CN107884388A - A kind of micro-Raman spectroscopy and its application method of fast automatic focusing - Google Patents
A kind of micro-Raman spectroscopy and its application method of fast automatic focusing Download PDFInfo
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- 238000001530 Raman microscopy Methods 0.000 title claims abstract description 17
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- 238000001069 Raman spectroscopy Methods 0.000 claims abstract description 21
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- 238000001228 spectrum Methods 0.000 description 5
- 238000004458 analytical method Methods 0.000 description 4
- 238000011160 research Methods 0.000 description 3
- 230000005540 biological transmission Effects 0.000 description 2
- 230000008878 coupling Effects 0.000 description 2
- 238000010168 coupling process Methods 0.000 description 2
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- 238000010183 spectrum analysis Methods 0.000 description 2
- 238000004364 calculation method Methods 0.000 description 1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/65—Raman scattering
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/01—Arrangements or apparatus for facilitating the optical investigation
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/84—Systems specially adapted for particular applications
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- G—PHYSICS
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- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
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- G02B21/241—Devices for focusing
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/01—Arrangements or apparatus for facilitating the optical investigation
- G01N2021/0106—General arrangement of respective parts
- G01N2021/0112—Apparatus in one mechanical, optical or electronic block
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/01—Arrangements or apparatus for facilitating the optical investigation
- G01N2021/0181—Memory or computer-assisted visual determination
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Abstract
The micro-Raman spectroscopy and its application method of a kind of fast automatic focusing disclosed by the invention, wherein the micro-Raman spectroscopy of the fast automatic focusing includes micro imaging system, Raman spectrum system, control device and computer system, control device is connected with computer system, micro imaging system is located above control device, and Raman spectrum system is located at micro imaging system side.The present invention is compact-sized, it is full-featured, can automatic focusing, obtain sample regional area spectral information and can obtain complete object image information under high-resolution microscope, and Raman spectral information is obtained, greatly improve the testing efficiency and test effect of Raman spectrometer.
Description
Technical field
The present invention relates to Raman spectrum detection technique field, relates more specifically to a kind of microscopic Raman of fast automatic focusing
Spectrometer and its application method.
Background technology
Raman spectrum is the vibration and the information rotated by obtaining molecule, and applied to one kind point of molecular structure research
Analysis method, Raman spectrum can realize the qualitative analysis and quantitative analysis to material by the vibration of material and rotation information.Draw
Its is simple in construction for graceful spectrometer, and measurement quickly, efficiently and accurately easy to operate can obtain high-resolution spectral information, although can be with
Get the spectral information of sample surfaces, but Raman spectrometer collection be sample surfaces averaged spectrum, can not get
Spectral information corresponding to regional area or partial points in sample.
Meanwhile traditional microscope can realize the observation to small items, but it is needed by eyepiece subjective observation mesh
Mark thing and manually adjusting knob is focused, this manually focusing mode wastes time and energy and easily produces human error.And
Although the clearly image information under object high-resolution can be got under high resolution microscope, high score can not be got
Object regional area or the internal structural information of partial points under resolution, and can not obtain under high-resolution microscope one it is complete
Whole object image information.Therefore, be badly in need of one kind can automatic focusing, obtain sample regional area spectral information and can be in height
The Raman spectrometer of complete object image information is obtained under the microscope of resolution ratio.
The content of the invention
The present invention provides a kind of micro-Raman spectroscopy and its application method of fast automatic focusing, to solve existing Raman
Spectrometer needs manual focus, can not obtain testee topography information and can not be obtained under the microscope of high-resolution rate
The problem of taking whole object image information.
In order to solve the above technical problems, a kind of micro-Raman spectroscopy of fast automatic focusing provided by the invention, including
Micro imaging system, Raman spectrum system, control device and computer system, the control device are connected with computer system,
The micro imaging system is located above control device, and the Raman spectrum system is located at micro imaging system side.
