CN107328758B - A kind of background estimating device and method of micro- wide field Raman image - Google Patents
A kind of background estimating device and method of micro- wide field Raman image Download PDFInfo
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- 238000001069 Raman spectroscopy Methods 0.000 title claims abstract description 78
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- 238000003332 Raman imaging Methods 0.000 claims description 10
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Abstract
The invention discloses a kind of background estimating device and methods for the micro- wide field Raman image of time series.The background estimating device introduces spectrometer in the imaging system of wide field, and while the Single wavelength wide field Raman image of wide field imaging system acquisition sample, obtains full spectrum Raman signal.First under equal white light light field, the corresponding relationship between the obtained measurement data intensity of both spectrometer background and wide field imaging system is demarcated.It while obtaining the wide field Raman image of sample, is scanned by spectrometer and obtains background image, according to identified corresponding relationship is demarcated, spectrometer background image obtained is converted into background image corresponding to imaging device.The present invention provides a kind of background estimating device and methods of micro- wide field Raman image, are with a wide range of applications.
Description
Technical field
The present invention relates to a kind of background estimating device and methods of micro- wide field Raman image.
Background technique
Raman image technology is a kind of chemical characterization imaging technique of molecular level, and the imaging of microscopic Raman wide field is even more one kind
The high time, spatial resolution Raman image means.Wide field excitation is carried out to sample by one-wavelength laser, then passes through adjustable optical filtering
Piece carries out narrow-band filtering to Raman signal, obtains the Single wavelength wide field Raman image of sample.Currently, due to the high time,
Spatial resolving power, micro- wide field Raman image technology are widely used in biochemistry, in-situ characterization of electrochemical process etc.
Front line science field.
Since the background in Raman image causes the contrast of Raman image to reduce, more accurate Raman image is obtained
It needs to be deducted background.Different from scanning imagery, existing micro- wide field Raman Imaging System is carrying out time series
It, can full spectrum Raman signal for reference, it is difficult to which background estimating is carried out to its Raman image when Single wavelength wide field is imaged.For
Current micro- this technological deficiency of wide field Raman Imaging System is solved, the invention proposes in acquisition Single wavelength wide field Raman figure
While picture, the full spectrum Raman signal using spectrometer collection sample wide field excitation area is for estimating wide field imaging device institute
Obtain the background in Raman image.
Summary of the invention
It is an object of the invention to overcome the deficiency of the prior art, and the background for providing a kind of micro- wide field Raman image is estimated
Counter device and method, the wide field Raman image for obtaining sample simultaneously, background estimating is carried out to background Raman image, is had extensive
Application value.
The technical solution adopted by the present invention to solve the technical problems are as follows:
A kind of background estimating device of micro- wide field Raman image, for estimating the back of time series wide field Raman image
Scape；, wide field Raman Imaging System the micro- wide field Raman image background estimating device includes two parts: 1)；2), it is based on spectrum
The background estimating device of instrument.
The wide field Raman Imaging System includes wide field excitation light source, lens group, adjustable optical filter and imaging device；It is described
Wide field excitation light source carries out wide field excitation to sample, generates Raman signal；For the Raman signal by the lens group, process is adjustable
The imaging device is acted on after optical filter narrow-band filtering, obtains wide field Raman image；
The background estimating device includes spectrometer, lens group, scanning galvanometer and spectroscope；The spectroscope believes Raman
Number collecting optical path is divided into two tunnels, which acts on imaging device into the wide field imaging system and described respectively
Spectrometer；
Sample generates Raman signal after wide field excites, which passes sequentially through the spectroscope, lens group, described sweeps
It retouches galvanometer and is transmitted to the spectrograph slit；The scanning galvanometer is turntable, and is made by deflection within a certain angle
The corresponding picture of the spectrograph slit moves in the wide field excitation area.
The device further includes control system, and the control system is connect with the spectrometer and imaging device respectively, and is controlled
Make the time for exposure of the spectrometer and wide field imaging system and the deflection angle of the scanning galvanometer.
As a preference, the spectroscope can use semi-permeable and semi-reflecting mirror.
