CN107831157A - Based on the Raman spectrum fluorescence background subtraction method except spectrum - Google Patents
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Abstract
Based on the Raman spectrum fluorescence background subtraction method except spectrum, step is：Gather original Raman spectrum and the Raman shift of sample；Obtain displacement Raman spectrum, matching Raman spectrum and matching Raman shift；The matching Raman spectrum and displacement Raman spectrum obtained based on abovementioned steps, displacement Raman is calculated except spectrum；Position of the displacement Raman except positive negative peak in spectrum is observed, determines raman spectra region, and is some SPECTRAL REGIONs by the non-raman spectra region division matched in Raman spectrum；Raman spectra region is removed in Raman spectrum is matched, piecewise fitting is then carried out to the matching Raman spectrum after having removed raman spectra region according to the division result of SPECTRAL REGION, obtains estimating fluorescence background；According to matching Raman spectrum and estimation fluorescence background, the Raman spectrum after deducting fluorescence background is calculated.The present invention realizes the deduction of Raman spectrum fluorescence background using displacement except spectrum, has the characteristics of having a wide range of application, and algorithm is simple, cheap, human error is smaller.
Description
Technical field
The invention belongs to technical field of data processing, the fluorescence background subtraction method of more particularly to a kind of Raman spectrum.
Background technology
In the practical application of Raman spectroscopy, Raman spectrum usually can be with very strong fluorescence background.Such as bone liquid,
The Raman spectrum of the biology such as human plasma, human skin and medicine/medical science associated sample is glimmering just along with very strong fluorescence background
The presence of light background can cause Raman signal to be submerged in fluorescence background.In order to obtain Raman signal, it is necessary to Raman spectrum
Fluorescence background be deducted.The hyperfluorescence background that existing many methods are suggested and are used to deduct in Raman spectrum at present.Often
Fluorescence background subtraction method mainly includes following several：Method based on sample pretreatment, based on Raman spectrum and fluorescence
The method of spectral characteristic difference and the method based on data analysis.
Method based on sample pretreatment mainly includes sample filtering method and Photobleaching (sample irradiates for a long time).Sample
Product filtration method needs to filter out the material that fluorescence is produced in sample, and Photobleaching is to destroy fluorescence by long-time illumination sample
Molecule.These methods may make the species composition of sample or the structure of matter change, and not be suitable for system sheet to be measured
Body just has the application scenarios of the very Raman signal of hyperfluorescence.
The method of property difference based on Raman spectrum and fluorescence spectrum is widely used and huge number.For example, based on drawing
The difference of graceful spectrum and fluorescence spectrum generation time, there has been proposed the method for time-resolved Raman spectro scopy --- usual fluorescence letter
Number lag behind Raman signal, therefore using apparatus for resolving time such as ultra-short pulse laser, ICCDs or Kerr-gates by fluorescence background
Deduct.Difference of the people also based on Raman spectrum and fluorescence background polarization characteristic proposes polarization Modulation resolved Raman spectra
Method --- Raman optical signal is usually polarised light, and fluorescence is non-polarized light, therefore using polarizer by fluorescence background
Deduct.Source difference of the people also based on Raman spectrum with fluorescence background, which proposes, changes excitation wavelength resolved Raman spectra
Method --- most of materials seldom absorb ultraviolet and infrared light, using ultraviolet or infrared ray excited can effectively deduct fluorescence
Background.Also based on when excitation wavelength moves a small amount, fluorescence spectrum is almost unchanged but Raman spectrum can have therewith
The characteristic of certain displacement, there has been proposed movement to excite Raman difference spectral method --- by excitation wavelength one very little of adjustment
Fluorescence background is deducted in displacement.But above method will be equipped with special experimental facilities, these experimental facilities are generally complicated,
It is expensive, it is difficult to popularization and application.
