CN101278191B - Fluorescence spectroscopy apparatus - Google Patents
Fluorescence spectroscopy apparatus Download PDFInfo
- Publication number
- CN101278191B CN101278191B CN2006800362769A CN200680036276A CN101278191B CN 101278191 B CN101278191 B CN 101278191B CN 2006800362769 A CN2006800362769 A CN 2006800362769A CN 200680036276 A CN200680036276 A CN 200680036276A CN 101278191 B CN101278191 B CN 101278191B
- Authority
- CN
- China
- Prior art keywords
- fluorescence
- data
- exciting light
- light
- weighting coefficient
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Images
Landscapes
- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
Abstract
A fluorescence spectral analyzer (100) comprising an exciting optical system (110), an exciting light control unit (130), a fluorescence detection unit (140), a signal processing unit (150) and an operation unit (160). The exciting optical system (110) has an exciting light irradiation unit (120) for generating an exciting light, and the exciting light control unit (130) controls the exciting light irradiation unit (120) so that a sample (S) is irradiated with exciting lights having different wavelengths exclusively and at different timings. The exciting light irradiation unit (120) applies exciting lights having different wavelengths to one portion on the sample (S). The fluorescence detection unit (140) detects fluorescence generated from the sample (S) according to the irradiation of an exciting light. The signal processing unit (150) processes a signal reflecting a fluorescence intensity obtained at the fluorescence detection unit (140). The operation unit (160) performs a correlation analysis on fluorescence fluctuations for respective different wavelengths based on a change in the wavelength of an exciting light applied to the sample (S) by the exciting light control unit (130) and the detection results by the fluorescence detection unit (140).
Description
Technical field
The present invention relates to a kind of fluorescence spectroscopy apparatus.
Background technology
The fluorescence spectrum analysis method (FCS method) of being correlated with is a kind of fluctuation of the light that Brownian movement caused by resolving fluorescence molecule in the small observation area of field of microscope, try to achieve the autocorrelation function of fluorescence intensity, resolve the diffusion time of each molecule and the method for average mark subnumber.For example, be described in detail among the vol.44N091431-1438 at " 1 Molecular Detection of fluorescence correlation spectrometry " Jin Chengzhu, protein nucleic acid ferment, 1999.
Fluorescence simple crosscorrelation spectrum analysis method (FCCS method) is a kind of by trying to achieve the cross correlation function between different fluorescence signals, resolve the method for both relevances, be used for intermolecular interactional parsing with the fluorchrome sign of 2 looks, for example, " Dual-Color Fluorescence Cross-CorrelationSpectroscopy for Multicomponent Diffusional Analysis inSolution ", Petra.Schwille et al, Biophysical journal 1997,72, be described in detail among 1878-1886 and " the Adynamic view of cellular processes by in vivo fluorescence auto-andcross-correlation spectroscopy; Petra.Schwille et al, Methods 29 (2003) 74-85 ".Fluorescence simple crosscorrelation spectrum analysis method is applicable to the protein interactions that diffusion time is few etc.Confocal consistent analysis of fluorescence (CFCA method) arranged in the same analytical method, it is at Confocalfluorescence coincidence analysis (CFCA), winkler etal., Proc.Natl.Acad.Sci.U.S.A.96:1375-1378 is described in detail in 1999.
In simple crosscorrelation spectrum analysis method (FCCS method), when having used when two overlapping fluorchromes are arranged in fluorescence spectrum, the influence of measurement error that caused by crosstalk can not correctly be carried out computing cross-correlation.
Summary of the invention
The present invention considers this actual state and invents that its purpose is to provide a kind of fluorescence spectroscopy apparatus, can not be subjected to the influence of measurement error that caused by crosstalk, correctly carries out computing cross-correlation.
Fluorescence spectroscopy apparatus of the present invention has the actuated optical unit, to the privileged site of sample stagger time-interleaved ground illumination wavelength or the different light of intensity; Fluorescence detection unit detects the fluorescence that produces from said sample; Signal processing unit carries out signal Processing to the detected signal of above-mentioned fluorescence detection unit; And arithmetic element, use many τ mode to make tables of data according to the signal that generates by above-mentioned signal processing unit, kind according to first fluorescence in each signal and second fluorescence is made weighting coefficient table, uses the data of first fluorescence and second fluorescence and each data among the weighting coefficient and weighting coefficient to carry out auto-correlation and calculates and cross-correlation calculation.
Description of drawings
Fig. 1 briefly represents the fluorescence spectroscopy apparatus of first embodiment of the present invention.
Fig. 2 represents the structure example of excitation illumination part shown in Figure 1.
Fig. 3 represents other structure example of excitation illumination part shown in Figure 1.
Fig. 4 represents the action flow chart of fluorescence spectroscopy apparatus shown in Figure 1.
Fig. 5 represents the time diagram of the signal of fluorescence spectroscopy apparatus shown in Figure 1.
Fig. 6 briefly represents the fluorescence spectroscopy apparatus of second embodiment of the present invention.
Fig. 7 briefly represents the fluorescence spectroscopy apparatus of the 3rd embodiment of the present invention.
The computing of the signal processing part of Fig. 8 A presentation graphs 7 generates with data and the part of the process flow diagram of the dissection process of operational part.
The computing of the signal processing part of Fig. 8 B presentation graphs 7 generates with data and the part of the process flow diagram of the dissection process of operational part.
Fig. 9 represents the structure and the numerical value of passage.
Figure 10 represents the data corresponding to first fluorescence of the first fluoroscopic examination signal of Fig. 5.
Figure 11 represents the weights assigned coefficient corresponding to first fluorescence of the data of Figure 10.