A kind of background estimating method of micro- wide field Raman image, using a kind of above-mentioned background estimating device, including with
Lower step:
Step 1, both spectrometer and imaging device intensity data corresponding relationship obtained are carried out using equal white light
Calibration, and following corresponding relationship is obtained by fitting of a polynomial:
IW=anIS n+an-1IS n-1+…+a1IS+a0+σ (1)
Wherein, IWThe spectrum that the Single wavelength wide field Raman image recorded by imaging device obtains after data conversion,
The wide field excitation area is scanned by spectrometer and obtains spectrum IS, a0,a1,…,anFor the multinomial coefficient that fitting obtains, σ is
Error of fitting item；
Step 2, it by carrying out wide field excitation to sample, and is set as the adjustable optical filter to pass through wavelength X1, and
Obtaining wavelength in the imaging device is λ1Wide field Raman image, the wide field at available face slit center on spectrometer
In excitation area on a line all the points raman spectral signal；
Scanning galvanometer deflection and inswept entire wide field excitation area are controlled, obtains spectrometer in entire wide field excitation area
High-spectral data collection Data (i, j), wherein (i, j) be sample on corresponding points Cartesian coordinate position；For λ1Place is special
The high-spectral data collection Data (i, j) in sign peak section makees baseline fitting, obtains the baseline background on this feature peak section, and time
After going through entire data set, background image b (i, j) measured by spectrometer is obtained；
B (i, j) is substituted into formula (1), as shown in formula (2), obtain corresponding in the Raman image of wide field background image B (i,
J):
B (i, j)=anb(i,j)n+an-1b(i,j)n-1+…+a1b(i,j)+a0 (2)
As a preference, the spectrometer is progressive scan, interlacing scan for the scanning mode of wide field excitation area
Or equidistant scanning.In the selection of specific scanning mode, it is adjusted according to different Raman systems.The mode of the progressive scan
Change slow Raman system suitable for background, it is very fast that the interlacing scan or the mode equidistantly scanned are suitable for background variation
Raman system.
As a preference, when the scanning mode is using interlacing scan or progressive scan, by Gaussian kernel interpolation or double
The interpolation algorithms such as linear interpolation restore the background image data of non-sweep test.
As a preference, the time for exposure of the imaging device is consistent with the time for exposure of spectrometer.
Beneficial effects of the present invention are as follows:
The background estimating device introduces spectrometer in the imaging system of wide field, and in wide field imaging system acquisition sample
While the Raman image of Single wavelength wide field, the full spectrum Raman signal of sample is obtained.First under uniform light field, calibration spectrometer and
Both imaging devices intensity data corresponding relationship obtained.While obtaining the Single wavelength wide field Raman image of sample, lead to
The full spectrum Raman signal that spectrometer scanning obtains sample is crossed, data processing is carried out to obtained full spectrum Raman signal and is obtained
Background image at specific peak position.Finally, according to identified corresponding relationship is demarcated, by spectrometer background image obtained
Be converted to background image corresponding with the Raman image that imaging device is recorded.
The background estimating device and method the wide field Raman image for obtaining sample simultaneously, to the background of Raman image into
Row assessment, enables the information of the more accurate response sample of wide field Raman image obtained, is with a wide range of applications.
Invention is further described in detail with reference to the accompanying drawings and embodiments；But a kind of micro- wide field of the invention is drawn
The background estimating device of graceful image is not limited to the embodiment.
Detailed description of the invention
Fig. 1 is structural schematic diagram of the invention.
Specific embodiment
Embodiment:
It is shown in Figure 1, the background estimating device of a kind of micro- wide field Raman image of the invention, for assessing time sequence
Background in the Raman image of col width field；, wide field Raman the micro- wide field Raman image background estimating device includes two parts: 1)
Imaging system；2), the background estimating device based on spectrometer.
