CN103411957B - High-space resolution twin shaft confocal spectrum micro imaging method and device - Google Patents
High-space resolution twin shaft confocal spectrum micro imaging method and device Download PDFInfo
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Abstract
The invention belongs to spectral measurement methods field, relate to a kind of high-space resolution twin shaft confocal spectrum formation method and device.Core concept of the present invention is organically blended at twin shaft confocal microscopy and spectrographic detection technology, normal outcast Reyleith scanttering light is utilized to carry out aided detection, the spatial resolution of raising system, and there are three dimension scale tomographic map, spectrographic detection and microcell collection of illustrative plates tomography three kinds of detection modes.It is high that the present invention has spatial resolution, accurate positioning, spectral detectivity advantages of higher, has wide practical use in fields such as biomedicine, physical material, petrochemical complex, environmental sciences, and the high-space resolution for microcell three-dimensional geometry position and spectrum detects and provides new way.
Description
Technical field
The invention belongs to spectral measurement methods field, relate to a kind of high-space resolution twin shaft confocal spectrum micro imaging method and device, can be used for three-dimensional appearance reconstruct and the microscopic spectrum detection of all kinds of sample.
Technical background
Confocal laser Raman spectrum microtechnic is the basic means of a kind of extremely important sample structure and constituent analysis.This technology had both inherited the high-resolution tomography feature of confocal microscopy, spectral analysis can be carried out again to sample, thus confocal laser Raman spectrum microtechnic is made to take the course of its own in spectrum test field, and the important means developed rapidly as a kind of extremely important sample structure and constituent analysis, makes it in the leading basic research of the subjects such as being widely used in ﹑ Sheng Wu ﹑ medical science, Wu Li ﹑ geology, court's evidence obtaining, criminal investigation.
Existing laser Raman spectrometer manufacturer such as the Horiba of France, the Reinshaw of Britain, the Sai Mo of the U.S. fly, adopt single shaft microscopic system, this causes Rayleigh scattering light light intensity excessive, reduce the observability of biological sample, according to Rayleigh scattering rule, when incident light is vertical with scattered light direction, Rayleigh scattering light intensity is the most weak, is conducive to the biological sample of systematic observation high scattering.
In traditional differential confocal light path, require pin hole, detector center and measuring system optical axis coincidence, thus ensure that the light beam that sample is reflected back can enter detector just, but, because pinhole size is less, usually at about 10 μm, pin hole is regulated and has certain difficulty.In addition, in differential confocal light path, the optimization axial defocusing amount of two pin holes is relevant to numerical aperture of objective N.A., the pin hole position of debuging for light path is only applicable to specific object lens N.A., thus, object lens cannot be changed per sample, thus cause differential confocal light path to there is larger limitation in actual applications.
Because Raman diffused light is very faint, in order to reduce the energy loss of Raman spectrum, pinhole size for focus location in existing confocal Raman technology is usually larger, usually, between φ 150 μm ~ φ 200 μm, expand the halfwidth that pinhole size then can increase confocal axial location curve, reduce its positioning precision, well can not play the effect of focusing, and system can only carry out spectrographic detection, and pattern is single, limits its application.
Existing Raman spectrum Detection Techniques, only make use of extremely faint Raman diffused light and position and spectrographic detection, given up and be better than Raman diffused light 10
3~ 10
6rayleigh light beam doubly, greatly reduces sensitivity and the positioning precision of system, and thus utilizing Reyleith scanttering light to carry out aided detection becomes the new approach of the positioning precision of improving spectrographic detection.
In addition, along with going deep into of raman study, Raman spectrum imaging has become a kind of important scientific research method.In the prior art, Raman spectrum imaging experiment needs to take a long time usually, and comparatively large by the impact of environment temperature, vibration, air shake etc. in the long-time imaging process of instrument, easily make instrument system produce drift, thus cause sample location to be detected out of focus; But existing confocal Raman spectra Detection Techniques do not possess real-time focal point follows the tracks of and aligning ability, thus in whole imaging process, cannot ensure that it excites the position of hot spot to be in objective focus positions, the actual hot spot that excites is much larger than object lens focused light focal spot, its result constrains the microminiaturization of detectable area, limits the microscopic spectrum detectivity of confocal Raman spectrometer device.