Figure 12 represents tables of data that the data reconstruction of first fluorescence and second fluorescence is obtained.
Figure 13 represents weighting coefficient table that the weighting coefficient reconstruct of first fluorescence and second fluorescence is obtained.
Figure 14 represents long-pending between the data of first fluorescence and calculates.
Figure 15 represents long-pending between the data of first fluorescence and second fluorescence and calculates.
Figure 16 briefly represents the fluorescence spectroscopy apparatus of the 4th embodiment of the present invention.
Figure 17 represents time series mixed signal fluctuation, that comprise fluctuation signal corresponding to first fluorescence that obtains and second fluorescence in the device of Figure 16.
Figure 18 represents the puppet first fluoroscopic examination signal that extracts from the time series mixed signal of Figure 17.
Figure 19 represents the puppet second fluoroscopic examination signal that extracts from the time series mixed signal of Figure 17.
Figure 20 is illustrated in fluorescence spectrum to be had in two overlapping fluorchromes, produces the situation of crosstalk.
Embodiment
Below, with reference to the description of drawings embodiments of the present invention.
Before embodiments of the present invention are described, at first the structure of the generation crosstalk of simple crosscorrelation spectrum analysis method (FCCS method) is described with reference to Figure 20.Figure 20 is illustrated in fluorescence spectrum the situation that produces crosstalk in two overlapping fluorchromes.In Figure 20, when first fluorchrome with first exiting spectrum was shone first exciting light, first fluorescence with first fluorescence spectrum produced from first fluorchrome.By using filtrator to detect the light of the wavelength in the first optical signal detecting scope selectively, detect first fluorescence.In addition, when second fluorchrome with second exiting spectrum was shone second exciting light, second fluorescence with second fluorescence spectrum produced from second fluorchrome.By using filtrator to detect the light of the wavelength in the second optical signal detecting scope selectively, detect second fluorescence.As expressing from Figure 20, the lower right corner of first fluorescence spectrum and the second optical signal detecting scope are overlapping.Therefore, when detecting second fluorescence, first fluorescence also is detected together, crosstalk that Here it is.Consequently, in simple crosscorrelation spectrum analysis method (FCCS method), the influence of measurement error that caused by crosstalk can not correctly be carried out computing cross-correlation.
(first embodiment)
Fig. 1 briefly represents the fluorescence spectroscopy apparatus of first embodiment of the present invention.As shown in Figure 1, the fluorescence spectroscopy apparatus 100 of present embodiment have that actuated optical is 110, fluoroscopic examination portion 140, signal processing part 150 and operational part 160.Actuated optical is 110 to have the excitation illumination part 120 that generates exciting light, objective table 112, the object lens 114 of carrying sample S and the dichronic mirror 116 that exciting light is separated with fluorescence.
Sample S comprises different pigments, and this pigment reacts respectively the exciting light of different wave length at the light-struck position of the excitation of different wave length and produces fluorescence.Fluorescence spectrum at least a portion of different pigments is overlapping.
The fluorescence that produces from sample S detects according to the exciting light of irradiation in fluoroscopic examination portion 140.
150 pairs of signals that reflected the fluorescence intensity that is obtained by fluoroscopic examination portion 140 of signal processing part are handled.The wavelength change of the exciting light that shines sample S that operational part 160 causes based on exciting light control part 130 and the testing result of fluoroscopic examination portion 140 are carried out correlation analysis to the fluctuation of the fluorescence of each different wave length.For example, operational part 160 is based on the comparison corresponding to the output signal of each fluorescence, resolves with autocorrelation analysis or cross-correlation analysis or confocal consistent analysis of fluorescence.
Fig. 2 represents to encourage the structure example of illumination part 120.In this embodiment, excitation illumination part 120 comprises the first light source 122a, secondary light source 122b, catoptron 124a, dichronic mirror 124b and acousto-optic element (AOTF) 126.The first light source 122a and secondary light source 122b produce first exciting light and second exciting light that wavelength band differs from one another respectively.Catoptron 124a will reflect to the object lens 114 of Fig. 1 from first exciting light that the first light source 122a produces.Dichronic mirror 124b sees through first exciting light by catoptron 124a reflection, will reflect to the object lens 114 of Fig. 1 from second exciting light that secondary light source 122b produces.The first light source 122a and secondary light source 122b are produced first exciting light and second exciting light respectively continuously by continuous drive.Acousto-optic element 126 is configured in the public light path that first exciting light and second exciting light pass through, can control and pass through frequency band, according to the exciting light operation signal that the exciting light control part 130 from Fig. 1 provides, make some the seeing through in first exciting light and second exciting light selectively.In other words, acousto-optic element 126 selects to shine the light of sample S from the light of the different wave length of the first light source 122a and secondary light source 122b generation.The exciting light operation signal is the signal that time series changes, and acousto-optic element 126 alternately sees through first exciting light and second exciting light.
Fig. 3 represents to encourage other structure example of illumination part 120.In this embodiment, excitation illumination part 120 comprises the first light source 122a, secondary light source 122b, catoptron 124a, dichronic mirror 124b and switch 128.The function of the first light source 122a, secondary light source 122b, catoptron 124a and dichronic mirror 124b is identical with the example of Fig. 2.And the first light source 122a and secondary light source 122b can control conducting and end.Switch 128 makes some conductings of the first light source 122a and secondary light source 122b selectively according to the exciting light operation signal that the exciting light control part 130 from Fig. 1 provides, and makes ending of other.That is, switch 128 is selected luminous light source.The exciting light operation signal is the signal that time series changes, the first light source 122a and secondary light source 122b alternate conduction.Consequently, first exciting light and second exciting light alternately shine sample S.That is, switch 128 selects to shine the light of sample S from the light of the different wave length of the first light source 122a and secondary light source 122b generation.