Preferably, the control device includes objective table, the first motor, the second motor, automatic focusing mechanism, coarse adjustment rotation
Turn round, control cabinet and base, the control cabinet are connected with computer system, first motor, the second motor and auto-focusing
Device is connected with control cabinet circuit respectively, the objective table and the first motor and the second motor connection and is installed on base
Side, the coarse adjustment turn-knob are connected with objective table.
Preferably, the control cabinet includes communication module, the single-chip microcomputer being connected with the communication module, with the single-chip microcomputer
The driver of connection, and the power supply being connected with the driver.
Preferably, the micro imaging system includes LED, the first collimating mirror, the first light splitting piece, the second light splitting piece, pipe
Road, CCD camera and microcobjective, the microcobjective are located above the objective table, and first light splitting piece is located at micro- thing
Above mirror and horizontal by 45 degree of angles, second light splitting piece is located above the first light splitting piece and is mutually with the first light splitting piece
90 degree of angles, the pipeline are located above the second light splitting piece, and the CCD camera is located at directly over pipeline, first collimating mirror
The second light splitting piece downside side is vertically arranged in, the LED is arranged on the first collimating mirror and deviates from the second light splitting piece side.
Preferably, the Raman spectrum system includes processing unit, speculum, the 3rd light splitting piece, the second collimating mirror, coupling
Lens, the first fibre-optical probe, the second fibre-optical probe and optical filter, the processing unit is connected with the computer system, described
3rd light splitting piece is located at the first light splitting piece downside side, and the optical filter, coupled lens and the first fibre-optical probe are successively
It is arranged on the 3rd light splitting piece and deviates from the first light splitting piece side, the speculum is located at directly over the 3rd light splitting piece, and described second is accurate
Straight mirror, the second fibre-optical probe are successively set on speculum downside side, first fibre-optical probe and the second fibre-optical probe with
The processing unit connection.
Preferably, the computer system includes display screen and connected computer.
In order to solve the above technical problems, present invention also offers a kind of microscopic Raman of fast automatic focusing as described above
The application method of spectrometer, comprises the following steps：
Step 1：Sample is placed on objective table, starts computer and corresponding software, selects IMAQ auto-focusing
Pattern；
Step 2：Start LED and CCD camera；
Step 3：Automatic data collection image information, the computer judge whether the evaluation function value of image is maximum；If it is not,
Into step 4；If so, into step 5；
Step 4：PLC drives the objective table movement, into step 3；
Step 5：Optimal focus image is stored, by splicing to several optimal focus images, obtains complete image；
Step 6：Select area-of-interest or point to be used as measured zone from the complete image, drive the objective table
The measured zone is moved under microcobjective；
Step 7：Start Raman spectrum system, gather the Raman spectral information of the measured zone.
A kind of micro-Raman spectroscopy of fast automatic focusing provided by the present invention, mainly including micro imaging system,
Raman spectrum system, control device and computer system, pass through mutual synthetic operation between them, you can to pass through micro-imaging
The microcobjective of auto-focusing obtains high-resolution picture rich in detail as research object in system, can pass through image selection again
Regional area or partial points carry out the corresponding spectral information of quick and easy acquisition sample, are obtained by auto-focusing microscope
Spectral information can be with accurately detecting to the different layer depth of sample and the Raman signal of focal plane, and it has good spatial resolution
And control the microcobjective of auto-focusing to be accurately positioned the ad-hoc location of sample by control device, gather the region
Raman signal, it is achieved thereby that the combination of collection and the spectrum analysis of high-definition picture to sample under the microscope.This hair
It is bright compact-sized, it is full-featured, can automatic focusing, obtain sample regional area spectral information and can be high-resolution aobvious
Complete object image information is obtained under micro mirror, and carries out Raman spectrum analysis, greatly improves the test effect of Raman spectrometer
Fruit.