Method based on data analysis includes frequency domain filtering method, Wavelet Transform and curve-fitting method.Wherein, frequency domain filtering
Method and Wavelet Transform are changed by domain-frequency domain, are deducted fluorescence background in a manner of filtering, the both approaches nearly more than ten years
Any development and application are not almost obtained inside.Curve-fitting method is presently the most one kind side common and that application is most wide
Method, it is fitted background baseline by the way of polynomial curve is by iteration optimization, will finally be fitted the baseline of gained from original
Deducted in Raman spectrum.Error can be produced in background estimating based on the method for data analysis, be not suitable for needing to some light
Spectrum details does situation about further discussing.
The content of the invention
It is an object of the invention to provide a kind of Raman spectrum fluorescence background subtraction method based on except spectrum, can solve existing side
The problem of cost existing for method is high, narrow application range.
To achieve these goals, the present invention adopts the following technical scheme that：
Based on the Raman spectrum fluorescence background subtraction method except spectrum, comprise the following steps：
Gather original Raman spectrum I (λ) and the Raman shift λ of sample；
Obtain displacement Raman spectrum Is(λs), matching Raman spectrum Ic(λs) and matching Raman shift λs；By original Raman
Spectrum I (λ) top n data are removed or rear N number of data are removed to obtain displacement Raman spectrum Is(λs), it is corresponding by original Raman light
Spectrum I (λ) rear N number of data are removed or top n data are removed to obtain matching Raman spectrum Ic(λs), by the rear N number of of Raman shift λ
Data are removed or top n data are removed to obtain matching Raman shift λs；
The matching Raman spectrum I obtained based on abovementioned stepsc(λs) and displacement Raman spectrum Is(λs), calculate displacement Raman
Except spectrum R (λs)=Ic(λs)/Is(λs)；
Displacement Raman is observed except spectrum R (λs) in positive negative peak position, determine raman spectra region, and Raman spectrum will be matched
Ic(λs) in non-raman spectra region division be some SPECTRAL REGIONs；
In matching Raman spectrum Ic(λs) in remove raman spectra region, then according to the division result of SPECTRAL REGION to
Remove the matching Raman spectrum behind raman spectra region and carry out piecewise fitting, obtain estimating fluorescence background B (λs)；
According to matching Raman spectrum Ic(λs) and estimation fluorescence background B (λs), the Raman after deducting fluorescence background is calculated
Spectrum Ir(λs)=Ic(λs)-B(λs)。
More specifically, data deduct number N less than 10.
More specifically, when wanting to allow displacement Raman spectrum to move to lower wave number, the top n of original Raman spectrum I (λ) is removed
Data obtain displacement Raman spectrum Is(λs), the corresponding rear N number of data for removing original Raman spectrum I (λ) obtain matching Raman spectrum
Ic(λs), the rear N number of data for removing Raman shift λ obtain matching Raman shift λs；When wanting to allow displacement Raman spectrum to be moved to high wave number
When dynamic, the rear N number of data for removing original Raman spectrum I (λ) obtain displacement Raman spectrum Is(λs), it is corresponding to remove original Raman light
Spectrum I (λ) top n data obtain matching Raman spectrum Ic(λs), the top n data for removing Raman shift λ obtain matching Raman position
Move λs。
More specifically, so that whether each section of Raman shift is except spectrum R (λ when dividing SPECTRAL REGIONs) one can be fitted to
Straight line is foundation, will match Raman spectrum Ic(λs) in non-raman spectra region division be some SPECTRAL REGIONs, each section can be with
The Raman shift of straight line is fitted to except spectrum is a SPECTRAL REGION.
More specifically, it is fitted using Gaussian linear and segmentation plan is carried out to the matching Raman spectrum behind removal raman spectra region
Close.
From above technical scheme, the present invention does not have to pre-process sample, directly gathers the original Raman of sample
Spectrum, you can realize and observe the Raman signal for producing fluorescence background structure, widened the scope of application of Raman spectrum；Together
When, fluorescence background is estimated except the method for spectrum using displacement due to the present invention, with the method based on data analysis in the prior art
Compare, algorithm is simple, reduces human error, reduces the dispute in discussing Raman signal details.And the present invention adopts
Raman spectroscopy device is built with conventional device, experimental provision is simple and price is relatively low, has widened Raman spectrum measurement system
Application, can be applied in the fields such as basic science, biomedicine and life science, to deduct in Raman spectrum
Fluorescence background.