Return Fig. 1, fluoroscopic examination portion 140 comprises dichronic mirror 142, the first fluorescence filter 144a, the second fluorescence filter 144b, the first photo detector 146a and the second photo detector 146b.The first fluorescence filter 144a sees through first fluorescence selectively, and the second fluorescence filter 144b sees through second fluorescence selectively.The first photo detector 146a has light sensitivity to the wavelength band of first fluorescence, and the second photo detector 146b has light sensitivity to the wavelength band of second fluorescence.That is, the first photo detector 146a and the second photo detector 146b have light sensitivity to the different wave length frequency band.
Below, with reference to the action of the fluorescence spectroscopy apparatus of the flowchart text present embodiment of Fig. 4.
Exciting light control part 130 generates the exciting light operation signal, and this signal is outputed to excitation illumination part 120.As shown in Figure 5, the exciting light operation signal is the binary signal of periodically variable " 0 " and " 1 ".
At excitation illumination part 120 be under the situation of structure of Fig. 2, and acousto-optic element 126 sees through first exciting light at the exciting light operation signal during for " 0 " selectively, when the exciting light operation signal is " 1 " second exciting light is seen through selectively.
In addition, be under the situation of structure of Fig. 3 at excitation illumination part 120, switch 128 makes the first light source 122a conducting selectively at the exciting light operation signal during for " 0 ", makes secondary light source 122b conducting selectively when the exciting light operation signal is " 1 ".
Its result, as shown in Figure 5, the exciting light that shines sample S alternately switches first exciting light and second exciting light.
According to the exciting light of irradiation, produce fluorescence from sample S.The first photo detector 146a detects first fluorescence that produces from sample S according to first exciting light of irradiation, and the first fluoroscopic examination signal shown in Figure 5 is outputed to signal processing part 150.The second photo detector 146b detects second fluorescence that produces from sample S according to second exciting light of irradiation, and the second fluoroscopic examination signal shown in Figure 5 is outputed to signal processing part 150.
The first fluoroscopic examination signal and the second fluoroscopic examination signal transformation that signal processing part 150 will provide respectively from the first photo detector 146a and the second photo detector 146b of fluoroscopic examination portion 140 are each fluorescence intensity signals of fixing time, this fluorescence intensity signals and exciting light control signal are made up with optimal form, generate the computing data, these data are outputed to operational part 160.
In the present embodiment, first exciting light of different wave length and second exciting light time of staggering shines sample S exclusively.According to first exciting light and second exciting light first fluorescence and second fluorescence that produce respectively from sample S of irradiation, the first photo detector 146a and the second photo detector 146b by fluoroscopic examination portion 140 detects respectively.Therefore, the influence of measurement error that can not caused by crosstalk is carried out correct computing cross-correlation.
(second embodiment)
Fig. 6 roughly represents the fluorescence spectroscopy apparatus of second embodiment of the present invention.Except that fluoroscopic examination portion 240, the fluorescence spectroscopy apparatus 200 of present embodiment is identical with the fluorescence spectroscopy apparatus 100 of first embodiment.
In the present embodiment, first exciting light of different wave length and second exciting light time of staggering shines sample S exclusively.According to first exciting light and second exciting light of irradiation, first fluorescence that produces respectively from sample S and second fluorescence detect by the photo detector 244 timesharing ground of a fluoroscopic examination portion 240.Therefore, the influence of measurement error that can not caused by crosstalk is carried out correct computing cross-correlation.
(the 3rd embodiment)
Fig. 7 roughly represents the fluorescence spectroscopy apparatus of the 3rd embodiment of the present invention.As shown in Figure 7, the fluorescence spectroscopy apparatus 300 of present embodiment have that actuated optical is 310, fluoroscopic examination portion 340, signal processing part 350 and operational part 360.Actuated optical is 310 to have the excitation illumination part 320 that generates exciting light, objective table 312, the object lens 314 of carrying sample S and the dichronic mirror 316 that exciting light is separated with fluorescence.
Fluorescence spectroscopy apparatus 300 also has the exciting light control part 330 of control excitation illumination part 320, makes the exciting light of different wave length time of staggering shine sample S exclusively.Excitation illumination part 320 shines wavelength or the different exciting light of intensity the privileged site of sample S selectively.Excitation light wavelength or intensity are by the change of time series ground.
Sample S comprises different pigments, and this pigment reacts respectively the exciting light of different wave length at the light-struck position of the excitation of different wave length and produces fluorescence.Fluorescence spectrum at least a portion of different pigments is overlapping.
The fluorescence that produces from sample S detects according to the exciting light of irradiation in fluoroscopic examination portion 340.Fluoroscopic examination portion 340 separates each fluorescence that produces respectively and detection according to the different wave length of irradiation or the exciting light of intensity.
Fluoroscopic examination portion 340 comprises dichronic mirror 342, the first fluorescence filter 344a, the second fluorescence filter 344b, the first photo detector 346a and the second photo detector 346b.The first fluorescence filter 344a sees through first fluorescence selectively, and the second fluorescence filter 344b sees through second fluorescence selectively.The first photo detector 346a has light sensitivity to the wavelength band of first fluorescence, and the second photo detector 346b has light sensitivity to the wavelength band of second fluorescence.In other words, the first photo detector 346a has light sensitivity with the second photo detector 346b to different wavelength bands.
The fluorescence spectroscopy apparatus of present embodiment, for irradiation and the detection of fluorescence and the generation that data are used in computing of exciting light, identical with first embodiment, roughly the process flow diagram according to Fig. 4 moves.
