CN106404642B - A kind of fluidic cell system fluorescence lifetime Gauss curve fitting measurement method - Google Patents
A kind of fluidic cell system fluorescence lifetime Gauss curve fitting measurement method Download PDFInfo
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- 238000000691 measurement method Methods 0.000 title claims abstract description 5
- 238000000034 method Methods 0.000 claims abstract description 30
- 230000001052 transient effect Effects 0.000 claims abstract description 4
- 239000004005 microsphere Substances 0.000 claims description 6
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- 230000022131 cell cycle Effects 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- MURGITYSBWUQTI-UHFFFAOYSA-N fluorescin Chemical compound OC(=O)C1=CC=CC=C1C1C2=CC=C(O)C=C2OC2=CC(O)=CC=C21 MURGITYSBWUQTI-UHFFFAOYSA-N 0.000 description 2
- 239000002245 particle Substances 0.000 description 2
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- 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 1
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- 238000000053 physical method Methods 0.000 description 1
- 238000003672 processing method Methods 0.000 description 1
- 238000010183 spectrum analysis Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
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Abstract
The present invention provides a kind of fluidic cell system fluorescence lifetime Gauss curve fitting measurement methods, it the described method comprises the following steps: 1) finding out transient Scattering luminous intensity and fluorescence signal intensity using time imfinitesimal method, 2) variation of peak position is analyzed using convolution method, 3) scattered light signal and fluorescence signal are indicated using Gauss curve fitting, obtained pulse signal carries out Gauss curve fitting.
Description
Technical field
The present invention relates to fluorescence detection and spectrum analysis fields, and in particular to the fluorescence intensity of flow cytometer detects and glimmering
Light service life time domain estimates field.
Background technique
Flow cytometer be a kind of collection laser technology, electronics physical technique, photoelectric measurement technology, electronic computer technology,
The novel high-tech instrument that cell fluorescence chemical technology and monoclonal antibody technique are integrated.To in suspension in high speed, straight line
Unicellular or other particles of flowing realize high speed one by one by detection scattered light signal and (or) the fluorescence signal of label
Multi-parameter quantitative analysis.Have in fields such as cell biology, cell cycle kinetics, immunology, hematology and oncology wide
General application.
Flow cytometer scatters light to unicellular or other particles in suspension in high speed, streamlined flow, by detection
The fluorescence signal of signal and (or) label realizes the multi-parameter quantitative analysis of high speed one by one.It is dynamic in cell biology, cell cycle
The fields such as mechanics, immunology, hematology and oncology have a wide range of applications.Flow cytometer realization forward scattering light (FS),
The collection and photoelectric conversion of side scattered light (SS) and assorted fluorescence signal (FLn), and the pulse signal after conversion is carried out more
Parameter extraction, the multi-parameter finally obtained according to extraction are realized to cell/microballoon statistical analysis.Fluorescence lifetime measurement can
The information such as organic fluorescein, fluorescin fluorescence relaxation relevant with the inorganic molecule that other can fluoresce, strength retrogression are provided.
And there is no nonlinear problems existing during fluorescence intensity measurement for fluorescence lifetime, so fluorescence lifetime is introduced in streaming
A multi-parameter information is used as in cell instrument measurement in single cell.
The fluorescence life measuring method for being applied to flow cytometer showed mainly has frequency domain method and two kinds of time domain approach.Frequency domain method
In system, excitation light source is modulated by high frequency sinusoidal signal, so that the fluorescence signal inspired and exciting light be made to have
Identical modulating frequency, and there is certain phase shift between the two.In time domain approach system, using femto-second laser as sharp
Light emitting source excites sample or cell, is seen by single photon counter and exponential decay model to fluorescence decay time
It surveys.Frequency domain method and time domain approach can all greatly increase the cost and complexity of conventional flow cytometer.
When C.Ruofan in 2014 et al. is proposed using digital signal processing method to conventional flow cytometer fluorescence signal
Prolong and analyzed, fluorescence signal is carried out using 250Mbps high-speed ADC chip in the case where not changing flow cytometer structure
It samples and fluorescence lifetime is counted using (Direct method, Gaussian Fitting, Half Area) 3 kinds of methods
It calculates, the time domain resolution capability of calculated result is limited to the sample frequency of ADC.