Brief description of the drawings
Fig. 1 is the step flow chart of the inventive method；
The structural representation of light collecting system in the Raman spectrum measurement system that Fig. 2 is built by the embodiment of the present invention；
Fig. 3 a are the original Raman spectrogram of R6G/ ethanol systems；
Fig. 3 b are the displacement Raman spectrogram after the original Raman spectrum of R6G/ ethanol systems moves to lower wave number direction；
Fig. 4 a are to deduct the Raman spectrum after fluorescence background to the Raman spectrum of R6G/ ethanol systems using the inventive method
Figure；
Fig. 4 b are straight alcohol Raman spectrogram；
Fig. 5 a are the original Raman spectrogram of human plasma；
Fig. 5 b are to deduct the Raman spectrogram after fluorescence background to the Raman spectrum of human plasma using the inventive method；
Fig. 6 a are the original Raman spectrogram of mouse skin living；
Fig. 6 b are to deduct the Raman spectrogram after fluorescence background to the Raman spectrum of mouse skin using the inventive method.
The embodiment of the present invention is described in more detail below in conjunction with accompanying drawing.
Embodiment
Below in conjunction with the accompanying drawing in the embodiment of the present invention, the technical scheme of the embodiment of the present invention is carried out clear, complete
Ground describes, it is clear that described embodiment is only part of the embodiment of the present invention, rather than whole embodiments.Based on this
Embodiment in invention, the every other implementation that those of ordinary skill in the art obtain under the premise of creative work is not made
Example, belongs to the scope of protection of the invention.
Fig. 1 is the flow chart of the inventive method, as shown in figure 1, the inventive method comprises the following steps：
The original Raman spectrum I (λ) and Raman shift λ, Raman shift for gathering sample are the abscissas in Raman spectrum,
Original Raman spectrum I (λ) and Raman shift λ using Raman spectroscopy device collection sample, one in original Raman spectrum I (λ)
As all there is very strong fluorescence background, but there is also the raman spectra for being better than system noise simultaneously；
Obtain displacement Raman spectrum Is(λs), matching Raman spectrum Ic(λs) and matching Raman shift λs；By original Raman
Spectrum I (λ) top n data are removed or rear N number of data are removed and can obtain displacement Raman spectrum Is(λs), it is corresponding by original drawing
Rear N number of data of graceful spectrum I (λ) are removed or top n data are removed to obtain matching Raman spectrum Ic(λs), after Raman shift λ
N number of data are removed or top n data are removed to obtain matching Raman shift λs, it is specific as follows：When wanting to allow displacement Raman spectrum to low
When wave number moves, the top n data for removing original Raman spectrum I (λ) obtain displacement Raman spectrum Is(λs), it is corresponding, remove original
Rear N number of data of beginning Raman spectrum I (λ) obtain matching Raman spectrum Ic(λs)；When wanting to allow displacement Raman spectrum to be moved to high wave number
When, the rear N number of data for removing original Raman spectrum I (λ) obtain displacement Raman spectrum Is(λs), it is corresponding, remove original Raman light
Spectrum I (λ) top n data obtain matching Raman spectrum Ic(λs), as displacement Raman spectrum Is(λs) to lower wave number move when, go
Fall Raman shift λ rear N number of data, obtain λs, as displacement Raman spectrum Is(λs) to high wave number move when, then remove Raman position
λ top n data are moved, obtain λs, no matter displacement Raman spectrum Is(λs) it is to be moved to the movement of high wave number or to lower wave number, λs
