T is trembled along disappearingse_i；
Step 7: detection impedance channel failing edge and peak value obtain impedance channel failing edge triggering moment Tes_i, impedance it is logical
Road failing edge, which disappears, trembles moment Te_iWith impedance channel peak value moment Tmax_i；
Step 8: optical channel and the same cell signal characteristic synchronization of impedance channel: T is readmax_i, work as Tmax_iIn Ts_ex+D
To Te_exWhen+D the period occurs, indicate to recognize optical channel signal and impedance signal that same cell generates；
Wherein, S is the sample rate of analog-digital converter ADC, and v flow to optical detection center from gem hole center for cell and is averaged
Flow velocity, L are the distance at gem hole center to optical detection center.
Preferably, after step 8 further include:
Step 9: system stability judgement: when pulse delay feature Pdl is less than alarm interval limit or is greater than zone of alarm
Between the upper limit, wherein Pdl=Ts_rs/ (Ts_ex-Te_ex), Ts_rs=(Tmax_i Ts_ex-D).
Flow cytometry device disclosed by the utility model and compensation method have the effect that (1) is clear in structure,
Assembling is simple, safeguards, high reliablity low with manufacturing cost；(2) optics is integrated with impedance detection, and test sample is high-efficient；
(3) secondary sheath stream protection sample will not contaminant sensor, synchronous signal is high-quality；(4) reliable backoff algorithm, makes flow passage structure
Complexity reduces.
Detailed description of the invention
Fig. 1 is a kind of structural schematic diagram of flow cytometry device provided by the utility model；
Fig. 2 is a kind of operation principle schematic diagram of flow cytometry device provided by the utility model；
Fig. 3 is the effective impulse schematic diagram in a kind of expanded window provided by the embodiment of the utility model；
Fig. 4 is the abnormal pulsers figure abandoned in a kind of expanded window provided by the embodiment of the utility model；
Fig. 5 is a kind of scattering light pulse signal identification figure provided by the embodiment of the utility model；
Fig. 6 is a kind of schematic diagram that expanded window is generated according to scattering light pulse signal provided by the embodiment of the utility model；
Fig. 7 is a kind of schematic diagram for finding impedance pulse signal section provided by the embodiment of the utility model；
Fig. 8 is a kind of schematic diagram for identifying impedance pulse signal peak value provided by the embodiment of the utility model；
Fig. 9 is a kind of schematic diagram of impedance pulse signal recognition methods provided by the embodiment of the utility model；
Figure 10 is the schematic diagram of the effective impulse in a kind of impedance signal expanded window provided by the embodiment of the utility model；
Figure 11 is a kind of schematic diagram of method judgement of stability provided by the embodiment of the utility model.
Specific embodiment
With reference to the accompanying drawings and examples, the technical solution of the utility model is described in further detail.
Such as Fig. 1-2, flow cytometry device provided by the embodiment of the utility model, including flow chamber rectify room 1, impedance
Rectify room 2, impedance aperture 3, impedance positive electrode 4, specimen needle 5, flow chamber 6, laser light source module 7 and scattering light collection module 8；
Specimen needle 5 is located inside impedance rectification room 2, and impedance aperture 3 is located at the rear end of impedance rectification room 2, and the outlet end of impedance aperture 3 connects
Impedance positive electrode 4 is connect, forward voltage is provided, specimen needle 5 connects cathode, provides negative voltage；The rear end of impedance rectification room 2 is located at
Flow chamber rectifies the inside of room 1, and flow chamber 6 is located at the rear end of impedance aperture 3, laser light source module 7 and scattering light collection module 8
It is located at the opposite two sides of flow chamber 6.Specific work process is as follows:
Cell suspension is entered by the pipeline that specimen needle 5 connects, meanwhile, impedance rectification is passed through sheath fluid in room 2, works as cell stream
Out when specimen needle 5, impedance aperture 3 is flowed to by sheath fluid package in impedance rectification room 2, wherein 3 outlet end of impedance aperture connects impedance
Positive electrode 4, specimen needle 5 connect cathode, and generate constant current in the aperture of impedance aperture 3 and detect electric field.Cell is small by impedance
When hole 3, the sheath fluid in impedance aperture 3 is displaced, 3 internal conductance rate of impedance aperture is caused to decline, resistance rises, while generating cell
Pulse, wherein cell size is different, and resistance is also different, therefore impulse amplitude and width are also different, by count number of pulses,
Amplitude distinguishes cell quantity and size.When cell flows out impedance aperture 3, by the secondary sheath stream packet in flow chamber rectification room 1
It wraps up in and to form unicellular stream and flow to flow chamber 6, laser light source module 7 forms a certain size hot spot in the determination position of flow chamber 6,
And it is received by scattering light collection module 8.When cell passes through hot spot, different size of cell blocks facula area difference, scattering
The scattering light that light collection module 8 is collected is also different, and each cell can trigger by hot spot and generate a scattering light, and be scattered
Light collection module 8 is collected, and the scattering light of collection is converted into pulse signal by photoelectric conversion by scattering light collection module 8, is passed through
Number of pulses, amplitude are analyzed to distinguish cell quantity and size.