Based on above-mentioned situation, the present invention proposes twin shaft confocal spectrum microscopic system, adopts double-shaft way to reduce Rayleigh scattering light intensity, improves system space resolving power; The Rayleigh light beam abandoned in existing confocal Raman spectra detection system sample scattering light is utilized to carry out detected with high accuracy, itself and spectrum investigating system are organically blended, thus, detect while carrying out spatial positional information and spectral information, to realizing high-space resolution " collection of illustrative plates unification " imaging and detection.
Summary of the invention
The object of the invention is to overcome in existing confocal Raman detection instrument that collection efficiency is low, spatial resolution is not enough, and be unfavorable for defects such as the suppression of the factors such as environmental background light, intensity of light source fluctuation, propose one and there is high-space resolution twin shaft confocal spectrum detection method and device.
The object of the invention is to be achieved through the following technical solutions:
A kind of high-space resolution twin shaft confocal spectrum micro imaging method of the present invention, concrete steps are:
1) adopt the mode of twin shaft confocal microscopy to scan sample, illumination objective lens is symmetrically distributed in measuring surface normal both sides with collection object lens, and the angle of lighting optical axis and measuring surface normal is θ
1, the angle gathering optical axis and measuring surface normal is θ
2, with measuring surface normal direction for measuring axis, set up system coordinate system (x, y, z), wherein θ
1=θ
2;
2) exciting light focuses on sample via illumination objective lens, inspire Reyleith scanttering light and the Raman diffused light being loaded with sample spectral characteristic, and collected object lens converge to dichroic optical system, light beam is after dichroic optical system light splitting, Raman diffused light and Reyleith scanttering light are separated from each other Raman scattering Transmission light and enter spectrum investigating system, Reyleith scanttering light is entered confocal detection system by reflection, the Reyleith scanttering light signal fitting of acquisition is confocal curves by data handling system, utilize the characteristic that confocal curves extreme point is accurately corresponding with focal position, the spectral information of accurately catching and exciting hot spot focal position is triggered by extreme value, realize the spectrographic detection of high-space resolution,
3), when processing separately the signal of the Reyleith scanttering light obtained, the three dimension scale tomographic map of high-space resolution is obtained; When independent process obtains the signal of Raman diffused light, obtain spectrum picture; When processing the signal of the Reyleith scanttering light of acquisition and Raman diffused light, obtain the microcell collection of illustrative plates tomography of high-space resolution, i.e. " the collection of illustrative plates unification " of sample geometric position information and spectral information simultaneously;
4) according to the characteristic that confocal curves extreme point is accurately corresponding with focal position, accurate tracking can be carried out to sample in real time in measuring process to focus, ensure that sample is in focal position all the time in whole measuring process, suppress the factor such as environment temperature and vibration on the impact of spectral measurement, thus improve measuring accuracy.
In illustrated measuring method, in confocal detection system, data handling system obtains after focal spot pattern from image capturing system, calculates the center of now focal spot pattern, using this center as true origin, sets up the coordinate system (x in detector image planes
d', y
d'), arrange a circular pin hole at initial point place and confocal dummy pinhole focal spot image detects, when sample scans, data handling system calculates pixel grey scale summation within the scope of confocal dummy pinhole, obtains intensity response.
In illustrated measuring method, excitation beam can be the light beams such as line polarisation, rotatory polarization; Can also be the structure light beam generated by pupil filtering technology, polarized light and pupil filtering combine with technique can compress measurement focused spot size, improve the transverse resolution of system.
In illustrated measuring method, this system can also detect the scattering spectrums such as fluorescence, Brillouin scattering, Compton scattering light.
In illustrated measuring method, before illuminating lens or after gathering lens, add illumination end iris filter, shaping is carried out to illuminating bundle or collection light beam, improves the transverse resolution of system.
Present invention also offers a kind of high-space resolution twin shaft confocal spectrum microscopic imaging device, comprise light source, illumination objective lens, gather object lens and 3-D scanning worktable, also comprise collimator and extender mirror, dichroic light-dividing device, confocal detection system and spectrum investigating system; Wherein, layout is in measuring surface normal both sides symmetrically for illumination objective lens and collection object lens, and the angle of lighting optical axis and measuring surface normal is θ
1, the angle gathering optical axis and measuring surface normal is θ
2, wherein θ
1=θ
2collimator and extender mirror and illumination objective lens are successively placed on the emergent ray direction of light source, collection object lens and dichroic light-dividing device are successively placed on the reflection ray direction of sample, confocal detection device is placed on the reflection direction of dichroic light-dividing device, and spectral detection device is placed on dichroic light-dividing device transmission direction.
