CN107063979A - A kind of flow cytometer fluorescence lifetime ime-domain measuring method - Google Patents
A kind of flow cytometer fluorescence lifetime ime-domain measuring method Download PDFInfo
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Abstract
The invention provides a kind of flow cytometer fluorescence lifetime ime-domain measuring method, it the described method comprises the following steps：1) coherence spectra of fluorescent pulse signal and forward scattering light pulse signal is refined using MCZT methods, 2) calculating of time domain cross-correlation function is realized using FICP algorithms, and time resolution is improved by appropriate interpolation.
Description
Technical field
The present invention relates to fluoroscopic examination and spectrum analysis field, and in particular to the fluorescence intensity of flow cytometer is detected and glimmering
Light life-span time domain estimates field.
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 detecting the fluorescence signal of scattered light signal and (or) mark, are realized 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.
Flow cytometer is to unicellular or other particles in high speed, streamlined flow in suspension, by detecting scattered light
The fluorescence signal of signal and (or) mark, realizes multi-parameter quantitative analysis at a high speed one by one.Moved in cell biology, cell cycle
The fields such as mechanics, immunology, hematology and oncology have a wide range of applications.Flow cytometer realize forward scattering light (FS),
The collection of side scattered light (SS) and assorted fluorescence signal (FLn) and opto-electronic conversion, and the pulse signal after conversion is carried out many
Parameter extraction, the multi-parameter finally obtained according to extracting realizes the statistical analysis to cell/microballoon.Fluorescence lifetime measurement can
The information such as fluorescence relaxation, the strength retrogression that to provide organic fluorescein, fluorescin related to the inorganic molecule that other can fluoresce.
And nonlinear problem present in fluorescence intensity measurement process is not present in fluorescence lifetime, so fluorescence lifetime is introduced in streaming
A multi-parameter information is used as in cell instrument measurement in single cell.
Being applied to the fluorescence life measuring method of flow cytometer showed mainly has two kinds of frequency domain method and time domain approach.Frequency domain method
In system, excitation source is modulated by high frequency sinusoidal signal, so that the fluorescence signal inspired has with exciting light
Identical modulating frequency, and there is certain phase shift between the two.In time domain approach system, by the use of femto-second laser as
Excitation source is excited to sample or cell, and fluorescence decay time is carried out by single photon counter and exponential decay model
Observation.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 ADCs chip in the case where not changing flow cytometer structure
Sample and fluorescence lifetime is counted using (Direct method, Gaussian Fitting, Half Area) 3 kinds of methods
Calculate, the time domain resolution capability of result of calculation is limited to ADC sample frequency.
The content of the invention
In order to solve the above problems, it is an object of the invention to provide a kind of flow cytometer fluorescence lifetime time domain measurement side
Method, the described method comprises the following steps：
1) coherence spectra of fluorescent pulse signal and forward scattering light pulse signal is refined using MCZT methods,
Wherein, fs (n) represents forward scattering light pulse signal, flm(n) each road fluorescent pulse signal, m=1,2,3 are represented；
K=0,1 ..., N-1；
2) calculating of time domain cross-correlation function is realized using FICP algorithms, and time resolution is improved by appropriate interpolation：
By FS (k) and FLm(k) complete coherence spectra Rm (k) is constructed such as shown in formula (2)：
First half and latter half time domain correlation function are solved respectively according to formula (3) and (4)：
Wherein k=0,1 ..., N-1,
Complete time domain correlation function such as formula (5) can be constructed by r1 (n) and r2 (n), when any setting N2 can make
The resolution ratio at domain cross-correlation function peak improves N2/N1 times,
It should be appreciated that foregoing description substantially and follow-up description in detail are exemplary illustration and explanation, should not
As the limitation to claimed content of the invention.
Brief description of the drawings
With reference to the accompanying drawing enclosed, the present invention more purpose, function and advantages will pass through the as follows of embodiment of the present invention
Description is illustrated, wherein：
Fig. 1 is that fluorescent pulse is produced and time delay principle schematic；
The coherence spectra oscillogram of Tu2Wei Ge roads fluorescence signal and forward-scattering signal；Wherein Fig. 2 (a) is direct profit
Coherence spectra is calculated with FFT, Fig. 2 (b) calculates coherence spectra using MCZT frequency spectrum refinements；
Fig. 3 is the time domain cross-correlation function figure of each channel fluorescence signal and forward scattering light, and wherein Fig. 3 (a) calculates for IFFT
The time domain cross-correlation function that method is obtained；Fig. 3 (b) is the time domain cross-correlation function that 10 times of interpolation of FICP algorithms are obtained；Fig. 3 (c) is
FL2 relevant peaks details in Fig. 3 (a) that IFFT algorithms are obtained；Fig. 3 (d) is in 10 times of interpolation calculation result (b) of FICP algorithms
FL2 relevant peaks details.
Fig. 4 is fluorescent pulse signal histogram, wherein, Fig. 4 (a) is FL1 peak value histograms, and Fig. 4 (b) is that FL2 peak values are straight
Fang Tu, Fig. 4 (c) are FL3 peak value histograms, and Fig. 4 (d) is FL1 time delay histograms, and Fig. 4 (e) is FL2 time delay histograms, Fig. 4 (f)
For FL3 time delay histograms.
Embodiment
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 as 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.
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.
The invention provides a kind of high-resolution fluorescence delay time estimation method, the measurement to fluorescence lifetime is realized.First, it is sharp
The coherence spectra of fluorescent pulse signal and forward scattering light pulse signal is carried out carefully with improved chirp z transform (MCZT)
Change, it is to avoid information caused by standard FFT fence effects is lost.Then, FICP (fine interpolation of are utilized
Correlation peak) algorithm realizes the calculating of time domain cross-correlation function, and time resolution is improved by appropriate interpolation.It is high
Resolution ratio fluorescence delay time estimation method has：(1) ware circuit is simple in construction, it is easy to accomplish；(2) intensity modulation is avoided
And the problems such as synchronization between high speed processing module；(3) completed completely by digital signal processing chip, calculating speed is fast, flexibly
Property is good；(4) time resolution for calculating gained fluorescence lifetime the advantage such as is not limited by ADC sample frequencys.
During flow cytomery, the generation principle of scattered light and fluorescent pulse signal is as shown in figure 1, microballoon is arrived
Up to when exciting hot spot upper marginal position a, starting to produce forward-scattering signal and each road fluorescence signal after shaping, and with light
Light intensity gradually strengthens in spot, and output signal increases therewith；When microballoon reaches hot spot light intensity maximum position b, corresponding scattered light
And each road fluorescence signal reaches maximum；When microballoon reaches hot spot lower edge position c, forward-scattering signal and each road fluorescence
Signal value output will be 0.
The present invention is analyzed the frequency spectrum of forward scattering light pulse signal and each road fluorescent pulse signal, and according to formula
(1) spectrum information in the range of certain frequency is refined using MCZT algorithms, the high frequency spectral resolution frequency after being refined
Domain information.The frequency spectrum of signal is calculated using MCZT, the frequency spectrum of refinement, its spectral resolution can be calculated for ultrashort sampled signal
Do not restricted by sample length and sample rate, refinement calculating can also be carried out to a certain section of frequency spectrum.
Forward scattering light pulse signal fs (n) and each road fluorescent pulse signal flm (n) are the pulse for including N point datas
Sequence, corresponding zoom FFT is respectively FS (k) and FLm (k).
Wherein m=1,2,3；K=0,1 ..., N-1.
Fft algorithm can not differentiate the detailed information of effective spectrum scope.Relative to the cross-correlation obtained using FFT calculating
Spectrum, MCZT carries out the fence effect that N1/N times of zoom FFT can avoid fft algorithm from producing, and is kept away with higher spectral resolution
Exempt from the loss of spectrum information, so that coherence spectra curve is more smooth.
FS (k) is known by sampling theorem and FLm (k) be N1 in the cycle periodic function, and left-half and right half part
With conjugate symmetry property.From FICP algorithms, in frequency spectrum entering row interpolation makes spectrum cycle expand to N2, can not increase
Corresponding time-domain sampling frequency is risen to N2/N1 times of original sample frequency, so as to improve fluorescence arteries and veins on the premise of amount of calculation
Rush the accuracy of signal time delay estimation.Complete coherence spectra Rm (k) is constructed such as shown in formula (2) by FS (k) and FLm (k).
The time delay of each channel fluorescence pulse signal is always in limited range, and it is attached that the main peak of cross-correlation function is in null value
Closely, in this case, it is only necessary to calculate the left side finite point (latter half) and the right finite point (first half of cross-correlation waveform
Point).First half and latter half time domain correlation function are solved respectively according to formula (3) and (4).
Wherein k=0,1 ..., N-1.
Complete time domain correlation function such as formula (5) can be constructed by r1 (n) and r2 (n), when any setting N2 can make
The resolution ratio at domain cross-correlation function peak improves N2/N1 times.
The specific embodiment of the present invention is handled the fluorescent pulse signal that pulsewidth is 4 μ s or so, FS, FLm's
Sample frequency is 100MHz, i.e., the time interval between pulse train consecutive number strong point is 10ns.
Obtained coherence spectra is calculated as shown in Fig. 2 wherein Fig. 2 (a) calculates obtained cross-correlation to be direct using FFT
Spectrum, Fig. 2 (b) refines obtained frequency spectrum to carry out N1/N times using MCZT.As shown in Figure 2, fft algorithm can obtain -4≤k≤4
In the range of effective spectrum information, but the detailed information of the spectral range can not be differentiated.Relative to using FFT calculate obtain it is mutual
Correlated Spectroscopy, MCZT carries out the fence effect that N1/N times of zoom FFT can avoid fft algorithm from producing, it can be obtained -8 ×
Effective spectrum information in the range of N1/N≤k≤8 × N1/N (N1/N=10 is taken in Fig. 2), keeps away with higher spectral resolution
Exempt from the loss of spectrum information, so that coherence spectra curve is more smooth.
Present invention setting N2/N1=10, is utilized respectively IFFT algorithms and FICP algorithms calculate time domain correlation function curve such as
Shown in Fig. 3.Improving the spectral resolution of coherence spectra can make cross-correlation waveform more smooth, and related summit is more round, and frequency spectrum
Refinement do not influenceed by sampling length N；Meanwhile, the multiple of interpolation is improved, the resolution ratio raising of relevant peaks top can be made, used
FICP algorithms, can any ' amplification ' cross-correlation peak so that the precision estimated time delay is higher, especially for the letter of low sampling
Number, its advantage becomes apparent from.Fig. 3 (a) calculates the obtained impulse time delay of FL1, FL2 and FL3 relative to FS using IFFT
760ns (76 × 10ns), 750ns (75 × 10ns) and 790ns (79 × 10ns), temporal resolution is 10ns；Fig. 3 (b) is utilized
10 times of interpolation FICP algorithms, temporal resolution isCalculating obtained corresponding fluorescent pulse time delay is respectively
765ns (765 × 1ns), 748ns (748 × 1ns) and 772ns (772 × 1ns).Compare as can be seen that FICP algorithms can be carried
The temporal resolution of high time domain cross-correlation function, impulse time delay estimated accuracy is higher.
The present invention performs calibration operation using flow cytometer and standard fluorescence microballoon is measured, and to 1000
Events fluorescence signal carries out the calculating of peak value and fluorescence lifetime respectively.Statistical analysis is carried out to 1000 groups of data, obtained
The peak Distribution histogram and impulse time delay distribution histogram of each fluorescence signal are as shown in Figure 4.Wherein, Fig. 4 (a) is FL1 peak values
Histogram, Fig. 4 (b) is FL2 peak value histograms, and Fig. 4 (c) is FL3 peak value histograms, and Fig. 4 (d) is FL1 time delay histograms, Fig. 4
(e) it is FL2 time delay histograms, Fig. 4 (f) is FL3 time delay histograms.Fluorescence FL1 intensity distribution ranges are 0.74~1.38V, are put down
Average is 1.23V, and standard deviation is 0.118；Fluorescence FL2 intensity distribution ranges are 1.44~3.07V, and average value is 2.56V, standard
Difference is 0.292；Fluorescence FL3 intensity distribution ranges are 0.66~1.57V, and average value is 1.22V, and standard deviation is 0.210.Fluorescence
FL1 pulse delays distribution is 0.740~0.796 μ s, and average value is 0.759 μ s, and standard deviation is 0.009；Fluorescence FL2 pulses
Delay distribution is 0.722~0.782 μ s, and average value is 0.751 μ s, and standard deviation is 0.011；Fluorescence FL3 pulse delays point
Cloth scope is 0.752~0.804 μ s, and average value is 0.773 μ s, and standard deviation is 0.008.It can be seen from Fig. 4 and above analysis
Relative to photoluminescence peak characterizing method, fluorescent pulse time delay estimation characterizing method has smaller distribution, smaller standard
Difference.So as to improve the distribution density of corresponding cell population in conventional scatter diagram, while improving the accurate of cell point cluster analysis
Property.
With reference to the explanation of the invention disclosed here and practice, other embodiment of the invention is for those skilled in the art
It all will be readily apparent and understand.Illustrate and embodiment is to be considered only as exemplary, of the invention true scope and purport is equal
It is defined in the claims.
Claims (1)
1. a kind of flow cytometer fluorescence lifetime ime-domain measuring method, the described method comprises the following steps：
1) coherence spectra of fluorescent pulse signal and forward scattering light pulse signal is refined using MCZT methods,
Wherein, fs (n) represents forward scattering light pulse signal, flm(n) each road fluorescent pulse signal, m=1,2,3 are represented；K=0,
1,…,N-1；
2) calculating of time domain cross-correlation function is realized using FICP algorithms, and time resolution is improved by appropriate interpolation：
By FS (k) and FLm(k) complete coherence spectra Rm (k) is constructed such as shown in formula (2)：
First half and latter half time domain correlation function are solved respectively according to formula (3) and (4)：
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Complete time domain correlation function such as formula (5) can be constructed by r1 (n) and r2 (n), it is any to set N2 time domain be made mutual
The resolution ratio at correlation function peak improves N2/N1 times,
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