Below, the data parsing of many τ mode related function of having used weighting coefficient (parameter that is used for correlation analysis) is narrated.In this data parsing, make tables of data and weighting coefficient table according to each fluoroscopic examination signal, use the data and the weighting coefficient of data, first fluorescence in the weighting coefficient and second fluorescence of each fluorescence, carry out auto-correlation and cross-correlation calculation.
When carrying out the computing of related function, the passage (Channel) of the calculating usefulness of data and weighting coefficient uses octave (Octave) mode, the calculating of data and weighting coefficient is limited in the result of limited less passage of number, realizes drawing (Plot) with result of calculation at interval.And,, calculate data mean value and the weighting coefficient mean value suitable with different time delays in different at first τ zones.The various processing of the data of fluorescence and weighting coefficient are the least unit of calculating with data or weighting coefficient.
Below, as concrete example, many τ mode is described by the process flow diagram of Fig. 8 A and Fig. 8 B.
(step S0)
As measurement data, obtain the time series measurement data of first fluorescence and second fluorescence to sample.The continuous signal of the insertion method of the time series data that obtains as shown in Figure 5.
(step S1)
Whether judgement obtains data and exists, and under the situation of "Yes", advances to step S2, under the situation of "No", enters into imaging (Imaging) determining step S18.
(step S2)
Count reading in the data number.This data sum is used for passage calculating, summation calculating etc.
(step S3)
Calculate the drawing τ value (channel value) of many τ mode and port number etc.Many τ mode is according to the sum decision port number of the data of reading in.Concrete computing method as shown in Figure 9, with time to peak (bin time) τ
0Be reference value, set 16 initial channel value; Later per 8 channel value are with time to peak τ
0The value that is increased to 2 times is a reference value.
In other words, 16 initial passages are made as 0 section, after, per 8 passages are divided into 1 section, 2 sections ....It is that hop count is made as n that the channel value of each section gets increment (reference value), is 2
nτ
0For example, the increment of 0 section channel value is τ
0, the increment of 2 sections channel value is 4 τ
0
What necessary port number was based on time to peak and the sum of the data of reading in calculates.
(step S4 and S5)
Carry out the identification of fluorescence.At step S4, for the first fluoroscopic examination signal, at exciting light is under the situation of first exciting light, the input data are processed after step S6 as the active data of first fluorescence, at exciting light is under the situation of second exciting light, and the input data are processed as zero in step S5, at exciting light neither first exciting light neither the situation of second exciting light under, same, the data of first fluorescence are inserted into as zero in step S5.In addition, for the second fluoroscopic examination signal, at exciting light is under the situation of second exciting light, the input data are processed after step S6 as the active data of second fluorescence, at exciting light is under the situation of first exciting light, and the data of first fluorescence are processed as zero in step S5, at exciting light neither second exciting light neither the situation of first exciting light under, same, the data of second fluorescence are inserted into as zero in step S5.
(step S6)
Carry out data extract.At first,, extract the data of first fluorescence, it be stored in the position suitable with first fluorescence for the first fluoroscopic examination signal, with other the suitable position store data 0 of fluorescence (second fluorescence).Consequently, make the tables of data of first fluorescence shown in Figure 10.Equally, second fluorescence is also made the another one tables of data.Consequently, two tables of data constitute respectively first fluorescence and second fluorescence.
(step S7)
For during anti-stop signal or the data disappearance to the influence of analysis result, during during signal or the data disappearance and beyond it between, carry out different weightings.Make weighting coefficient table for this reason.When the excitation plain edge being switched the limit measurement, detect the information that comprises the kind (being the kind of fluorescence) of the exciting light except that the information of data size in the data with time series.In many τ mode, the kinds of information of fluorescence is used for calculating as weighting coefficient.When not carrying out DATA REASONING, be that 1 (weighting coefficient=1) is represented with the data number to first fluorescence and second fluorescence.The first fluoroscopic examination signal is stored in the position suitable with first fluorescence with weighting coefficient 1, weighting coefficient 0 is stored in the position suitable with other fluorescence.Consequently, make the weighting coefficient table of first fluorescence shown in Figure 11.Equally, second fluorescence is made another weighting coefficient table.That is, make the table that has changed weighting coefficient, this weighting coefficient is as the parameter that is used for each data that corresponds respectively to first fluorescence or second fluorescence are carried out computing.Consequently, two weighting coefficient tables constitute respectively first fluorescence and second fluorescence.
(step S8)
Carry out data reconstruction.That is, calculate the initial channel data of each different passage of reference value (increment).By the tables of data of first fluorescence and second fluorescence, carry out each fluorescence and computing.Passage 16 later per 8 passages of delay time T make reference value (increment) be increased to twice, and therefore, the data of each passage are increased to two data and formation before 2 times by reference value (increment).The variation of concrete data as shown in figure 12.In first fluorescence and second fluorescence, by carrying out each and computing, form the data of passage with constantly new reference value (increment) according to data distribution list, constitute new tables of data.
In other words, the arrangement of the total data of reading in is made as the data rows of the 0th row,, this is arranged data rows as the 1st row each wherein adjacent two addition.Afterwards, carry out same operation repeatedly, generate the 2nd the row data rows, the 3rd the row data rows ....This operation lasts till the data rows that obtains the line number identical with the hop count of passage always.In the tables of data that obtains like this, the passage with each section is corresponding respectively for the data of each row.For example, the data of the 2nd row 0 row are corresponding with the passage of the 2nd section 0 row.
(step S9)
Be weighted coefficient reconstruct.In other words, the weighting coefficient to the initial passage of each different passage of reference value (increment) calculates.Weighting coefficient table by first fluorescence and second fluorescence carries out and computing each fluorescence.The variation of weighting coefficient table is identical with step S8, passes through and computing, and forms the weighting coefficient (Figure 13) of the passage that constantly has new reference value (increment), constitutes new weighting coefficient table.