Data number of the data number all than λ it is few；Preferably, data deduct number N less than 10；
The displacement Raman spectrum I obtained based on abovementioned stepss(λs) and matching Raman spectrum Ic(λs), calculate displacement Raman
Except spectrum R (λs)：R(λs)=Ic(λs)/Is(λs)；
Displacement Raman is observed except spectrum R (λs) in positive negative peak position, raman spectra region is determined by the position of positive negative peak,
That is Raman signal region, in matching Raman spectrum Ic(λs) corresponding Raman signal region is found, Raman spectrum I will be matchedc(λs)
In region division in addition to raman spectra region be some SPECTRAL REGIONs, Raman spectrum I will be matchedc(λs) in non-Raman
Spectral peak region division is some SPECTRAL REGIONs；Whether to match Raman spectrum I when dividing SPECTRAL REGIONc(λs) non-raman spectra
Each section in region moves Raman except spectrum R (λs) can be fitted to straight line be according to being divided；
In matching Raman spectrum Ic(λs) in remove raman spectra region, then according to the division result of SPECTRAL REGION to
Remove the matching Raman spectrum behind raman spectra region and carry out piecewise fitting, obtain estimating fluorescence background B (λs)；Preferably, may be used
Piecewise fitting is carried out to the matching Raman spectrum after removing raman spectra region using Gaussian linear fitting；
According to matching Raman spectrum Ic(λs) and estimation fluorescence background B (λs), calculate the Raman spectrum after deducting fluorescence background
Ir(λs)=Ic(λs)-B(λs)。
The inventive method is described in detail with a specific embodiment below.The Raman light of the present embodiment is built first
Spectral apparatus, Raman spectroscopy device include four light source, light collecting system, beam splitting system and detector parts, build Raman light
Light source, light collecting system, beam splitting system and detector used in spectral apparatus can all use commercially available prod, wherein, light source uses
Model Genesis MX532-1000MTM, the laser that wavelength is 532nm, light collecting system are the back scattering voluntarily built
Pattern light collecting system, beam splitting system cascade spectrometer using model Omni- λ 5008i color dispersion-type three, and detector uses type
Number be DR-316B-LDC-DD CCD camera；The light irradiation that light source is sent is collected anti-by sample on sample by light collecting system
The light penetrated, and after being divided by beam splitting system, spectrum is gathered by detector.
Fig. 2 be the present embodiment backscatter mode light collecting system structural representation, the backscatter mode light collecting system
Including Glan prism 1, half-wave plate 2, right-angle prism 3, the first lens 4 and the second lens 5 being arranged in casing 7, on casing 1
It is provided with incidence hole a and light hole b.The model PGL5015 of the Glan prism 1 of the present embodiment, the model of half-wave plate 2
WPZ4212-532-M25.4, the model RAP0105 of right-angle prism 3, the first lens 4 use focal length as 50mm, a diameter of 50mm
K9 planoconvex spotlights, the second lens 5 use focal length as 75mm, a diameter of 50mm K9 planoconvex spotlights.The light that light source is sent is through entering
Unthreaded hole a enters in casing 7, and successively by Glan prism 1, half-wave plate 2 and right-angle prism 3, right-angle prism 3 makes light path transfer 90 °
After be incident to the first lens 4, converged to by the first lens 4 on the second lens 5, and reflected by the second lens 5 by the first lens 4
Afterwards, it is radiated on sample 6, the light that final sample 6 reflects projects through light hole b.