Brief description of the drawings
Fig. 1 is a kind of micro-Raman spectroscopy structural representation of fast automatic focusing provided by the invention；
Fig. 2 is a kind of application method flow chart of the micro-Raman spectroscopy of fast automatic focusing provided by the invention.
Wherein, micro imaging system 1, Raman spectrum system 2, control device 3, computer system 4, LED 11, first are accurate
Straight mirror 12, the first light splitting piece 13, the second light splitting piece 14, pipeline 15, CCD camera 16, microcobjective 17, processing unit 21, reflection
Mirror 22, the 3rd light splitting piece 23, the second collimating mirror 24, coupled lens 25, the first fibre-optical probe 26, the second fibre-optical probe 27, optical filtering
Piece 28, objective table 31, the first motor 32, the second motor 33, automatic focusing mechanism 34, coarse adjustment turn-knob 35, control cabinet 36, base
37th, display screen 41, computer 42, sample a.
Embodiment
With reference to the accompanying drawings and examples, the embodiment of the present invention is described in further detail.Implement below
Example is used to illustrate the present invention, but is not limited to the scope of the present invention.
Refer to shown in Fig. 1, a kind of micro-Raman spectroscopy of fast automatic focusing provided by the invention, including it is micro- into
As system 1, Raman spectrum system 2, control device 3 and computer system 4, control device 3 is connected with computer system 4, micro-
Imaging system 1 is high-resolution micro imaging system, and above control device 3, Raman spectrum system 2 is located at micro-imaging system
Unite 1 side.Wherein, control device 3 includes objective table 31, the first motor 32, the second motor 33, automatic focusing mechanism 34, coarse adjustment
Turn-knob 35, control cabinet 36 and base 37, control cabinet 36 are connected with computer system 4, the first motor 32, the second motor 33 and automatic
Focusing mechanism 34 is connected with the circuit of control cabinet 36 respectively, and objective table 31 is connected and is installed in the first motor 32 and the second motor 33
The top of base 37, coarse adjustment turn-knob 35 are connected with objective table 31.
Further, control cabinet 36 includes communication module, the single-chip microcomputer being connected with communication module, the drive being connected with single-chip microcomputer
Dynamic device, and the power supply being connected with driver.
Micro imaging system 1 includes LED 11, the first collimating mirror 12, the first light splitting piece 13, the second light splitting piece 14, pipeline
15th, CCD camera 16 and microcobjective 17, microcobjective 17 are located at the top of objective table 31, and the first light splitting piece 13 is located at microcobjective
Above in the of 17 and horizontal by 45 degree of angles, the second light splitting piece 14 is located above the first light splitting piece 13 and mutual with the first light splitting piece 13
Into 90 degree of angles, pipeline 15 is located at the top of the second light splitting piece 14, and CCD camera 16 is located at directly over pipeline 15, the first collimating mirror 12
The downside side of the second light splitting piece 14 is vertically arranged in, LED is arranged on the first collimating mirror 12 and deviates from the side of the second light splitting piece 14.
Raman spectrum system 2 includes processing unit 21, speculum 22, the 3rd light splitting piece 23, the second collimating mirror 24, coupling thoroughly
Mirror 25, the first fibre-optical probe 26, the second fibre-optical probe 27 and optical filter 28, processing unit 21 are connected with computer system 4, and the 3rd
Light splitting piece 23 is located at the downside side of the first light splitting piece 13, and optical filter 28, the fibre-optical probe 26 of coupled lens 25 and first are set successively
Put and deviate from the side of the first light splitting piece 13 in the 3rd light splitting piece 23, speculum 22 is located at directly over the 3rd light splitting piece 23, and both are parallel
Set, the second collimating mirror 24, the second fibre-optical probe 27 are successively set on the downside side of speculum 22, the He of the first fibre-optical probe 26
Second fibre-optical probe 27 is connected with processing unit 21.
Computer system 4 includes display screen 41 and connected computer 42.