In other words, the arrangement of all weighting coefficients is made as the weighting coefficient row of the 0th row, to wherein adjacent each two carry out addition, this arrangement is made as the weighting coefficient row of the 1st row.After this, carry out same operation repeatedly, the weighting coefficient that produces the 2nd row is listed as, the weighting coefficient of the 3rd row is listed as ....This operation lasts till the weighting coefficient row that obtain the line number identical with the passage hop count always.In the weighting coefficient table that obtains like this, the passage with each section is corresponding respectively for the weighting coefficient of each row.
(step S10)
Data I to first fluorescence
D1Carry out the long-pending and calculating between data.In other words, as shown in figure 14, the data and the 0th column data of the channel position with same datum value (increment) of first fluorescence multiplied each other, calculate they and.In other words, in the data rows of first fluorescence corresponding with each section passage, try to achieve initial data and other each data product and.Secondly, to the second fluorescence data I
D2Carry out the long-pending and calculating between data.In other words, second fluorescence is carried out same processing, the data and the 0th column data of the channel position with same datum value (increment) of second fluorescence is multiplied each other, calculate they and.In other words, in the data rows of second fluorescence corresponding with each section passage, try to achieve initial data and other each data product and.
(step S11)
Weighting coefficient W to first fluorescence
D1Be weighted the long-pending and calculating between coefficient.In other words, the weighting coefficient and the 0th row weighting coefficient of the channel position with same datum value (increment) of first fluorescence multiplied each other, calculate they and.In other words, in the weighting coefficient of first fluorescence corresponding row with each section passage, try to achieve initial weighting coefficient and other each weighting coefficient product and.Secondly, to the second fluorescence weighting coefficient W
D2Be weighted the long-pending and calculating between coefficient.In other words, second fluorescence is carried out same processing, the weighting coefficient and the 0th row weighting coefficient of the channel position with same datum value (increment) of second fluorescence is multiplied each other, calculate they and.In other words, in the weighting coefficient of second fluorescence corresponding row with each section passage, try to achieve initial weighting coefficient and other each weighting coefficient product and.
(step S12)
Data I to first fluorescence
D1With weighting coefficient W
D1, carry out the long-pending and calculating of the 0th column data and weighting coefficient.In other words, the weighting coefficient and the 0th column data of the channel position with same datum value (increment) of first fluorescence multiplied each other, calculate they and.In other words, in the data rows of first fluorescence corresponding and weighting coefficient row with each section passage, try to achieve the original date of data rows and weighting coefficient row each weighting coefficient product and.Secondly, to the data I of second fluorescence
D2With weighting coefficient W
D2Carry out the long-pending and calculating of data and weighting coefficient.In other words, second fluorescence is carried out same processing, the weighting coefficient and the 0th column data of the channel position with same datum value (increment) of second fluorescence is multiplied each other, calculate they and.In other words, in the data rows of first fluorescence corresponding and weighting coefficient row with each section passage, try to achieve the original date of data rows and weighting coefficient row each weighting coefficient product and.
(step S13)
Weighting coefficient W to first fluorescence
D1And data I
D1, carry out the long-pending and calculating of the 0th row weighting coefficient and data.In other words, the data and the 0th row weighting coefficient of the channel position with same datum value (increment) of first fluorescence multiplied each other, calculate they and.In other words, in the data rows of first fluorescence corresponding and weighting coefficient row with each section passage, try to achieve the initial weighting coefficient of weighting coefficient row and data rows each data product and.Secondly, to the weighting coefficient W of second fluorescence
D2And data I
D2Be weighted the long-pending and calculating of coefficient and data.In other words, second fluorescence is carried out same processing, the data and the 0th row weighting coefficient of the channel position with same datum value (increment) of second fluorescence is multiplied each other, calculate they and.In other words, in the data rows of second fluorescence corresponding and weighting coefficient row with each section passage, try to achieve the initial weighting coefficient of weighting coefficient row and data rows each data product and.
(step S14)
Data I to first fluorescence and second fluorescence
D1And I
D2Carry out long-pending between the data of first fluorescence and second fluorescence and calculate.In other words, as shown in figure 15, the data of the channel position of second fluorescence with same datum value (increment) and the 0th column data of the 1st fluorescence are multiplied each other, calculate they and.In other words, in the data rows of the data rows of first fluorescence corresponding and second fluorescence with each section passage, try to achieve the initial data of data rows of first fluorescence and second fluorescence data rows each data product and.
(step S15)
Weighting coefficient W to first fluorescence and second fluorescence
D1And W
D2Carry out long-pending between the weighting coefficient of first fluorescence and second fluorescence and calculate.In other words, the weighting coefficient of the channel position of second fluorescence with same datum value (increment) and the 0th row weighting coefficient of the 1st fluorescence are multiplied each other, calculate they and.In other words, in the weighting coefficient row of the weighting coefficient of the first fluorescence corresponding row and second fluorescence with each section passage, try to achieve the initial weighting coefficient of weighting coefficient row of first fluorescence and second fluorescence the weighting coefficient row each weighting coefficient product with.
(step S16)
Data I to first fluorescence
D1Weighting coefficient W with second fluorescence
D2, carry out long-pending between the weighting coefficient of the 0th column data of first fluorescence and second fluorescence and calculate.In other words, the weighting coefficient of the channel position of second fluorescence with same datum value (increment) and the 0th column data of first fluorescence are multiplied each other, calculate they and.In other words, in the weighting coefficient row of the data rows of first fluorescence corresponding and second fluorescence with each section passage, try to achieve the original date of data rows of first fluorescence and second fluorescence the weighting coefficient row each weighting coefficient product and.