Summary of the invention
To solve the above-mentioned problems, the purpose of the present invention is to provide a kind of fluidic cell system fluorescence lifetime Gauss curve fittings
Measurement method the described method comprises the following steps:
1) transient Scattering luminous intensity and fluorescence signal intensity are found out using time imfinitesimal method,
T moment scattered light intensity is obtained by following formula:
Wherein, R is the radius of fluorescent microsphere, and dx is the width of infinitesimal, and x is distance of the infinitesimal away from position origin；
T moment fluorescence signal intensity is obtained by following formula:
Wherein, τ is damped cycle；
2) variation of peak position is analyzed using convolution method:
3) scattered light signal and fluorescence signal are indicated using Gauss curve fitting, it is quasi- that obtained pulse signal carries out Gauss
It closes.
Preferably, to the evaluation index of fitting result in the step 3) are as follows:
Side divides error: RMSE (root-mean-square-error)
Determine coefficient: R-square
The quadratic sum SSR of the difference of prediction data and initial data mean value.
The quadratic sum SST of the difference of initial data and mean value.
It should be appreciated that aforementioned description substantially and subsequent detailed description are exemplary illustration and explanation, it should not
As the limitation to the claimed content of the present invention.
Detailed description of the invention
With reference to the attached drawing of accompanying, the more purposes of the present invention, function and advantage are by the as follows of embodiment through the invention
Description is illustrated, in which:
Fig. 1 is scattering light pulse production principle schematic diagram；
Fig. 2 is scattering light mechanism of production schematic diagram；The scattering light fs_S1 that wherein Fig. 2 (a) is first infinitesimal S1 was generated
Journey schematic diagram；The scattering light fs_Sn that Fig. 2 (b) is the last one infinitesimal Sn generates process schematic；
Fig. 3 is fluorescence mechanism of production schematic diagram；
Fig. 4 is the result figure analyzed using convolution method and time imfinitesimal method；
Fig. 5 is forward-scattering signal fs and Gaussian fitting result figure；
Fig. 6 is fluorescence signal fl1 and Gaussian fitting result figure；
Fig. 7 is fluorescence signal fl2 and Gaussian fitting result figure；
Fig. 8 is fluorescence signal fl3 and Gaussian fitting result figure；
Tri- road fluorescence signal fluorescence time delay histogram of Tu9Wei.
Specific embodiment
By reference to 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 exemplary embodiment as disclosed below；Can by different form come
It is realized.The essence of specification is only to aid in those skilled in the relevant arts' Integrated Understanding detail of the invention.
Hereinafter, the embodiment of the present invention will be described with reference to the drawings.In the accompanying drawings, identical appended drawing reference represents identical
Or similar component or same or like step.
The present invention provides the delay time estimation methods that a kind of pair of fluorescent pulse signal carries out Gauss curve fitting, utilize fluorescent pulse
Time delay between signal and scattering light pulse signal, realizes the measurement to fluorescence lifetime.The delay time estimation method of Gauss curve fitting has
Have: (1) ware circuit has the advantages of simple structure and easy realization；(2) synchronization between intensity modulation and high speed processing module is avoided
The problems such as；(3) it is completed completely by digital signal processing chip, calculating speed is fast, and flexibility is good；(4) gained fluorescence lifetime is calculated
Time resolution the advantages such as do not limited by ADC sample frequency.
During flow cytomery, production principle such as Fig. 1 institute of scattered light signal intensity and facula position relationship
Show, fluorescent microsphere is divided into the infinitesimal of n radial equal in width, width dx, enabling distance of the infinitesimal away from position origin (l=0) is x
(0≤x≤2R), then the corresponding surface area of the infinitesimal be
The scattering light fs_S1 of first infinitesimal S1 is generated shown in process such as Fig. 2 (a), scatters light within the scope of time infinitesimal
There is no decaying for intensity, therefore are indicated with rectangle.Fs_S1 initial time is 0, end time L/v.The last one infinitesimal Sn
Scattering light fs_Sn generate process such as Fig. 2 (b) shown in.Fs_Sn initial time is 2R/v, and end time is (L+2R)/v.Hot spot
The scattering optical superposition of each infinitesimal can must correspond to the transient Scattering luminous intensity at moment in range.T moment scattered light intensity are as follows:
Fluorescence signal has attenuation characteristic, it is assumed that is single exponent ring-downWherein τ is damped cycle (fluorescent signal decay
For the 1/e of initial value).Scattered light signal fs (t) includes the information such as microballoon position, corresponding excitation hot spot intensity distribution, therefore sharp
Use fs (t) as the initial time light intensity signal of fluorescence signal time infitesimal analysis.Fig. 3 is fluorescence signal according to time infinitesimal point
Analyse obtained effect picture.T moment fluorescence signal intensity are as follows:
In addition, being analyzed using convolution method the variation of peak position:
The phase frequency difference of fl (t) and fs (t) isPhase frequencyThat is, corresponding
Time domain time delay is τ.Scattered light signal:Fluorescence signal:
One embodiment of the present of invention excites L=22 μm of hot spot minor axis length, and the intensity distribution along short axle is
R=10 μm of fluorescent microsphere radius, microballoon flow velocity v=10.5m/s.The sample frequency of FS, FLm are 100MHz, i.e. pulse train phase
Time interval between adjacent data point is 10ns.