In order to solve the above technical problems, present invention also offers a kind of microscopic Raman of fast automatic focusing as described above
The application method of spectrometer, comprises the following steps：
Step 1 S1：Sample is placed on objective table, starts computer and corresponding software, selects IMAQ automatically right
Burnt pattern；
Step 2 S2：Start LED and CCD camera；
Step 3 S3：Automatic data collection image information, the computer judge whether the evaluation function value of image is maximum；If
It is no, into step 4；If so, into step 5；
Step 4 S4：PLC drives the objective table movement, into step 3；
Step 5 S5：Optimal focus image is stored, by splicing to several optimal focus images, obtains complete graph
Picture；
Step 6 S6：Select area-of-interest or point to be used as measured zone from the complete image, drive the loading
The measured zone is moved under microcobjective by platform；
Step 7 S7：Start Raman spectrum system, gather the Raman spectral information of the measured zone.
The specific work process of the present invention is as follows：Autofocus mode is first turned on, clicks on the computer system of display screen
Autofocus mode, start LED 11, incident light passes through the first collimating mirror 12, the second light splitting piece 14 and the first light splitting piece 13
After being reflected into microcobjective 17, light source converges to sample surfaces, and caused reflected light is after micro- thing 17 through the first light splitting
After piece 13, the transmission of the second light splitting piece 14, the image that the progress moment of CCD camera 16 with high-pass filter is entered through pipeline 15 is adopted
Collection, the picture signal of collection are shown on computer display 41 by the processing of computer 42；Computer 42 is according to the clear of image
Clear degree function evaluation of estimate, evaluation of estimate is bigger, and image is more clear, and instruction is transferred to control cabinet 36 with computer 42 and driving is automatic right
Coke installation 34 drives the objective table 31 for being loaded with sample a to move up or down, and now micro imaging system 1 gathers sample image and passed
It is defeated by computer system 4 to calculate, generates image function evaluation of estimate, is contrasted by the size of function evaluation of estimate, 42, computer refers to
Order is transferred to control device 3 and drives the objective table 31 for being loaded with sample to move up and down to obtain different focal planes, therefrom finds
The maximum focal plane position of function evaluation of estimate can obtain optimal focus image, now close focal modes, auto-focusing
Terminate.The optimal focal plane collection of several different images is single-chip microcomputer instruction being transferred to by computer 42 in control cabinet 36,
MCU driving the first motor 32 and the second motor 33 drive the left-right and front-back of objective table 31 movement for being loaded with sample a, now calculate
Machine 42 issues instructions to micro imaging system 1 and gathers optimal focus image under microscope and preserved by computer 42.
Secondly, image mosaic pattern is opened：The image mosaic pattern of the computer system of selection display screen 41 is clicked on, is used
Several optimal focus images that computer picture splicing function preserves computer are spliced into the complete full resolution pricture letter of a width
Breath.
Finally, spectra collection pattern is opened：The spectra collection pattern of the computer system of selection display screen 41 is clicked on, is passed through
The image information of upper two steps collection, selects local region or point on image, now the output signal of computer 42 and instruction
It is transferred to Raman spectrum system 2 and control device 3, communication module in control device 3, single-chip microcomputer, driver, the control of power supply composition
Case 36 processed receive computer instruction motor 19 and motor 20 with dynamic object stage the regional area selected on image or
The position of point is moved under microcobjective 17, now, Raman module and the processing unit of spectrometer composition in Raman spectrum system 2
21 export continuous laser after the second collimating mirror 24 collimates, speculum 22 reflects by the 3rd light splitting by the second fibre-optical probe 27
The light splitting piece 13 of piece 23 and first is reflected into microcobjective 17 and converges to sample a surfaces, and caused reflected light passes through microcobjective
17 reflex to the 3rd light splitting piece 23 through the first light splitting piece 13, by optical filter 28 and coupled lens after the transmission of the 3rd light splitting piece 23
25 reach the first fibre-optical probe 26, finally enter Raman module and the processing unit 21 of spectrometer composition, the spectral information of acquisition
Computer 42 is transferred to, computer 42 is shown on display screen 41 by processing, completes the collection of spectral information.