The wide field Raman Imaging System includes that example platform 1, object lens 3, combined filter piece 4, divides wide field excitation light source 2
Light microscopic 5, tube lens 6,4f lens group 7, adjustable optical filter 8, imaging device 9；The background estimating device includes 4f lens group
10, scanning galvanometer 11, collecting lens 12, spectrometer 13.The scanning galvanometer 11 is turntable, control scanning galvanometer 11
Deflection is two-way on direction such as figure example platform 1 so that as moving in the excitation area of wide field corresponding to spectrograph slit 14
Shown in arrow.The wide field excitation light source 2 carries out excitation to sample and generates Raman signal；The wide field Raman signal is by diagram light
Road is divided by the spectroscope 5, is transmitted separately to imaging device 9 and spectrometer 13；
Apparatus of the present invention further include control system, the control system respectively with the imaging device 9 and spectrometer 13,
And control the time for exposure of the imaging device 9 and spectrometer 13 and the deflection angle of the scanning galvanometer 11.
A kind of background estimating method of micro- wide field Raman image, using a kind of above-mentioned background estimating device, including with
Lower step:
Step 1, using equal white light, to both spectrometer 13 and imaging device 9 intensity data corresponding relationship obtained
It is demarcated；And following corresponding relationship is obtained by fitting of a polynomial:
IW=anIS n+an-1IS n-1+…+a1IS+a0+σ (1)
Wherein, IWThe spectrum that the Single wavelength wide field Raman image recorded by imaging device obtains after data conversion,
The wide field excitation area is scanned by spectrometer and obtains spectrum IS, a0,a1,…,anFor the multinomial coefficient that fitting obtains, σ is
Error of fitting item；
Step 2, wide field excitation is carried out to sample, and is set as the adjustable optical filter 8 to pass through wavelength X1, and institute
Stating imaging device 9 and obtaining wavelength is λ1Wide field Raman image, on spectrometer 13 in available face spectrograph slit 14
In the wide field excitation area of the heart on a line all the points raman spectral signal；
The deflection of scanning galvanometer 11 and inswept entire wide field excitation area are controlled, spectrometer 13 is made to obtain entire wide field lasing region
High-spectral data collection Data (i, j) on domain, wherein (i, j) is the Cartesian coordinate position of corresponding points on sample；For λ1Institute
High-spectral data collection Data (i, j) in characteristic peak section makees baseline fitting, obtains the baseline background on this feature peak section, and
After traversing entire data set, obtain background image b (i, j)；
B (i, j) is substituted into formula (1), as shown in formula (2), obtain corresponding in the Raman image of wide field background image B (i,
J):
B (i, j)=anb(i,j)n+an-1b(i,j)n-1+…+a1b(i,j)+a0 (2)
Scanning mode of the spectrograph slit 14 in the excitation area of wide field is progressive scan, interlacing scan or equidistant
Scanning.The scanning mode using interlacing scan or it is equidistant when, pass through the interpolation algorithms such as Gaussian kernel interpolation or bilinear interpolation
The background image data of non-sweep test is supplemented.When the exposure of time for exposure of the imaging device 9 and spectrometer 13
Between it is consistent.
In attached drawing 1, the arrow direction along figure of wide field exciting light 2 is incident, the 4 (Edge of edge filter installed by 45 degree
Filter reach sample surfaces by object lens after) reflecting.Combined filter piece 4 (Edge filter and Long pass filter)
For filtering out stray light and Reyleith scanttering light.The Raman signal carries out narrowband filter by adjustable optical filter (Tunable Filter)
Wave can obtain Single wavelength wide field Raman image on imaging device.
4f lens group 7 is used to carry out image planes transfer to by microscope sleeve lens (Tube lens), facilitates the adjustable filter of installation
Mating plate 8 and imaging device 9.4f lens group 10 is mainly used for increasing the adjustable-angle of scanning galvanometer 11, i.e. increase spectrometer is narrow
Scanning range of the picture of seam on sample surfaces.When scanning galvanometer 11 is in initial position, i.e., optical path is collected with spectrometer 13
It is shown in solid on example platform 1 in the position of spectral slit imaging such as figure when primary optical axis is in 45 degree of Angle Positions；When scanning is shaken
After mirror 11 deflects certain angle, shown in position as dashed lines, sample in the position of 14 imaging of spectrograph slit such as figure at this time
Shown in dotted line on platform 1.