In one example, flow chamber 6 include side length be 200 be micron square fluid channel 9.
The utility model embodiment also provides a kind of compensation method of above-mentioned apparatus, defines analog-digital converter (analog to
Digital converter, ADC) measurable range of signal be 0V~+4V, effective impulse signal width be 0.5uS~2uS,
It is denoted as (wmin, wmax), the sample rate of ADC is 40MHz, is denoted as S；It is flat that cell from gem hole center 10 flow to optical detection center 11
Equal flow velocity is v, and gem hole center 10 to optical detection center 11 is apart from for L.The compensation method the following steps are included:
Step 1: impedance signal is cached using the FIFO memory of programmable storage depth, wherein caching depth
For D=(L/v) * S；
Step 2: detection optical channel rising edge: setting optical channel rising edge triggering level Vsp is 0.01V to 0.3V
Between, setting optical channel rising edge disappear tremble level Vsn be 0.02V between 0.45V；
A) moment is recorded as the triggering of optical channel rising edge when scattering light detection channel levels greater than Vsp such as Fig. 5
Moment Ts, first, which counts accumulator ∑ T1, is set as 1.
B) continue next collection point Ts+n electrical level judging, n is n-th electrical level judging, the level of detection when being less than Vsn,
1 is added to the first counting accumulator ∑ T1.When ∑ T1 is greater than maximum effective pulse width Wmax=(wmax* S)/2 when, mark this arteries and veins
Punching is abnormal pulsers, and is added up to the first abnormal pulsers counter ∑ Cerror_o, and the detection of this rising edge of a pulse is terminated.When
When ∑ T1 is less than or equal to Wmax, repeat b) to execute；When the level of collection point Ts+n is greater than level Vsn, this is marked to acquire the moment
Disappear for optical channel rising edge and trembles moment Tse, rising edge detects successfully, and record collection point Ts+n level is to random access memory
In (random access memory, RAM), it is denoted as Vmax.
Step 3: such as Fig. 5, optical channel failing edge and peak value, optical channel failing edge and peak detection are detected.Light is set
Learning channel failing edge triggering level Vep is 0.015V between 0.4V, and setting optical channel failing edge, which disappears, to be trembled level Ven and be
0.01V is between 0.3V；Usually setting Vep level is less than Vsn.
A) when scattering light detection channel levels less than Vep, being denoted as the moment is optical channel failing edge triggering moment Tes,
Second counting accumulator ∑ T2 is set as 1.
B) continue next collection point Tes+m electrical level judging, m is the m times electrical level judging, when the level detected is greater than level
When Ven, 1 is added to the second counting accumulator ∑ T2.When ∑ T2 is greater than maximum effective pulse width Wmax=(wmax*S)/2, mark
Remember that this pulse is abnormal pulsers, and add up to the first abnormal pulsers counter ∑ Cerror_o, terminates this pulse falling edge
Detection repeats b) to execute when ∑ T2 is less than or equal to Wmax；When the level of collection point Tes+m is less than level Ven, to counting
Accumulator ∑ T2 is reset, and is marked the acquisition moment to disappear for optical channel failing edge and is trembled moment Te, failing edge detects successfully.
When executing b-stage, acquisition level point each time is compared with the Vmax in RAM, when this acquires level point
When greater than Vmax in RAM, by Vmax of this collection point level recording into RAM, while it is logical for optics to record this acquisition moment
Road peak value moment Tmax_o.
Step 4: such as Fig. 6, expanded window before and after optical channel pulse is arranged: setting rising edge of a pulse expanded window Wnd_s is
1uS, failing edge expanded window Wnd_e are 1uS.Rising edge expanded window sampled point is S*Wnd_s, and failing edge expanded window sampled point is S*
Wnd_e.The first moment of expanded window Ts_ex is recorded as to acquire moment Ts to Wnd_s is pushed forward；To acquire moment Te to pusher Wnd_
E is recorded as the second moment of expanded window Te_ex.
Step 5: such as Fig. 7, impedance effective impulse section is set: starting to the Te_ex+D moment to terminate with the Ts_ex+D moment
As optical channel effective impulse in the impedance operator recognition time section of impedance channel.
Step 6: such as Fig. 9, impedance channel rising edge is detected, setting impedance channel rising edge triggering level Vsp_i is
0.01V is between 0.3V, and setting impedance channel rising edge disappears, and to tremble level Vsn_i be 0.02V between 0.45V；
It obtains impedance channel rising edge triggering moment Ts_i and impedance channel rising edge disappears and trembles moment Tse_i；
A) such as Fig. 4, when impedance detection channel levels are greater than Vsn_i value range, to the second abnormal pulsers counter ∑
Cerror_i adds up, and terminates the detection of this rising edge of a pulse.When impedance detection channel levels are greater than Vsp_i, when marking this
Carving is impedance channel rising edge triggering moment Ts_i, and third counts accumulator ∑ T3_i and is set as 1.
B) continuing next collection point Ts_i+n electrical level judging, n is n-th, when the level of detection is less than level Vsn_i,
Accumulator ∑ T3_i is counted to third and adds 1.When ∑ T3_i is greater than maximum effective pulse width Wmax=(wmax*S)/2, label
This pulse is abnormal pulsers, and is added up to the second abnormal pulsers counter ∑ Cerror_i, and the inspection of this rising edge of a pulse is terminated
It surveys.When ∑ T3_i is less than or equal to Wmax, repeat b) to execute；When the level of collection point Ts_i+n is greater than level Vsn_i, label
This acquisition moment disappears for impedance channel rising edge trembles moment Tse_i, and rising edge detects successfully, and record collection point Ts_i+n level is extremely
Vmax_i in RAM.
Step 7: detection impedance channel failing edge and peak value, setting triggering level Vep_i are 0.015V between 0.4V,
Setting, which disappears, trembles level Ven_i between 0.01V to 0.3；Usually setting Vep_i level is less than Vsn_i.
A) when impedance detection channel levels are less than Vep_i, marking the moment is impedance channel failing edge triggering moment
Tes_i, the 4th counting accumulator ∑ T4_i are set as 1.
B) continue next collection point Tes_i+m electrical level judging, m is the m times electrical level judging, when the level detected is greater than electricity
When flat Ven_i, 1 is added to the 4th counting accumulator ∑ T4_i.When ∑ T4_i is greater than maximum effective pulse width Wmax=(wmax*
When S)/2, marking this pulse is abnormal pulsers, and is added up to the second abnormal pulsers counter ∑ Cerror_i, terminates this arteries and veins
Rising edge detection is rushed, when ∑ T4_i is less than or equal to Wmax_i, repeats b) to execute；When collection point Tes_i+m level is less than level
When Ven_i, accumulator ∑ T4_i is counted to the 4th and is reset, mark this acquisition moment to disappear for impedance channel failing edge and tremble moment Te_
I, failing edge detect successfully, such as Fig. 3.
As Fig. 8 is compared acquisition level point each time with the Vmax_i in RAM, when executing b-stage when this is adopted
When collecting the Vmax_i that level point is greater than in RAM, by Vmax_i of this collection point level recording into RAM, while this acquisition is recorded
Moment is impedance channel peak value moment Tmax_i.As identified in Fig. 9.
Step 8: as shown in Figure 10, optical channel and the same cell signal characteristic synchronization of impedance channel: reading Tmax_i,
When Tmax_i is when Ts_ex+D occurs to the Te_ex+D period, indicate to recognize optical channel signal that same cell generates and
Impedance signal.
In one example, further include step 9 after the step 8: system stability judgement: being arranged to step 8
The signal time recognized carries out interpretation, and label (Ts_ex+D-Tmax_i) is Ts_rs, records this pulse delay feature Pdl=
Ts_rs/(Ts_ex-Te_ex).For each sample measurement, the delay feature of all pulses is counted, calculates its mean value A, when mean value is big
It when 0.5, indicates that cell shortens from impedance channel to optical channel flow time, when mean value is less than 0.5, indicates cell from resistance
Anti- channel is elongated to optical channel flow time.It is 0.4 that time alarm interval limit, which is arranged, and the setting upper limit is 0.6, such as Figure 11.
When test sample mean value A is not in section of alarming, alarm instrument.It should be appreciated that according to design requirement, section of alarming
Lower and upper limit can change.
Above specific embodiment has carried out further the purpose of this utility model, technical scheme and beneficial effects
It is described in detail, it should be understood that the above is only the specific embodiments of the utility model, is not used to limit originally practical
Novel protection scope, within the spirit and principle of the utility model, any modification, equivalent substitution, improvement and etc. done,
It should be included within the scope of protection of this utility model.
Claims (3)
1. a kind of flow cytometry device, which is characterized in that rectify room including flow chamber, impedance rectifies room, impedance aperture, resistance
Anti- positive electrode, specimen needle, flow chamber；
The specimen needle is located at impedance rectification chamber interior, and the impedance aperture is located at the rear end of impedance rectification room, institute
The outlet end for stating impedance aperture connects the impedance positive electrode, and the specimen needle connects cathode；
The rear end of impedance rectification room is located at the inside of flow chamber rectification room, after flow chamber is located at the impedance aperture
End.
2. flow cytometry device according to claim 1, which is characterized in that the flow cytometry device also wraps
Laser light source module and scattering light collection module are included, the laser light source module and the scattering light collection module are located at institute
State the opposite two sides of flow chamber.
3. flow cytometry device according to claim 1, which is characterized in that the flow chamber includes that side length is 200
The square fluid channel of micron.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108663306A (en) * | 2018-07-27 | 2018-10-16 | 北京指真生物科技有限公司 | A kind of flow cytometry device and compensation method |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108663306A (en) * | 2018-07-27 | 2018-10-16 | 北京指真生物科技有限公司 | A kind of flow cytometry device and compensation method |
| CN108663306B (en) * | 2018-07-27 | 2024-02-09 | 北京指真生物科技有限公司 | Flow cytometry analysis device and compensation method |
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