Because traditional micro- auto-focusing is that operating personnel are focused by eyepiece manual coarse adjustment and fine tuning knob, the present invention
It is that processing calculating is carried out according to the objective definition evaluating of image by computer, can be realized to sample by control system
Product carry out completely fast automatic focusing device, due to being digitized processing, thus the device have objectivity accuracy and
The realization focusing of speed per hour and the full automation that need not manually participate in, the image for focusing to obtain is accurate and clear, obtains
The clear sample image of high-resolution can also save as the subsequent analysis of research and processing is prepared.Gathered by auto-focusing
To after optimal focus image, at the same by image mosaic obtain micro- lower sample a panel height differentiate complete display image believe
Breath.In the present invention except sample need be manually loaded in addition to, from focusing, IMAQ can to spectra collection and image mosaic
It is automatically performed.
Because Raman spectrum can be quick, simple, repeatable and undamaged qualitative fixed to organic matter and inorganic matter progress
The analysis of amount and required sample size is small, although auto-focusing microscope can obtain high-resolution image information automatically, not
The internal structural information of the corresponding sample of interest in image information can be obtained, although Raman spectrum can obtain the spectrum of sample
Information is internal structural information, but can not observe the image information corresponding to sample.The present invention uses the microscope of auto-focusing
Device is combined with Raman spectrometer, passes through Raman spectrum system 2, high-resolution micro imaging system 1, control device 3 and meter
4 mutual synthetic operation of calculation machine system, you can to obtain high-resolution picture rich in detail as grinding by the microscope of auto-focusing
Study carefully object, area-of-interest or point-of-interest can be selected be used as sample by Raman spectrum system 2 again, and obtaining quickly and easily
Spectral information corresponding to sampling condition, realize that the spectral information that auto-focusing microscope obtains can be different to sample with accurately detecting
Layer depth and focal plane Raman signal.The present invention has good spatial resolution and adjusted by the microscope of auto-focusing
Section system can be accurately positioned the ad-hoc location of sample, and gather the Raman signal of the microcell, it is achieved thereby that to sample aobvious
The combination of collection and the spectrum analysis of high-definition picture under micro mirror.
Finally, the present processes are only preferable embodiment, are not intended to limit the scope of the present invention.It is all
Within the spirit and principles in the present invention, any modification, equivalent substitution and improvements made etc., the protection of the present invention should be included in
Within the scope of.
Claims (7)
1. a kind of micro-Raman spectroscopy of fast automatic focusing, it is characterised in that including micro imaging system, Raman light pedigree
System, control device and computer system, the control device are connected with the computer system, and the micro imaging system is located at
Above the control device, the Raman spectrum system is located at the micro imaging system side.
A kind of 2. micro-Raman spectroscopy of fast automatic focusing according to claim 1, it is characterised in that the control
Device includes objective table, the first motor, the second motor, automatic focusing mechanism, coarse adjustment turn-knob, control cabinet and base, the control
Case is connected with the computer system, first motor, the second motor and automatic focusing mechanism respectively with the control cabinet
Circuit connects, and the objective table and first motor and the second motor connection and is installed in above the base, described thick
Turn-knob is adjusted to be connected with the objective table.
A kind of 3. micro-Raman spectroscopy of fast automatic focusing according to claim 2, it is characterised in that the control
Case includes communication module, the single-chip microcomputer being connected with the communication module, the driver being connected with the single-chip microcomputer, and with it is described
The power supply of driver connection.
4. the micro-Raman spectroscopy of a kind of fast automatic focusing according to claim 3, it is characterised in that described micro-
Imaging system includes LED, the first collimating mirror, the first light splitting piece, the second light splitting piece, pipeline, CCD camera and microcobjective, institute
State microcobjective to be located above the objective table, first light splitting piece is located above the microcobjective and horizontal by 45
Angle is spent, second light splitting piece is located above first light splitting piece and is mutually 90 degree of angles, institute with first light splitting piece
State pipeline to be located above second light splitting piece, the CCD camera is located at directly over the pipeline, and first collimating mirror is vertical
The second light splitting piece downside side is arranged on, the LED is arranged on first collimating mirror away from the described second light splitting
Piece side.