Above-described embodiment is only used to further illustrate a kind of background estimating device of micro- wide field Raman image of the invention
And method, but the invention is not limited to embodiments, according to the technical essence of the invention to above embodiments optical path, optics
Any simple modification, equivalent change and modification made by element, fall within the scope of protection of technical solution of the present invention.
Claims (5)
1. a kind of background estimating device of micro- wide field Raman image, for estimating the back of time series wide field Raman Imaging System
Scape intensity；, wide field Raman Imaging System the micro- wide field Raman image background estimating device includes two parts: 1)；2) it, is based on
The background estimating device of spectrometer；It is characterized by:
The background estimating device includes spectrometer, lens group, scanning galvanometer and spectroscope；The spectroscope draws the wide field
The Raman signal of graceful imaging system collects optical path and is divided into two tunnels, bis- road Raman signal of Shi Gai act on respectively to the wide field Raman at
As the imaging device and the spectrometer in system；
Sample generates Raman signal after wide field excites, which passes sequentially through the spectroscope, lens group, described sweeps
Retouch the spectrograph slit that galvanometer is transmitted to the spectrometer；The scanning galvanometer is turntable, and by certain angle
Intrinsic deflection moves the corresponding picture of the spectrograph slit of the spectrometer in the excitation area of wide field.
2. a kind of background estimating device of micro- wide field Raman image according to claim 1, it is characterised in that: further include
Control system, the control system are connect with the imaging device of the spectrometer and the wide field Raman Imaging System respectively, and
Control the imaging device of the spectrometer and the wide field Raman Imaging System time for exposure and the scanning galvanometer it is inclined
Gyration.
3. a kind of background estimating method of micro- wide field Raman image, which is characterized in that including any one of claim 1 to 2 institute
The background estimating device stated, comprising the following steps:
Step 1, both spectrometer and imaging device intensity data corresponding relationship obtained are demarcated using equal white light；
And following corresponding relationship is obtained by fitting of a polynomial:
IW=anIS n+an-1IS n-1+…+a1IS+a0+σ (1)；
Wherein, IWThe spectrum that the Single wavelength wide field Raman image recorded by imaging device obtains after data conversion, passes through light
Spectrometer scans the wide field excitation area and obtains spectrum IS, a0,a1,…,anFor the multinomial coefficient that fitting obtains, σ is that fitting misses
Poor item；
Step 2, it by carrying out wide field excitation to sample, and sets the adjustable optical filter of the wide field Raman Imaging System to
Pass through wavelength X1, and obtaining wavelength in the imaging device is λ1Wide field Raman image, available face is narrow on spectrometer
In the wide field excitation area at seam center on a line all the points raman spectral signal；
Scanning galvanometer deflection and inswept entire wide field excitation area are controlled, the high-spectral data in entire wide field excitation area is obtained
Collect Data (i, j), wherein (i, j) is the Cartesian coordinate position of corresponding points on sample；For characteristic peak λ1Place characteristic peak area
Between high-spectral data collection Data (i, j) make baseline fitting, obtain the baseline background on this feature peak section, and entire in traversal
After data set, background image b (i, j) measured by spectrometer is obtained；
B (i, j) is substituted into formula (1), as shown in formula (2), obtains the background image B (i, j) corresponded in the Raman image of wide field:
B (i, j)=anb(i,j)n+an-1b(i,j)n-1+…+a1b(i,j)+a0 (2)。
4. a kind of background estimating method of micro- wide field Raman image according to claim 3, it is characterised in that: the light
Spectrometer is progressive scan, interlacing scan or equidistant scanning for the scanning mode of wide field excitation area.
5. a kind of background estimating method of micro- wide field Raman image according to claim 4, it is characterised in that: described to sweep
When retouching mode using interlacing scan or equidistant scanning, by Gaussian kernel interpolation or bilinear interpolation to the background of non-sweep test
Image data is restored.
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Effective date of registration: 20231130 Address after: Unit 205, No. 1-1 Pugong Shanxi Road, Software Park Phase III, Xiamen City, Fujian Province, 361000 Patentee after: Xiamen Vision Technology Co.,Ltd. Address before: 361000 22, No. 422, Siming South Road, Siming District, Xiamen City, Fujian Province Patentee before: Wang Lei |