Original Raman spectrum I (λ) and Raman shift λ using Raman spectroscopy device collection sample；
The top n data for removing original Raman spectrum I (λ) obtain displacement Raman spectrum Is(λs), it is corresponding, remove original
Raman spectrum I (λ) rear N number of data obtain matching Raman spectrum Ic(λs), the rear N number of data for removing Raman shift λ are matched
Raman shift λs, now displacement Raman spectrum Is(λs) moved to lower wave number；
According to matching Raman spectrum Ic(λs) and displacement Raman spectrum Is(λs) displacement Raman is calculated except spectrum R (λs)=Ic
(λs)/Is(λs)；
In displacement Raman except spectrum R (λs) in the positive and negative peak position of observation, so that it is determined that raman spectra region, and with whether each
Section Raman shift is except spectrum R (λs) can be fitted well as foundation by the use of straight line, Raman spectrum I will be matchedc(λs) in
Non- raman spectra region division is some SPECTRAL REGIONs, matching Raman spectrum Ic(λs) non-raman spectra region in each section
The Raman shift of straight line can be fitted to except spectrum is a SPECTRAL REGION；The present embodiment is carried out using Igor pro softwares
Fitting a straight line, fitting sentence are：CurveFit line ratio/X=position/D, ratio are to want fit-spectra region
For displacement Raman except spectrum, position is the SPECTRAL REGION corresponding to ratio；
The raman spectra region determined according to previous step, in matching Raman spectrum Ic(λs) in remove raman spectra region,
Then the matching Raman spectrum after having removed raman spectra region is carried out according to the division result of SPECTRAL REGION being segmented Gauss plan
Close, obtain estimating fluorescence background B (λs)；The present embodiment straight line fitting procedure is equally completed using Igor pro softwares, is fitted language
Sentence be：CurveFit gauss spectrum/X=position/D, wherein, spectrum is for wanting fit-spectra region
With Raman spectrum, position is the SPECTRAL REGION corresponding to spectrum；The mode of fitting has a variety of, can also use other
Software is fitted；
With matching Raman spectrum Ic(λs) subtract estimation fluorescence background B (λs), that is, obtain deducting the Raman light after fluorescence background
Compose Ir(λs)=Ic(λs)-B(λs)。
Fig. 3 a are the original Raman spectrum of R6G/ ethanol systems, and wherein R6G concentration as little as nanomole is per rising amount level, from figure
3a can be seen that the Raman spectrum of ethanol is almost submerged in hyperfluorescence background completely；Using the inventive method by Fig. 3 a original
Beginning Raman spectrum moves 4cm to lower wave number direction-1After obtain displacement Raman spectrum, with shown in Fig. 3 a original Raman spectrum remove
Displacement Raman spectrum, displacement Raman as shown in Figure 3 b is obtained except spectrum, be can be seen that from Fig. 3 b in the position that marked " * " number
There is apparent positive and negative peak shape, the SPECTRAL REGION corresponding to whole positive negative peak is raman spectra region, i.e. Raman signal area
Domain；The displacement Raman shown in Fig. 3 b is observed except spectrum as can be seen that for displacement Raman except the non-raman spectra region in spectrum is general
It can be fitted with 4 to 6 straight lines, it is contemplated that to deduct raman spectra region, when to carrying out Gauss curve fitting, 9 can be divided into
Section is fitted.
Fig. 4 a are to carry out the result after fluorescence background deduction to the original Raman spectrum shown in Fig. 3 a using the inventive method
Figure, Fig. 4 b are the straight alcohol Raman spectrum of same Raman spectroscopy device unified test amount, are drawn in comparison diagram 4a and Fig. 4 b, Fig. 4 a and Fig. 4 b
The shape of graceful spectrum is almost consistent, illustrates that the human error of the inventive method is relatively low, reduces to Raman signal details discussion
When dispute, using the inventive method deduct Raman spectrum in fluorescence background be effective and reliable.
Fig. 5 a are the original Raman spectrum of human plasma, and Fig. 5 b are to be deducted using the inventive method in original Raman spectrum
Raman spectrum after fluorescence background.Fig. 6 a are the original Raman spectrum of mouse skin living, and Fig. 6 b are to be deducted using the inventive method
Raman spectrum after original Raman spectrum fluorescence background, the deduction effect of the inventive method is can be seen that from Fig. 5 a, 5b, 6a and 6b
Fruit is good, and the inventive method to measurement sample without pre-processing, you can realizes the drawing to producing fluorescence background structure
Graceful signal is observed, and has widened the structure species that Raman spectrum can be studied.
Described above is only presently preferred embodiments of the present invention, not does any formal limitation to the present invention, although this
Invention is disclosed above with preferred embodiment, but is not limited to the present invention, any person skilled in the art,
Do not depart from the range of technical solution of the present invention, when the technology contents using the disclosure above make a little change or are modified to equivalent
The equivalent embodiment of change, as long as being the content without departing from technical solution of the present invention, the technical spirit according to the present invention is to the above
Any simple modification, equivalent change and modification that embodiment is made, in the range of still falling within technical solution of the present invention.