In illustrated measurement mechanism, also be included between collimator and extender mirror and illumination objective lens and add illumination end iris filter, or between collection object lens and confocal detection device, add collection terminal iris filter, or add illumination end iris filter and collection terminal iris filter simultaneously between collimator and extender mirror and illumination objective lens and between collection object lens and confocal detection device.
In illustrated measurement mechanism, be also included between collimator and extender mirror and illumination objective lens and add polarization modulating arrangement, or add polarization modulating arrangement between illumination objective lens and illumination end iris filter.
In illustrated measurement mechanism, also comprise the data handling system of finally carrying out Data Fusion.
In illustrated measurement mechanism, spectral detection device can be confocal spectroscopic sniffer, comprise the first condenser, be positioned at the pin hole at the first condenser focus place, second condenser lens, the spectrometer being positioned at second condenser lens focus place and spectrometer after detector; The detector that can also be common spectrum investigating system, comprise second condenser lens, be positioned at after the spectrometer at second condenser lens focus place and spectrometer.
In illustrated measurement mechanism, by increasing the Aili spot that image amplifying device enlarged image harvester detects, comprise place successively along light path measurement lens, with measure the confocal image amplifying device of lens and be positioned at the image collecting device at image amplifying device focus place, to improve the acquisition precision of confocal measuring apparatus.
Beneficial effect
The present invention contrasts prior art following remarkable innovative point:
1, twin shaft confocal microscopy and Raman spectrum Detection Techniques are organically combined, merge the high precision Objective rallying point position tracking ability of twin shaft confocal microscopy, confocal Raman spectra microscope can be made to detect the accurately corresponding minimum sample spectra characteristic exciting focal beam spot region, significantly improve the microscopical raman microspectroscopy spectroscopic probing capabilities of existing confocal Raman spectra;
2, utilize cross-compound arrangement, reduce Rayleigh scattering light intensity; Dichroic light-dividing device is utilized to carry out light splitting to Reyleith scanttering light and Raman diffused light, Reyleith scanttering light enters differential detection system, Raman diffused light enters Raman spectrum detection system, improve system spectrum detection sensitivity, dichroic light-dividing device can adjust angle as required, is conducive to the detection of lower wave number Raman spectrum;
3, twin shaft confocal microscope system and Raman spectrum imaging system are merged mutually on 26S Proteasome Structure and Function, both the tomography of sample microcell geometric parameter can have been realized, the spectrographic detection of sample microcell can be realized again, namely realize three dimension scale tomography, collection of illustrative plates tomography and spectrum test three kinds of imaging patterns simultaneously, and significantly improve the antijamming capability of imaging test system, linear and defocused property.
The present invention contrasts prior art and has following remarkable advantage:
1, compared with single shaft Raman spectrum system, Rayleigh scattering light intensity is reduced;
2, select single detector to realize dummy pinhole confocal detection in conjunction with computer software, detection mode is flexible, is conducive to the conversion of different rows N.A. object lens in practical application, is more conducive to the versatility realizing instrument.
3, dichroic light-dividing device can adjust according to demand, to improve system lower wave number detectivity.
Accompanying drawing explanation
Fig. 1 is high-space resolution twin shaft confocal spectrum micro imaging method schematic diagram;
Fig. 2 is high-space resolution twin shaft confocal spectrum microscopic imaging device schematic diagram;
Fig. 3 is the high-space resolution twin shaft confocal spectrum microscopic imaging device schematic diagram of band Polarization Modulation;
Fig. 4 is the high-space resolution twin shaft confocal spectrum microscopic imaging device schematic diagram with detection focal spot amplification system;
Fig. 5 is high-space resolution twin shaft confocal spectrum micro imaging method and device embodiment schematic diagram;
Wherein, 1-light source, 2-illumination objective lens, 3-sample, 4-lighting optical axis, 5-measuring surface normal, 6-θ
1, 7-gathers object lens, 8-dichroic optical system, 9-measures lens, 10-image enhancement system, 11-image capturing system, the confocal dummy pinhole of 12-, 13-measures optical axis, the confocal Raman curve of 14-, 15-confocal detection system, 16-focal spot pattern, 17-first condenser, 18-pin hole, 19-spectrum investigating system, 20-gathers optical axis, 21-data handling system, 22-second condenser lens, 23-spectrometer, 24-detector, 25-collimating and beam expanding system, 26-3-D scanning worktable, 27-confocal curves, 28-illumination end iris filter, 29-Polarization Modulation system, 30-collection terminal iris filter, 31-θ
2.