The present invention is as shown in Figure 4 using the fluorescence signal intensity curve that convolution method and time imfinitesimal method are calculated.Time
Imfinitesimal method envelope of curves is consistent with convolution method result, and the two peak point is damped cycle relative to fs (t) peak point time delay
τ.Scattered light signal has centre symmetry, and symmetrical centre is spot center position l=L/2, and the corresponding moment is t=(L+
2R)/2.Fluorescence signal is the damped cycle τ of attenuation function relative to the time delay of scattered light signal, while attenuation function can cause
The asymmetry of fluorescence signal waveform, and the asymmetry of the bigger waveform of τ is more obvious.Since fluorescence signal width is about τ's
102~103 times, therefore caused asymmetry is not very significant.
The present invention is indicated scattered light signal and fluorescence signal using Gauss curve fitting, the pulse obtained to 100MSPS
Signal carries out Gauss curve fitting, and fitting result is as shown in table 1.Specific waveform is as shown in figures 5-8.The evaluation index of fitting result are as follows:
Side divides error: RMSE (root-mean-square-error)
Determine coefficient: R-square
Square of the difference of SSR:Sum of squares of the regression. prediction data and initial data mean value
With.
The quadratic sum of the difference of SST:Total sum of squares. initial data and mean value.
Fitting result are as follows: a1=10.51；B1=2.01；C1=0.5；Evaluation index: RMSE=2.21 × 10-5；R-
Square=1.000；That is, the fluorescence signal comprising single exponent ring-down characteristic can effectively be characterized with Gauss model.
The present invention carries out Gauss curve fitting analysis to 1000 group pulse data respectively.Finally obtain average value and standard deviation and change
Different coefficient(CV, coefficient of variation), as shown in table 2, respective histogram such as Fig. 9
It is shown.
1. Gaussian fitting result of table and evaluation index
2 fluorescence time-delay calculation result of table statistical analysis
In conjunction with the explanation and practice of the invention disclosed here, the other embodiment of the present invention is for those skilled in the art
It all will be readily apparent and understand.Illustrate and embodiment is regarded only as being exemplary, true scope of the invention and purport are equal
It is defined in the claims.
Claims (1)
1. a kind of fluidic cell system fluorescence lifetime Gauss curve fitting measurement method, the described method comprises the following steps:
1) transient Scattering luminous intensity and fluorescence signal intensity are found out using time imfinitesimal method,
T moment scattered light intensity is obtained by following formula:
Wherein, R is the radius of fluorescent microsphere, and dx is the width of infinitesimal, and x is distance of the infinitesimal away from position origin, when fs (t) is t
Scattered light intensity is carved, v is the flow velocity of fluorescent microsphere, and L is excitation hot spot minor axis length, and σ is standard deviation；
T moment fluorescence signal intensity is obtained by following formula:
Wherein, τ is damped cycle, and fl (t) is t moment fluorescence signal intensity, and δ is attenuation amplitude, and t-n τ is die-away time, and n is
Fluorescent microsphere is divided into the quantity of the infinitesimal of radial equal in width；
2) variation of peak position is analyzed using convolution method:
3) scattered light signal and fluorescence signal are indicated using Gauss curve fitting, obtained pulse signal carries out Gauss curve fitting；
Wherein to the evaluation index of fitting result are as follows:
Root-mean-square error: RMSE (root-mean-square-error)
XObs, iIndicate initial data, XModel, iIndicate prediction data；N is sample number；Determine coefficient: R-square
The error sum of squares of SSE prediction data and initial data；
The quadratic sum SSR of the difference of prediction data and initial data mean value；
Indicate the mean value of initial data；N is sample number；
The quadratic sum SST of the difference of initial data and mean value；
N is sample number.
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