(step S17)
Weighting coefficient W to first fluorescence
D1Data I with second fluorescence
D2, carry out data long-pending of the 0th row weighting coefficient of first fluorescence and second fluorescence and calculate.In other words, the weighting coefficient of the 0th row of the data of the channel position of second fluorescence with same datum value (increment) and first fluorescence is multiplied each other, calculate they and.In other words, in the weighting coefficient row of the data rows of second fluorescence corresponding and first fluorescence with each section passage, try to achieve the original date of data rows of second fluorescence and first fluorescence the weighting coefficient row each weighting coefficient product and.
(step S18)
Carry out the judgement of computing end and imaging.Under the situation of "Yes", enter the summation correlation computations, under the situation of "No", turn back to data and obtain step S1.
(step S19)
When data are read in end (when step S21 is "Yes"),, carry out the supposition of autocorrelation function and the supposition of cross correlation function based on above-mentioned each result of calculation.In other words, to each related side of D1 → D2, D1 → D1 and D2 → D2 to, infer related function with different respectively analytic expressions.
For example, to D1 → D1 and D2 → D2, with the calculating formula of (S10*S11)/(S12*S13), to the calculating formula of D1 → D2 with (S14*S15)/(S16*S17).
For example, the analytic expression of cross correlation function can show as following (1) formula.
(several 1)
mlF
D1R
D2Sum(τ)＝way[2]mlFRSum[k]
＝mlF[0][mlrow][0]*mlF[1][mlrow][k]
mlW
D1V
D2Sum(τ)＝way[2]mlWVSum[k] ...(1)
＝mlW[0][mlrow][0]*mlW[1][mlrow][k]
mlF
D1V
D2Sum(τ)＝way[2]mlFSum[k]
＝mlF[0][mlrow][0]*mlW[1][mlrow][k]
mlW
D1R
D2Sum(τ)＝way[2]mlFRSum[k]
＝mlW[0][mlrow][0]*mlF[1][mlrow][k]
Above-mentioned various in, mlF
DR
DSum (τ
v) expression long-pending between data and calculate mlW
DV
DSum (τ
v) expression weighting coefficient long-pending and calculating.In addition, mlF
DV
DSum (τ
v) represent the long-pending of the 0th column data and weighting coefficient and calculate mlW
DR
DSum (τ
v) expression the 0th row weighting coefficient and data long-pending and calculating.At this, subscript D is D1 or D2, corresponding to the data of calculating object, promptly corresponding to data corresponding with first fluorescence or the data corresponding with second fluorescence.And, F
D1, R
D2The expression reconstruct and the data D1 of calculating, the data of D2, W
D1, V
D2Expression is used for computational data F
D1, R
D2Data number (weighting coefficient).
(1) formula is based on the simple crosscorrelation analytic expression of following (2) formula.In addition, the simple crosscorrelation analytic expression of (2) formula is to use weighting to derive by the general cross correlation function to (3) formula.If N
1=N
2=N
12, then (3) formula is expressed as (9) formula.
(several 2)
(several 3)
(several 4)
(step S20)
Based on each final calculation result, carry out the processing of the curve display etc. of cross correlation function.
In the present embodiment, the correlation analysis computing of fluorescence fluctuation is in the signal or data that signal processing part 350 is generated, according to signal or data corresponding to fluorescence that the wavelength or the different exciting light of intensity of irradiation produces respectively, carries out parameter modification.Therefore, be not subjected to the influence of measurement error of crosstalk, carry out correct computing cross-correlation.
(the 4th embodiment)
Figure 16 briefly represents the fluorescence spectroscopy apparatus of the 4th embodiment of the present invention.Except that fluoroscopic examination portion 440 and signal processing part 450, the fluorescence spectroscopy apparatus 400 of present embodiment is identical with the fluorescence spectroscopy apparatus 300 of the 3rd embodiment.
In the present embodiment, from the detection signal of fluoroscopic examination portion 440 output as shown in figure 17, be the time series mixed signal that first fluorescence and second fluorescence alternately mix.In Figure 17, D1 and D2 represent scope detection time of first fluorescence and second fluorescence respectively.In other words, alternately comprise the detection signal of first fluorescence and second fluorescence in this signal, can cut apart with the time.In fact, except that detection time of first fluorescence and second fluorescence the scope, also there is the time range of switching first exciting light and second exciting light, but in Figure 17, omits in the time series mixed signal.
The time series mixed signal of this moment is sent to signal processing part 450, is divided into the signal of each fluorescence and handles.In other words, signal processing part 450 extracts puppet first fluoroscopic examination signal shown in Figure 180 and the puppet second fluoroscopic examination signal shown in Figure 19 from the time series mixed signal of Figure 17.For the first fluoroscopic examination signal, only the fluorescence intensity during extraction excitation illumination part 320 irradiations first fluorescence is as the information of first fluorescence, and the information during other is made as zero.Equally, for the first fluoroscopic examination signal, only the fluorescence intensity during extraction excitation illumination part 320 irradiations second fluorescence is as the information of second fluorescence, and the information during other is made as zero.Like this, signal processing part 450 generates pseudo-first fluoroscopic examination signal and the pseudo-second fluoroscopic examination signal based on the fluctuation signal from 440 outputs of fluoroscopic examination portion.
Pseudo-first fluoroscopic examination signal and the pseudo-second fluoroscopic examination signal carry out same processing with the first fluoroscopic examination signal and the second fluoroscopic examination signal that illustrate afterwards in the 3rd embodiment.