Embodiment
Below in conjunction with drawings and Examples, the invention will be further described.
Embodiment
In the present embodiment, light source 1 is laser instrument, Polarization Modulation system 29 is radial polarisation photogenerator, dichroic optical system 8 is Notch filter, data handling system 21 is computing machine, image capturing system 11 is ccd detector, and detector 24 is ccd detector, and image enhancement system 10 is enlarging objective.
As shown in Figure 1, Figure 2 with shown in Fig. 5, illumination objective lens 2 is symmetrically distributed in measuring surface normal 5 both sides with collection object lens 7, and lighting optical axis 4 is θ with the angle of measuring surface normal 5
16, gathering optical axis 20 with the angle of measuring surface normal 5 is θ
231, wherein θ
1=θ
2, high-space resolution twin shaft confocal spectrum micro imaging method, its measuring process is:
First, the light beam that laser instrument 1 sends is after collimating and beam expanding system 25, the directional light equal with illumination objective lens 2 Entry pupil diameters is become after carrying out expanding outgoing, radial polarisation light is become after radial polarisation photogenerator 29, radial polarisation light light beam after illumination end iris filter 28 is modulated, form compression hot spot via illumination objective lens 2 and focus on sample 3 surface be placed on 3-D scanning worktable 26, and inspire Reyleith scanttering light and the Raman diffused light being loaded with sample 3 spectral characteristic, sample 3 processes by strengthening the Raman enhancing technology such as Raman spectrum nano particle, to improve the intensity of Raman diffused light.
Axially (namely in figure z to) mobile sample 3, the Raman diffused light of Reyleith scanttering light and corresponding sample 3 zones of different is entered by reflection and gathers object lens 7, the light beam collected through gathering object lens 7 is carried out light splitting by Notch filter8, wherein, Raman diffused light enters spectrum investigating system 19 transmitted through Notch filter8, spectrum investigating system 19 is confocal Raman spectra detection system, Raman diffused light is converged to pin hole 18 by the first condenser 17, assemble through second condenser lens 22 and enter spectrometer 23, finally incide ccd detector 24, obtain the Raman spectrum I (λ) (λ is wavelength) of sample 3, as shown in confocal Raman curve 14, Reyleith scanttering light is reflected by Notch filter8, after collection terminal iris filter 30 is modulated, enters confocal detection system 15, converging, converging hot spot and amplify through enlarging objective 10 and be imaged on the first ccd detector 11 through measuring lens 9.
Computing machine 21 obtains after focal spot pattern 16 from ccd detector 11, calculates the center of now focal spot pattern 16, using this center as true origin, sets up the coordinate system (x in CCD image planes
d', y
d'), a circle confocal dummy pinhole 12 focal spot pattern 16 is set at initial point place and detects; When sample 3 scans, computing machine 21 calculates pixel grey scale summation in confocal dummy pinhole 12 scope, obtains confocal intensity response I
c(x, y, z), as shown in confocal curves 27, is obtained the elevation information of sample surfaces, reconstructs the three-dimensional surface shape of sample 3 by " extreme point " of confocal curves 27.。
According to the characteristic that the extreme point of confocal curves 27 is accurately corresponding with system focus, accurately obtain system focus position, sample 3 is moved to focus O position, again obtain the spectral signal I (λ) of focus O position.
By I (λ), I
c(x, y, z) is sent to computing machine 21 and carries out data processing, thus acquisition comprises sample 3 positional information I
cthe four-dimensional metrical information I (x, y, z, λ) of (x, y, z) and spectral information I (λ).
After completing above-mentioned steps, 3-D scanning worktable 26 pairs of samples 3 are utilized to carry out transversal scanning (x, y direction namely in figure), after moving to next point, 3-D scanning worktable 26 pairs of samples 3 are utilized to carry out axial scan (the z direction namely in figure), after obtaining the position of system focus O, sample 3 is moved to focus O place, and obtains spectral information.