A kind of 5. micro-Raman spectroscopy of fast automatic focusing according to claim 4, it is characterised in that the Raman
Spectroscopic system includes processing unit, speculum, the 3rd light splitting piece, the second collimating mirror, coupled lens, the first fibre-optical probe, second
Fibre-optical probe and optical filter, the processing unit are connected with the computer system, and the 3rd light splitting piece is located at described first
Light splitting piece downside side, the optical filter, coupled lens and the first fibre-optical probe are successively set on the 3rd light splitting piece back of the body
From the first light splitting piece side, the speculum is located at directly over the 3rd light splitting piece, and second collimating mirror, the second optical fiber are visited
Head be successively set on towards speculum downside side, first fibre-optical probe and second fibre-optical probe with it is described
Processing unit connects.
A kind of 6. micro-Raman spectroscopy of fast automatic focusing according to claim 1, it is characterised in that the calculating
Machine system includes display screen and connected computer.
7. a kind of application method of the micro-Raman spectroscopy of fast automatic focusing as described in claim 1 to 6, its feature exist
In comprising the following steps：
Step 1：Sample is placed on objective table, starts computer and corresponding software, selects IMAQ auto-focusing mould
Formula；
Step 2：Start LED and CCD camera；
Step 3：Automatic data collection image information, the computer judge whether the evaluation function value of image is maximum；If it is not, into
Step 4；If so, into step 5；
Step 4：PLC drives the objective table movement, into step 2；
Step 5：Optimal focus image is stored, by splicing to several optimal focus images, obtains complete image；
Step 6：Select area-of-interest or point to be used as measured zone from the complete image, drive the objective table by institute
Measured zone is stated to be moved under microcobjective；
Step 7：Start Raman spectrum system, gather the Raman spectral information of the measured zone.
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Cited By (10)
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|---|---|---|---|---|
| CN108259749A (en) * | 2018-02-05 | 2018-07-06 | 西安工业大学 | A kind of auto-focusing experimental system |
| CN108717057A (en) * | 2018-05-31 | 2018-10-30 | 中央民族大学 | A kind of portable surface enhancing Raman spectrometer and its measurement method |
| CN109459848A (en) * | 2018-10-31 | 2019-03-12 | 精微视达医疗科技（武汉）有限公司 | Sonde-type co-focusing micro-endoscope, its focusing mechanism and method |
| CN112825622A (en) * | 2020-08-31 | 2021-05-21 | 深圳迈瑞生物医疗电子股份有限公司 | Sample image capturing method and sample image capturing apparatus |
| CN112835630A (en) * | 2021-02-04 | 2021-05-25 | 佛山格捷锐信息技术有限公司 | Unattended quick starting method and device for handheld XRF spectrometer |
| CN113008862A (en) * | 2021-02-03 | 2021-06-22 | 中国海洋大学 | Underwater Raman probe and underwater detection system |
| CN113467067A (en) * | 2021-05-24 | 2021-10-01 | 南京工程学院 | Automatic focusing method and device of microscopic imaging system based on multi-image area relation |
| CN113552712A (en) * | 2021-08-23 | 2021-10-26 | 奥谱天成（厦门）光电有限公司 | Automatic focusing system and focusing method for micro-Raman spectrometer |
| US11874526B2 (en) | 2020-11-02 | 2024-01-16 | Industrial Technology Research Institute | Textile detection module, textile sorting system and using method thereof |
| WO2024119480A1 (en) * | 2022-12-09 | 2024-06-13 | 深圳华大智造科技股份有限公司 | Focusing control method and related apparatus |
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