In the present embodiment, the correlation analysis computing of the fluctuation of fluorescence is in the signal or data that signal processing part 350 is generated, according to signal or data corresponding to fluorescence that the wavelength or the different exciting light of intensity of irradiation produces respectively, carries out parameter modification.Therefore, be not subjected to the influence of measurement error of crosstalk, carry out correct computing cross-correlation.
So far, with reference to description of drawings embodiments of the present invention, but the present invention is not limited to these embodiments, as long as in the scope that does not break away from this aim, can carry out various distortion or change.
Utilizability on the industry
According to the present invention, a kind of fluorescence spectroscopy apparatus is provided, can not be subjected to the impact of the measure error that crosstalk causes, correctly carry out computing cross-correlation.
Claims (9)
1. fluorescence spectroscopy apparatus is characterized in that having:
The actuated optical unit is to the privileged site of sample stagger time-interleaved ground illumination wavelength or the different light of intensity;
Fluorescence detection unit detects the fluorescence that produces from said sample;
Signal processing unit carries out signal Processing to the detected signal of above-mentioned fluorescence detection unit; And
Arithmetic element, use many τ mode to make tables of data according to the signal that generates by above-mentioned signal processing unit, kind according to first fluorescence in each signal and second fluorescence is made weighting coefficient table, uses the data of first fluorescence among each data and the weighting coefficient and second fluorescence and weighting coefficient to carry out auto-correlation and calculates and cross-correlation calculation.
2. fluorescence spectroscopy apparatus according to claim 1 is characterized in that,
Said sample comprises different pigments, and this different pigment reacts and generation fluorescence the exciting light of above-mentioned different wave length respectively at the above-mentioned position that exciting light shone of above-mentioned different wave length, and fluorescence spectrum at least a portion of the pigment that these are different is overlapping.
3. fluorescence spectroscopy apparatus according to claim 1 is characterized in that,
Above-mentioned actuated optical unit shines the exciting light of different wave length repeatedly to said sample with predetermined timing.
4. fluorescence spectroscopy apparatus according to claim 1 is characterized in that,
Above-mentioned actuated optical unit comprises:
A plurality of light sources send the light of different wave length; And
Selected cell selects to shine the light of said sample the light of the different wave length that sends from this light source.
5. fluorescence spectroscopy apparatus according to claim 4 is characterized in that,
Be used to select the selected cell of above-mentioned irradiates light, be configured in the shared light path that a plurality of light passed through of above-mentioned different wave length.
6. fluorescence spectroscopy apparatus according to claim 4 is characterized in that,
Being used to select the selected cell of above-mentioned irradiates light, is to select the light source switch unit of illuminating source maybe can control acousto-optic element by frequency band.
7. fluorescence spectroscopy apparatus according to claim 1 is characterized in that,
Above-mentioned fluorescence detection unit is made of a plurality of photo detectors that the different wave length frequency band had light sensitivity.
8. fluorescence spectroscopy apparatus according to claim 1 is characterized in that,
Above-mentioned fluorescence detection unit is made of a photo detector, and what this photo detector had the fluorescence that can detect the above-mentioned different caused different wave length of exciting light is subjected to the optical frequency band.
9. fluorescence spectroscopy apparatus according to claim 1 is characterized in that, above-mentioned fluorescence detection unit is according to each fluorescence that produces respectively corresponding to the irradiation of the exciting light of above-mentioned different wavelength or intensity, separates and detects.
Applications Claiming Priority (5)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2005282687A JP4879543B2 (en) | 2005-09-28 | 2005-09-28 | Fluorescence spectrometer |
| JP282687/2005 | 2005-09-28 | ||
| JP2005282686A JP2007093370A (en) | 2005-09-28 | 2005-09-28 | Fluorescence spectroscopic analyzer |
| JP282686/2005 | 2005-09-28 | ||
| PCT/JP2006/319132 WO2007037252A1 (en) | 2005-09-28 | 2006-09-27 | Fluorescence spectral analyzer |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101278191A CN101278191A (en) | 2008-10-01 |
| CN101278191B true CN101278191B (en) | 2011-06-08 |
Family
ID=37979270
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2006800362769A Expired - Fee Related CN101278191B (en) | 2005-09-28 | 2006-09-27 | Fluorescence spectroscopy apparatus |
Country Status (2)
| Country | Link |
|---|---|
| JP (1) | JP2007093370A (en) |
| CN (1) | CN101278191B (en) |
Families Citing this family (14)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP4879543B2 (en) * | 2005-09-28 | 2012-02-22 | オリンパス株式会社 | Fluorescence spectrometer |
| JP5019116B2 (en) * | 2007-10-22 | 2012-09-05 | オリンパス株式会社 | Fluorescence spectrometer |
| JP5087068B2 (en) * | 2009-12-04 | 2012-11-28 | 株式会社電制 | Oil detection device and oil detection method |
| JP5419047B2 (en) * | 2010-03-19 | 2014-02-19 | 株式会社島津製作所 | Mass spectrometry data processing method and mass spectrometer |