Only process is carried out to the confocal detection system 15 acquisition detection hot spot receiving Reyleith scanttering light and obtain confocal response I
c(x, y, z), accurately catches by its " extreme point " focal position exciting hot spot, and system can carry out the three dimension scale tomography of high-space resolution; The spectral response I(λ that the Raman spectrum detection system 19 that only Raman diffused light is received in docking obtains) 14 when processing, system can carry out Raman spectrum detection; Simultaneously to the confocal response I that the ccd detector 11 receiving Reyleith scanttering light obtains
c(x, y, z) the spectral signal I(λ obtained with the Raman spectrum detection system 19 of Raman diffused light) when processing, system can carry out the microcell collection of illustrative plates tomography of high-space resolution, namely realizes " collection of illustrative plates unification " Effect on Detecting of the high-space resolution of sample 3 geometric position information and spectral information.
As shown in Figure 5, high-space resolution twin shaft confocal spectrum microscopic imaging device comprises the laser instrument 1 placed successively along light path, collimating and beam expanding system 25, radial polarisation photogenerator 29, illumination end iris filter 28, illumination objective lens 2, sample 3, 3-D scanning worktable 26, and the collection object lens 7 of light path reflection direction, Notch filter8, be positioned at the Raman spectrum detection system 19 of Notch filter8 transmission direction, be positioned at the collection terminal iris filter 30 of Notch filter8 reflection direction, confocal detection system 15, and connect the computing machine 21 of confocal detection system 15 and Raman spectrum detection system 19, wherein, Raman spectrum detection system 19 comprise the first condenser 17 placed successively along light path, the pin hole 18 being positioned at the first condenser 17 focal position, be positioned at pin hole 18 after second condenser lens 22, be positioned at second condenser lens 22 focal position spectrometer 23 and be positioned at the ccd detector after spectrometer 24, confocal detection system 15 comprises the enlarging objective 10 being positioned at and measuring lens 9 focus place, and is positioned at the ccd detector 22 at enlarging objective 10 focus place.
Radial polarisation photogenerator 29, illumination end iris filter 28 is added in collimator and extender device 25 in fig. 2 and illumination objective lens 2, collection terminal iris filter 30 is added between Notch Filter8 and measurement lens 9, i.e. pie graph 3, add radial polarisation photogenerator 29, illumination end iris filter 28, collection terminal iris filter 30 for beam shaping, improve transverse resolution.
In the confocal detection device 15 of Fig. 2, increasing the Aili spot that image amplifying device 10 enlarged image harvester 11 detects, i.e. pie graph 4, increasing image amplifying device 10 for improving the acquisition precision of confocal measuring apparatus 15.
Below by reference to the accompanying drawings the specific embodiment of the present invention is described; but these explanations can not be understood to limit scope of the present invention; protection scope of the present invention is limited by the claims of enclosing, and any change on the claims in the present invention basis is all protection scope of the present invention.
Claims (10)
1. a high-space resolution twin shaft confocal spectrum micro imaging method, is characterized in that:
1) mode of twin shaft confocal microscopy is adopted to scan sample (3), illumination objective lens (2) is symmetrically distributed in measuring surface normal (5) both sides with collection object lens (7), and lighting optical axis (4) is θ with the angle of measuring surface normal (5)
1(6), gathering optical axis (20) with the angle of measuring surface normal (5) is θ
2(31), wherein θ
1=θ
2;
2) exciting light focuses on sample (3) via illumination objective lens (2), inspire Reyleith scanttering light and the Raman diffused light being loaded with sample spectral characteristic, and collected object lens (7) converge to dichroic optical system (8), light beam is after dichroic optical system (8) light splitting, Raman diffused light and Reyleith scanttering light are separated from each other Raman scattering Transmission light and enter spectrum investigating system (19), Reyleith scanttering light is entered confocal detection system (15) by reflection, the Reyleith scanttering light signal fitting of acquisition is confocal curves (14) by data handling system (21), utilize the characteristic that confocal curves extreme point is accurately corresponding with focal position, the spectral information of accurately catching and exciting hot spot focal position is triggered by extreme value, realize the spectrographic detection of high-space resolution,
3), when processing separately the signal of the Reyleith scanttering light obtained, the three dimension scale tomographic map of high-space resolution is obtained; When independent process obtains the signal of Raman diffused light, obtain spectrum picture; When processing the signal of the Reyleith scanttering light of acquisition and Raman diffused light, obtain the microcell collection of illustrative plates tomography of high-space resolution, i.e. " the collection of illustrative plates unification " of sample geometric position information and spectral information simultaneously;
4) according to the characteristic that confocal curves extreme point is accurately corresponding with focal position, accurate tracking can be carried out to sample in real time in measuring process to focus, ensure that sample is in focal position all the time in whole measuring process, suppress environment temperature and the impact of vibration on spectral measurement, thus improve measuring accuracy.