Claims (5)
1. based on the Raman spectrum fluorescence background subtraction method except spectrum, it is characterised in that comprise the following steps：
Gather original Raman spectrum I (λ) and the Raman shift λ of sample；
Obtain displacement Raman spectrum Is(λs), matching Raman spectrum Ic(λs) and matching Raman shift λs, by original Raman spectrum I
The top n data of (λ) are removed or rear N number of data are removed to obtain displacement Raman spectrum Is(λs), it is corresponding by original Raman spectrum I (λ)
Rear N number of data remove or top n data remove to obtain matching Raman spectrum Ic(λs), Raman shift λ rear N number of data are gone
Fall or top n data are removed to obtain matching Raman shift λs；
The matching Raman spectrum I obtained based on abovementioned stepsc(λs) and displacement Raman spectrum Is(λs), displacement Raman is calculated except spectrum R
(λs)=Ic(λs)/Is(λs)；
Displacement Raman is observed except spectrum R (λs) in positive negative peak position, determine raman spectra region, and will matching Raman spectrum Ic
(λs) in non-raman spectra region division be some SPECTRAL REGIONs；
In matching Raman spectrum Ic(λs) in remove raman spectra region, then according to the division result of SPECTRAL REGION to having removed
Matching Raman spectrum behind raman spectra region carries out piecewise fitting, obtains estimating fluorescence background B (λs)；
According to matching Raman spectrum Ic(λs) and estimation fluorescence background B (λs), calculate the Raman spectrum I after deducting fluorescence backgroundr
(λs)=Ic(λs)-B(λs)。
2. the Raman spectrum fluorescence background subtraction method according to claim 1 based on except spectrum, it is characterised in that：Data button
Except number N is less than 10.
3. the Raman spectrum fluorescence background subtraction method according to claim 1 or 2 based on except spectrum, it is characterised in that：When
Want to allow displacement Raman spectrum to lower wave number move when, the top n data for removing original Raman spectrum I (λ) obtain displacement Raman light
Compose Is(λs), the corresponding rear N number of data for removing original Raman spectrum I (λ) obtain matching Raman spectrum Ic(λs), remove Raman position
The rear N number of data for moving λ obtain matching Raman shift λs；When wanting to allow displacement Raman spectrum to move to high wave number, remove original drawing
Rear N number of data of graceful spectrum I (λ) obtain displacement Raman spectrum Is(λs), the corresponding top n number for removing original Raman spectrum I (λ)
According to obtaining matching Raman spectrum Ic(λs), the top n data for removing Raman shift λ obtain matching Raman shift λs。
4. the Raman spectrum fluorescence background subtraction method according to claim 1 based on except spectrum, it is characterised in that：Divide light
So that whether each section of Raman shift is except spectrum R (λ when composing regions) can be fitted to straight line be foundation, Raman light will be matched
Compose Ic(λs) in non-raman spectra region division be some SPECTRAL REGIONs, each section of Raman position that can be fitted to straight line
It is a SPECTRAL REGION to remove spectrum.
5. the Raman spectrum fluorescence background subtraction method according to claim 1 based on except spectrum, it is characterised in that：Using height
This linear fit carries out piecewise fitting to the matching Raman spectrum after removing raman spectra region.
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| CN109001182A (en) * | 2018-09-29 | 2018-12-14 | 西安电子科技大学 | The Raman spectrum non-destructive determination method of alcohol content in closed container |
| CN110658178A (en) * | 2019-09-29 | 2020-01-07 | 江苏拉曼医疗设备有限公司 | Fluorescence background subtraction method for Raman spectrum |
| CN110987899A (en) * | 2019-12-09 | 2020-04-10 | 西安电子科技大学 | Method for measuring content of total acid total esters in white spirit and identifying quality grade of white spirit |
| CN112161966A (en) * | 2020-09-29 | 2021-01-01 | 中国科学院长春光学精密机械与物理研究所 | Method and device for separating Raman spectrum of sample containing fluorescence spectrum |
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