| EP2620763A4 (en) * | 2010-10-19 | 2015-05-27 | Olympus Corp | Optical analysis device for observing polarisation characteristics of single light-emitting particle, optical analysis method and optical analysis computer program therefor |
| WO2012056580A1 (en) * | 2010-10-29 | 2012-05-03 | オリンパス株式会社 | Image analysis method and image analysis device |
| DE102011017046A1 (en) * | 2011-04-14 | 2012-10-18 | Till Photonics Gmbh | Switchable microscope arrangement with multiple detectors |
| WO2013024650A1 (en) * | 2011-08-15 | 2013-02-21 | オリンパス株式会社 | Photometric analysis device using single light emitting particle detection, photometric analysis method and computer program for photometric analysis, |
| KR102126032B1 (en) * | 2013-07-31 | 2020-07-08 | 삼성전자주식회사 | Multi-channel fluorescence detecting module and nucleic acid analysis system having the same |
| KR101469138B1 (en) * | 2014-01-27 | 2014-12-05 | 주식회사 아쿠아테크 | Biosensor and method for sensing the same |
| CN105044164A (en) * | 2015-07-14 | 2015-11-11 | 泸州品创科技有限公司 | Method for analyzing and detecting liquor characteristic before and after formation of liquor sediment |
| CN106353295A (en) * | 2016-11-11 | 2017-01-25 | 中国科学院生物物理研究所 | Protein heat stability analysis meter |
| GB2573692B (en) * | 2017-02-20 | 2022-02-16 | Hitachi High Tech Corp | Analysis system and analysis method |
| CN114860003A (en) * | 2022-05-12 | 2022-08-05 | 中国科学院苏州生物医学工程技术研究所 | PCR thermal cycle system control method, device, equipment and storage medium |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5102625A (en) * | 1990-02-16 | 1992-04-07 | Boc Health Care, Inc. | Apparatus for monitoring a chemical concentration |
| US5491343A (en) * | 1994-03-25 | 1996-02-13 | Brooker; Gary | High-speed multiple wavelength illumination source, apparatus containing the same, and applications thereof to methods of irradiating luminescent samples and of quantitative luminescence ratio microscopy |
| US6433929B1 (en) * | 2000-06-12 | 2002-08-13 | Olympus Optical Co., Ltd. | Scanning optical microscope and method of acquiring image |
Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP3654458B2 (en) * | 1995-10-31 | 2005-06-02 | アークレイ株式会社 | Light source device |
| JPH1152252A (en) * | 1997-08-06 | 1999-02-26 | Nikon Corp | Fluorescent microscope |
| JP2004187581A (en) * | 2002-12-11 | 2004-07-08 | Olympus Corp | Method for determining polymorphism of target nucleic acid |
| JP4480976B2 (en) * | 2003-01-28 | 2010-06-16 | オリンパス株式会社 | Scanning optical microscope apparatus, control method therefor, and program |
| JP4879543B2 (en) * | 2005-09-28 | 2012-02-22 | オリンパス株式会社 | Fluorescence spectrometer |
-
2005
- 2005-09-28 JP JP2005282686A patent/JP2007093370A/en active Pending
-
2006
- 2006-09-27 CN CN2006800362769A patent/CN101278191B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5102625A (en) * | 1990-02-16 | 1992-04-07 | Boc Health Care, Inc. | Apparatus for monitoring a chemical concentration |
| US5491343A (en) * | 1994-03-25 | 1996-02-13 | Brooker; Gary | High-speed multiple wavelength illumination source, apparatus containing the same, and applications thereof to methods of irradiating luminescent samples and of quantitative luminescence ratio microscopy |
| US6433929B1 (en) * | 2000-06-12 | 2002-08-13 | Olympus Optical Co., Ltd. | Scanning optical microscope and method of acquiring image |
Non-Patent Citations (2)
| Title |
|---|
| JP特开2004-187581A 2004.07.08 |
| JP特开平9-126888A 1997.05.16 |
Also Published As
| Publication number | Publication date |
|---|---|
| CN101278191A (en) | 2008-10-01 |
| JP2007093370A (en) | 2007-04-12 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101278191B (en) | Fluorescence spectroscopy apparatus | |
| US6376843B1 (en) | Method of characterizing fluorescent molecules or other particles using generating functions | |
| Schie et al. | Rapid acquisition of mean Raman spectra of eukaryotic cells for a robust single cell classification | |
| WO2005093392A1 (en) | Fluorescence spectrometer | |
| US7456954B2 (en) | Modulated excitation fluorescence analysis | |
| JP2024019180A (en) | Multispectral sample imaging | |
| US20060176479A1 (en) | Monitoring molecular interactions using photon arrival-time interval distribution analysis | |
| EP0764844B1 (en) | Method for analysis by light scattering | |
| EP1454128A2 (en) | System and method for time correlated multi-photon counting measurements | |
| Hendrix et al. | Pulsed interleaved excitation: principles and applications | |
| WO2006028020A1 (en) | Fluorescent microscope and fluorescent correlation spectral analysis device | |
| JP4879543B2 (en) | Fluorescence spectrometer | |
| WO2008127434A2 (en) | Time-resolved and wavelength-resolved spectroscopy for characterizing biological materials | |
| EP1942334A1 (en) | Fluorescence spectral analyzer | |
| US7257289B2 (en) | Spectral microscope and method for data acquisition using a spectral microscope | |
| US20050046836A1 (en) | Method for separating fluorescence spectra of dyes present in a sample | |
| NL2017449B1 (en) | Simultaneous Hyperspectral Coherent Raman Microscope | |
| Wolf et al. | Multifocal hyperspectral Raman imaging setup for multi-well plates | |
| JP4830087B2 (en) | Optical signal analyzer | |
| JP2005526246A (en) | Method and apparatus for measuring spectral differential images of fluorescent light | |
| Gamari et al. | Inexpensive electronics and software for photon statistics and correlation spectroscopy | |
| DK0983510T3 (en) | Recognition of tumor-specific gene products in cancer | |
| CN114324205A (en) | Method and system for performing spectrometry on sample and flow cytometer | |
| Bulseco et al. | Fluorescence correlation spectroscopy: molecular complexing in solution and in living cells | |
| Lasch et al. | Data acquisition and analysis in biomedical vibrational spectroscopy |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20110608 Termination date: 20190927 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |