CN106644901A - Fluorescence compensation method for flow cytometer - Google Patents
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- CN106644901A CN106644901A CN201610890104.3A CN201610890104A CN106644901A CN 106644901 A CN106644901 A CN 106644901A CN 201610890104 A CN201610890104 A CN 201610890104A CN 106644901 A CN106644901 A CN 106644901A
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- 238000000034 method Methods 0.000 title claims abstract description 34
- 239000011159 matrix material Substances 0.000 claims abstract description 58
- 238000004458 analytical method Methods 0.000 claims abstract description 16
- 238000012360 testing method Methods 0.000 claims abstract description 15
- 238000012937 correction Methods 0.000 claims abstract description 13
- 238000001514 detection method Methods 0.000 claims description 6
- 238000004043 dyeing Methods 0.000 claims description 4
- 238000013459 approach Methods 0.000 claims description 3
- 239000000203 mixture Substances 0.000 claims description 3
- 239000007850 fluorescent dye Substances 0.000 abstract description 13
- 238000010586 diagram Methods 0.000 description 9
- 238000005259 measurement Methods 0.000 description 8
- 238000005516 engineering process Methods 0.000 description 5
- 230000003287 optical effect Effects 0.000 description 5
- 238000002189 fluorescence spectrum Methods 0.000 description 4
- 239000000975 dye Substances 0.000 description 3
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 description 3
- 238000000205 computational method Methods 0.000 description 2
- 238000009826 distribution Methods 0.000 description 2
- 238000000295 emission spectrum Methods 0.000 description 2
- 230000005622 photoelectricity Effects 0.000 description 2
- 238000001228 spectrum Methods 0.000 description 2
- 239000013598 vector Substances 0.000 description 2
- PLXMOAALOJOTIY-FPTXNFDTSA-N Aesculin Natural products OC[C@@H]1[C@@H](O)[C@H](O)[C@@H](O)[C@H](O)[C@H]1Oc2cc3C=CC(=O)Oc3cc2O PLXMOAALOJOTIY-FPTXNFDTSA-N 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 230000022131 cell cycle Effects 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 239000003086 colorant Substances 0.000 description 1
- 238000010924 continuous production Methods 0.000 description 1
- BFMYDTVEBKDAKJ-UHFFFAOYSA-L disodium;(2',7'-dibromo-3',6'-dioxido-3-oxospiro[2-benzofuran-1,9'-xanthene]-4'-yl)mercury;hydrate Chemical compound O.[Na+].[Na+].O1C(=O)C2=CC=CC=C2C21C1=CC(Br)=C([O-])C([Hg])=C1OC1=C2C=C(Br)C([O-])=C1 BFMYDTVEBKDAKJ-UHFFFAOYSA-L 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 1
- 238000012921 fluorescence analysis Methods 0.000 description 1
- 238000001917 fluorescence detection Methods 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000002715 modification method Methods 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 238000000053 physical method Methods 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 230000003595 spectral effect Effects 0.000 description 1
- 238000004611 spectroscopical analysis Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N15/14—Optical investigation techniques, e.g. flow cytometry
- G01N15/1429—Signal processing
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N15/14—Optical investigation techniques, e.g. flow cytometry
- G01N15/1434—Optical arrangements
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
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Abstract
The invention provides a fluorescence compensation method for a flow cytometer. The fluorescence compensation method comprises the following steps: step (1) performing single positive sample testing; step (2) calculating fluorescence leakage coefficients and fluorescence leakage matrixes of each channel; step (3) calculating the fluorescence leakage matrixes, and according to the fluorescence leakage matrix K<-1>, obtaining a compensation matrix K<-1>; step (4) performing compensation correction; step (5) performing multi-color analysis test: testing samples colored by a multi-color fluorescence dye, and recording the signal amplitude of each detecting channel; step (6) performing fluorescence compensation.
Description
Technical field
The present invention relates to the present invention relates to the fluoroscopic examination of flow cytometer and spectroscopic analysis methods, more particularly to a kind of stream
The fluorescence compensation method of formula cell instrument.
Background technology
Flow cytometer be a kind of collection laser technology, electronics physical technique, photoelectric measurement technology, electronic computer technology,
The new high-tech instrument that cell fluorescence chemical technology and monoclonal antibody technique are integrated.To being in high speed, straight line in suspension
Unicellular or other particles of flowing, by the fluorescence signal for detecting scattered light signal and (or) mark, realize at a high speed one by one
Multi-parameter quantitative analysis.Have in fields such as cell biology, cell cycle kinetics, immunology, hematology and oncology wide
General application.
The principle of flow cytometer is that single cell suspension enters streaming exposure cell Jing after specific fluorescence dye dyeing, in stream
The analysis site of formula exposure cell, laser is irradiated to cell be there is scattering and reflects, and launch scattered light, the fluorescein entrained by cell
It is excited and launches fluorescence, forward scattering light (FSC) and side scattered light (SSC).By electrooptical device by fluorescence and
Scattered light is converted into electric signal, and forward scattering light is converted into electric signal by photodiode, and side scattered light is by photoelectricity times
Increasing is converted into electric signal, and fluorescence is then collected by concentrator, and the fluorescence of different colours turns to different photoelectricity times by double-colored reflective mirror
Increase pipe detector, fluorescence signal be converted into into electric signal, scattered light signal and fluorescence signal after amplification, then through digitization
Process, be input into computer and store, be analyzed according to the scattered light and fluorescence of cell.
Flow cytometer multicolor fluorescence analysis are referred to detecting sample while marking various fluorescent dyes to detect, at present
Exciting or property of the launch wavelength with wide emission spectra for the various fluorescent dyes for being used, Jing after optical filter filters, launches
Spectral region still has certain interference to overlap.At present, it is light intensity mainly according to the signal strength signal intensity in each receiver wavelength range
Value carries out fluorescence compensation.However, light intensity have wavelength dependence cannot actual response exciting light and fluorogen corresponding relation.
Therefore, in order to solve the problems, such as flow cytometer multicolor analysis during Overlapping of fluorescence spectra cause measure error,
Need the flow cytometer fluoroscopic examination and analysis compensation method of a kind of energy effectively solving the problems referred to above.
The content of the invention
According to an aspect of the invention, there is provided a kind of flow cytometer fluorescence compensation method, comprises the following steps：Step
Rapid 1) single positive sample test；Step 2) calculate each channel fluorescence leadage coefficient and fluorescence leakage matrix；Step 3) calculate fluorescence
Compensation matrix, leaks matrix K and tries to achieve compensation matrix K K according to fluorescence-1；Step 4) compensating approach；Step 5) multicolor analysis test,
The sample of multicolor fluorescence dyeing is tested, the signal amplitude of each sense channel is recorded；Step 6) fluorescence compensation.
Preferably, in the step 1) in carry out fluoroscopic examination to each single positive sample, adjustment sensor gain and after
The gain of continuous process circuit makes the signal amplitude of sense channel in suitable scope, sensor gain and follow-up after the completion of adjustment
The gain of process circuit will keep constant in follow-up test process.Record sense channel signal amplitude and other leakage paths
Leakage signal amplitude.
Preferably, in the step 2) in fluoroscopic examination is carried out to single single positive sample, by step 1) in detection it is logical
Road signal amplitude obtains the sense channel of single single positive sample relative to other with the leakage signal amplitude of other leakage paths
The leadage coefficient k of leakage pathij；The leadage coefficient of multiple single positive samples obtains multiple single positive according to certain corresponding relation
The fluorescence leakage matrix K of pattern detection.
Preferably, the fluorescence leakage matrix K is：
K matrix is that fluorescence leaks matrix, wherein kij(i=1 ..., n；J=1 ..., n；) it is fluorescence signal FLiLeak into
Fluorescence detector PMTjFluorescence leak rates, FL matrixes are to wait real fluorescence signal matrix of looking for the truth.
Preferably, the leakage inverse of a matrix matrix K-1For " compensation matrix ".
Preferably, in the step 4) in byLight intensity and number of photons in the fluoroscopic examination wave-length coverage for obtaining
Corresponding relation, draw the correction factor of each sense channel, and by the correction factor of multiple sense channels according to certain right
Composition correction matrix should be related to, fluorescence compensation matrix is modified using correction matrix.
Preferably, the step 6) according to step 5) in each sense channel signal amplitude and step 4) obtain
Revised fluorescence compensation matrix carries out computing to testing result, obtains the fluoroscopic examination result after fluorescence compensation.
Fluorescence compensation method for flow cytometer of the invention, can solve flow cytometer multicolor analysis mistake
Overlapping of fluorescence spectra causes the problem of measure error in journey, flow cytometer fluoroscopic examination that can be in the existing field of effectively solving and
Analysis compensation.
It should be appreciated that aforementioned description substantially and follow-up description in detail are exemplary illustration and explanation, should not
As the restriction to claimed content of the invention.
Description of the drawings
With reference to the accompanying drawing enclosed, the present invention more purpose, function and advantages are by by the as follows of embodiment of the present invention
Description is illustrated, wherein：
Fig. 1 is a better embodiment schematic diagram of flow cytometer fluorescence compensation method of the present invention；
Fig. 2 is that the fluorescence during flow cytometer multicolor analysis overlaps schematic diagram；
Fig. 3 is that dichromatism fluorescence signal detects and leak schematic diagram；
Fig. 4 is that four color fluorescence signals detect and leak schematic diagram.
Specific embodiment
Hereinafter, embodiments of the invention will be described with reference to the drawings.In the accompanying drawings, identical reference represents identical
Or similar part, or same or like step.
By reference to one exemplary embodiment, the purpose of the present invention and function and the side for realizing these purposes and function
Method will be illustrated.However, the present invention is not limited to one exemplary embodiment disclosed below；Can by multi-form come
It is realized.The essence of specification is only to aid in the detail of the various equivalent modifications Integrated Understanding present invention.
It is described in detail with reference to schematic diagram for the present invention, when the embodiment of the present invention is described in detail, for purposes of illustration only, representing
The profile of device architecture can disobey general ratio and make partial enlargement, and the schematic diagram is example, and its here should not be limited
The scope of protection of the invention processed.Additionally, the three-dimensional space of length, width and depth should be included in actual fabrication.
Overlapping of fluorescence spectra causes measure error during in order to solve the problems, such as flow cytometer multicolor analysis, the present invention
Propose that a kind of utilization fluorescent photon number replaces the modification method of fluorescent intensity to carry out fluorescence compensation.
Fig. 1 is to be shown according to a better embodiment for carrying out fluorescence compensation method for flow cytometer of the present invention
It is intended to.As shown in figure 1, flow cytometer fluorescence compensation method of the invention, comprises the following steps：
Step 101：Single positive sample test；
Specifically, fluoroscopic examination is carried out to each single positive sample in the step 101, adjusts the biography of flow cytometer
Sensor gain and the gain of subsequent process circuit make the signal amplitude of sense channel in suitable scope, sense after the completion of adjustment
Device gain and the gain of subsequent process circuit will keep constant in follow-up test process.Record sense channel signal amplitude and
The leakage signal amplitude of other leakage paths.
Step 102：Calculate each channel fluorescence leadage coefficient and fluorescence leakage matrix；
Specifically, in the step 2) in fluoroscopic examination is carried out to single single positive sample, by detecting logical in step 101
Road signal amplitude obtains the sense channel of single single positive sample relative to other with the leakage signal amplitude of other leakage paths
The leadage coefficient k of leakage pathij；The leadage coefficient of multiple single positive samples obtains multiple single positive according to certain corresponding relation
The fluorescence leakage matrix K of pattern detection.The computational methods of fluorescence leakage matrix K are as follows.
When the different fluorescein molecule of flow cytomery n kinds of the invention, any fluorescein send out
Light is penetrated all possibly into other fluorescence detection channels, FL1,…,FLnRepresent n kind fluorescent dyes respectively to send out upon laser irradiation
The fluorescence signal penetrated, PMT1..., PMTn represents respectively the fluorescence signal of the actual measurement of n target fluorescent detector, then and any one
The fluorescence signal PMT that individual Air conduct measurement is arrived1..., PMTn can be expressed as：
PMT=K × FL (1)
Formula (1) shows that the fluorescence signal of the actual measurement of detector is equal to fluorescence and leaks matrix and fluorescent dye actual transmission letter
Number product (not considering to detect that sample autofluorescence produces noise signal).In formula (1)：
K matrix is that fluorescence leaks matrix, wherein kij(i=1 ..., n；J=1 ..., n；) it is fluorescence signal FLiLeak into
Fluorescence detector PMTjFluorescence leak rates, FL matrixes are to wait real fluorescence signal matrix of looking for the truth.
Step 103：Fluorescence compensation matrix is calculated, the fluorescence obtained according to step 102 leaks matrix K and tries to achieve compensation matrix K-1；Compensation matrix K-1Computational methods it is as follows：
The analysis by more than can obtain complete matrix backoff algorithm and measure the Mathematical Modeling of true fluorescence intensity and be：
FL=K-1×PMT (2)
Complete matrix compensation is by the actual fluorescence intensity measured by photodetector being defined as into matrix and letting out predetermined
Leakage inverse of a matrix acts on the matrix, so as to calculate the actual strength of the fluorescence sent from desired fluorescent dye.By dividing
The wavelength of fluorescence distribution of each particulate of analysis separate marking obtains leaking matrix, in leakage matrix, the fluorescence ripple of each fluorescent dye
Long distribution is represented as column vector, and these column vectors are arranged in order, and leaks inverse of a matrix matrix K-1As " compensation matrix ".
Step 104：Compensating approach；
Specifically, in the step 104 byLight intensity and number of photons in the fluoroscopic examination wave-length coverage for obtaining
Corresponding relation, draw the correction factor of each sense channel, and by the correction factor of multiple sense channels according to certain right
Composition correction matrix should be related to.Fluorescence compensation matrix is modified using correction matrix.
Step 105：Multicolor analysis test is carried out, the sample of multicolor fluorescence dyeing is tested, record each inspection
Survey the signal amplitude of passage；
Step 106：Carry out fluorescence compensation.
Specifically, to the signal amplitude and step 104 according to each sense channel in step 105 in the step 106
The revised fluorescence compensation matrix for obtaining carries out computing to testing result, obtains the fluoroscopic examination result after fluorescence compensation.
Many color method fluorescence measurements are carried out for finer analysis cell characteristics, i.e., using various fluorochrome labels
Particulate, and launched from every kind of fluorescent dye using having the different multiple photodetectors (such as PMT) for receiving optical band to measure
Fluorescence.During using many color method fluorescence measurements, by being selected in photodetector side according to the wavelength of fluorescence of the fluorescent dye for using
Optical filter carry out the detection of fluorescence.But the various fluorescent dyes for being used at present excite or launch wavelength has wide transmitting
The property of spectrum, Jing after optical filter filters, emission spectrum scope still has certain interference to overlap.Therefore, if by these dye sets
Conjunction carries out polychrome measurement, even if the fluorescence sent from fluorescent dye is divided into into each wave band using optical filter, from non-targeted fluorescence
The fluorescence that dyestuff sends is likely to leak into target fluorescent detector.In the event of fluorescence leakage, measure at photodetector
Fluorescence intensity and there will be difference between the true fluorescence intensity of target fluorescent dyestuff, cause measure error.
According to the present invention, for compensating measurement errors, the actual measured value to detector is needed to carry out fluorescence compensation.Fluorescence
Compensation is the process for correcting fluorescence signal seepage, its objective is the appointing in addition to matching fluorescence is removed from fluorescent target detector
What connection disturbs fluorescence signal.By fluorescent dye (FITC, PE, ECD, the PE-Cy5, PE-Cy7) fluorescence emission of 488nm laser excitations
Spectrum juxtaposition schematic diagram is as shown in Figure 2.Fig. 3 is that dichromatism fluorescence signal detects and leak schematic diagram；Fig. 4 is four color fluorescence signals
Detection and leakage schematic diagram.
Fluorescence compensation method for flow cytometer of the invention, can solve flow cytometer multicolor analysis mistake
Overlapping of fluorescence spectra causes the problem of measure error in journey, flow cytometer fluoroscopic examination that can be in the existing field of effectively solving and
Analysis compensation.
With reference to the explanation of the invention and practice that disclose here, the other embodiment of the present invention is for those skilled in the art
All will be readily apparent and understand.Illustrate and embodiment be to be considered only as it is exemplary, the present invention true scope and purport it is equal
It is defined in the claims.
Claims (7)
1. a kind of fluorescence compensation method for flow cytometer, it is characterised in that comprise the following steps：
Step 1) single positive sample test；
Step 2) calculate each channel fluorescence leadage coefficient and fluorescence leakage matrix；
Step 3) fluorescence compensation matrix is calculated, matrix K is leaked according to fluorescence and tries to achieve compensation matrix K-1；
Step 4) compensating approach；
Step 5) multicolor analysis test, the sample of multicolor fluorescence dyeing is tested, record the letter of each sense channel
Number amplitude；
Step 6) fluorescence compensation.
2. flow cytometer fluorescence compensation method as claimed in claim 1, wherein in the step 1) in it is single positive to each
Sample carries out fluoroscopic examination, adjusts the gain of sensor gain and subsequent process circuit and makes the signal amplitude of sense channel suitable
In the range of, the gain of sensor gain and subsequent process circuit after the completion of adjustment will keep not in follow-up test process
Become.The leakage signal amplitude of record sense channel signal amplitude and other leakage paths.
3. flow cytometer fluorescence compensation method as claimed in claim 1, wherein in the step 2) in single single positive
Sample carries out fluoroscopic examination, by step 1) in sense channel signal amplitude obtain with the leakage signal amplitude of other leakage paths
Leadage coefficient k of the sense channel of single single positive sample relative to other leakage pathsij；The leakage system of multiple single positive samples
Number obtains the fluorescence leakage matrix K of multiple single positive sample detections according to certain corresponding relation.
4. flow cytometer fluorescence compensation method as claimed in claim 3, wherein fluorescence leakage matrix K is：
K matrix is that fluorescence leaks matrix, wherein kij(i=1 ..., n；J=1 ..., n；) it is fluorescence signal FLiLeak into fluorescence
Detector PMTjFluorescence leak rates, FL matrixes are to wait real fluorescence signal matrix of looking for the truth.
5. flow cytometer fluorescence compensation method as claimed in claim 1, wherein the leakage inverse of a matrix matrix K-1" to mend
Repay matrix ".
6. flow cytometer fluorescence compensation method as claimed in claim 1, wherein in the step 4) in by
The corresponding relation of light intensity and number of photons in the fluoroscopic examination wave-length coverage for arriving, draws the correction factor of each sense channel, and will
The correction factor of multiple sense channels compensates square using correction matrix according to certain corresponding relation composition correction matrix to fluorescence
Battle array is modified.
7. flow cytometer fluorescence compensation method as claimed in claim 1, wherein in the step 6) according to step 5)
In each sense channel signal amplitude and step 4) obtain revised fluorescence compensation matrix computing is carried out to testing result,
Obtain the fluoroscopic examination result after fluorescence compensation.
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| CN108693099A (en) * | 2018-03-16 | 2018-10-23 | 中国科学院苏州生物医学工程技术研究所 | Full-automatic fluorescence compensation method for flow cytometer |
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| CN109490176A (en) * | 2018-11-30 | 2019-03-19 | 苏州深析智能科技有限公司 | FCM analysis data processing method, device and classifying method, device |
| CN109580458A (en) * | 2018-11-30 | 2019-04-05 | 苏州深析智能科技有限公司 | Fluidic cell intelligent immunity classifying method, device and electronic equipment |
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| CN112154318A (en) * | 2018-05-21 | 2020-12-29 | 厦泰生物科技公司 | Rapid re-compensation of flow cytometry data for spill reconditioning |
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| CN111033222A (en) * | 2017-08-08 | 2020-04-17 | 索尼公司 | Information processing apparatus, information processing method, and program |
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| CN112154318A (en) * | 2018-05-21 | 2020-12-29 | 厦泰生物科技公司 | Rapid re-compensation of flow cytometry data for spill reconditioning |
| CN109030321A (en) * | 2018-05-23 | 2018-12-18 | 中国科学院苏州生物医学工程技术研究所 | Data processing method for flow cytometer |
| CN109490176A (en) * | 2018-11-30 | 2019-03-19 | 苏州深析智能科技有限公司 | FCM analysis data processing method, device and classifying method, device |
| CN109580458A (en) * | 2018-11-30 | 2019-04-05 | 苏州深析智能科技有限公司 | Fluidic cell intelligent immunity classifying method, device and electronic equipment |
| CN109490176B (en) * | 2018-11-30 | 2019-11-22 | 苏州深析智能科技有限公司 | FCM analysis data processing method, device and classifying method, device |
| CN112240863A (en) * | 2019-07-18 | 2021-01-19 | 希森美康株式会社 | Measuring device, method for adjusting measuring device, and computer storage medium |
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