2. high-space resolution twin shaft confocal spectrum micro imaging method according to claim 1, it is characterized in that: in confocal detection system (15), data handling system (21) obtains after focal spot pattern (16) from image capturing system (11), calculate the center of now focal spot pattern (16), using this center as true origin, set up the coordinate system (x in detector image planes
d', y
d'), a circular pin hole is set at initial point place and confocal dummy pinhole (12) focal spot image detects, when sample (3) scans, data handling system calculates pixel grey scale summation within the scope of confocal dummy pinhole, obtains intensity response.
3. high-space resolution twin shaft confocal spectrum micro imaging method according to claim 1, it is characterized in that: excitation beam is line polarisation bundle or rotatory polarization bundle, or the structure light beam generated by pupil filtering technology, polarized light and pupil filtering combine with technique can compress measurement focused spot size, improve the transverse resolution of system.
4. high-space resolution twin shaft confocal spectrum micro imaging method according to claim 1, is characterized in that: spectrum investigating system detection comprises the scattering spectrum of fluorescence, Brillouin scattering, Compton scattering light.
5. a high-space resolution twin shaft confocal spectrum microscopic imaging device, comprise light source (1), illumination objective lens (2), gather object lens (7) and 3-D scanning worktable (26), it is characterized in that: also comprise collimator and extender mirror (25), dichroic optical system (8), confocal detection system (15) and spectrum investigating system (19); Wherein, layout is in measuring surface normal (5) both sides symmetrically for illumination objective lens (2) and collection object lens (7), and lighting optical axis (4) is θ with the angle of measuring surface normal (5)
1(6), gathering optical axis (20) with the angle of measuring surface normal (5) is θ
2(31), wherein θ
1=θ
2collimator and extender mirror (25) and illumination objective lens (2) are successively placed on the emergent ray direction of light source (1), collection object lens (7) and dichroic optical system (8) are successively placed on the reflection ray direction of sample (3), confocal detection system (15) is placed on the reflection direction of dichroic optical system (8), and spectrum investigating system (19) is placed on dichroic optical system (8) transmission direction.
6. high-space resolution twin shaft confocal spectrum microscopic imaging device according to claim 5, it is characterized in that: be also included between collimator and extender mirror (25) and illumination objective lens (2) and add illumination end iris filter (28), or between collection object lens (7) and confocal detection system (15), add collection terminal iris filter (30), or between collimator and extender mirror (25) and illumination objective lens (2) and between collection object lens (7) and confocal detection system (15), add illumination end iris filter (28) and collection terminal iris filter (30) simultaneously.
7. high-space resolution twin shaft confocal spectrum microscopic imaging device according to claim 6, it is characterized in that: be also included between collimator and extender mirror (25) and illumination objective lens (2) and add polarization modulating arrangement (29), or add polarization modulating arrangement (29) between illumination objective lens (2) and illumination end iris filter (28).
8. high-space resolution twin shaft confocal spectrum microscopic imaging device according to claim 5, is characterized in that: also comprise the data handling system (21) of finally carrying out Data Fusion.
9. the high-space resolution twin shaft confocal spectrum microscopic imaging device according to claim 5 or 6, the detector (24) that it is characterized in that: spectrum investigating system (19) is confocal spectroscopic sniffer, comprise the first condenser (17), be positioned at after the pin hole (18) at the first condenser focus place, second condenser lens (22), the spectrometer (23) being positioned at second condenser lens (22) focus place and spectrometer (23); The detector (24) that can also be common spectrum investigating system, comprise second condenser lens (22), be positioned at after the spectrometer (23) at second condenser lens (22) focus place and spectrometer (23).
10. the high-space resolution twin shaft confocal spectrum microscopic imaging device according to claim 5 or 6 or 8, it is characterized in that: by increasing the Aili spot that image amplifying device (10) enlarged image acquisition system (11) detects, comprise place successively along light path measurement lens (9), with measure lens (9) confocal image amplifying device (10) and be positioned at the image capturing system (11) at image amplifying device (10) focus place, to improve the acquisition precision of confocal measuring system (15).
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