CN101526477B - Laser differential confocal spectrum microscopy tomography device - Google Patents

Laser differential confocal spectrum microscopy tomography device Download PDF

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Publication number
CN101526477B
CN101526477B CN2009100822486A CN200910082248A CN101526477B CN 101526477 B CN101526477 B CN 101526477B CN 2009100822486 A CN2009100822486 A CN 2009100822486A CN 200910082248 A CN200910082248 A CN 200910082248A CN 101526477 B CN101526477 B CN 101526477B
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spectrum
sample
differential confocal
spectroscope
object lens
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CN2009100822486A
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CN101526477A (en
CN101526477K1 (en
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赵维谦
王允
邱丽荣
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北京理工大学
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Abstract

The invention belongs to the technical fields of optical microscopy imaging and optical precision measurement and relates to a laser differential confocal spectrum microscopy tomography device which mainly comprises a Raman spectrum analysis part (25) and an objective lens (6), a polarizing spectroscope (7), a one-quarter glass slide (8) and a measurement objective lens (9) which are sequentially arranged along the optical path; the laser differential confocal spectrum microscopy tomography device further comprises a differential confocal detection part (26) which is positioned in the reverse direction of the reflection direction of the polarizing beam splitter. The differential confocal detection part is used for measuring the geometric position of a micro-area of a sample and focusing the sample to obtain image information of the micro-area of the sample; the Raman spectrum analysis part is used for analyzing the material spectrum of the detected area of the sample to obtain component information of the micro-area of the sample; the combination of the two parts can realize the nano-level micro-area spectrum measurement of the sample and simultaneously obtain the geometric feature and the material component information of the micro-area of the sample. The laser differential confocal spectrum microscopy tomography device provides a powerful observation means for bio-medicine, material science, high-energy physics and other forefront subjects.

Description

Laser differential confocal spectrum microscopy tomography device

Technical field

The invention belongs to optical microphotograph imaging and spectral measurement methods field, relate to a kind of laser differential confocal spectrum microscopy tomography device, can be used for the spectral analysis of the high-precision three-dimensional topography measurement and the sample microcell material component of sample.

Technical background

Scholars such as nineteen ninety G.J.Puppels have at first invented the confocal Raman spectra microtechnic and successfully have been used for experiment when unicellular and chromosomal form of observation and composition.The microscopical light spectrum image-forming principle of confocal Raman spectra is the position that pointolite, some measured object and spectrum point probe three is placed on the conjugation that corresponds to each other, and has constituted the micro imaging system with chromatography function that the point in the optical imagery throws light on and point is surveyed.The basic structure of typical confocal microscope as shown in Figure 1, light that laser instrument sends expands behind the bundle through first spectroscope through expanding the bundle object lens, axial drive means, object lens converge, be focused into hot spot on the measured object surface and be reflected, contain the Raman light that contains sample material spectrum information that is ejected by incident laser in the reflected light, reflected light returns along former road,, handled after converge through compiling lens after filtering mating plate filtering Reyleith scanttering light and fluorescence etc. by first spectroscope reflection through receiving by the spectral detector behind the pin hole behind the pin hole at a focus place that is positioned at plus lens.Because confocal Raman microscope adopts is that therefore a kind of point-to-point detection mode has determined it to have higher spatial resolving power and have the chromatography detectivity.

Confocal microscope is widely used in front subject and fields such as biomedicine, material science and high-energy physics because having the ability of spectrum tomography, and because its resolution is very high, can carry out fine imaging to biological living sample and small industrial products, the micro-geometrical structure information of sampling and material spectrum information and become the strong instrument that medical observation and manufacturing industry detect; But if will improve the microcell detectivity of confocal Raman spectra microscopic system, just must accurately focus sample, sample is positioned near the focus O, yet near the characteristic response sensitivity of confocal microscopy the focus O is but extremely low, its result can not get both existing confocal Raman spectra microtechnic between " microscopic spectrum " detectivity and " microcell geometric position " detectivity, there is following principle defective in promptly existing confocal Raman spectra microtechnic:

1. focus O place spectrographic detection ability is strong, but the geometric position detectivity is poor;

2. the Raman spectrum that excites that is difficult to accurate focus capture O place is realized the microscopic spectrum detection;

3. be subjected to the restriction of diffraction limit, focus O place hot spot and depth of focus size all will restrict the axial and horizontal resolution characteristic of confocal Raman spectra microscopic system etc.

Obvious, the principle defective of above-mentioned confocal Raman spectra microtechnic has restricted the detectivity of its microscopic spectrum and geometric position, makes occasions such as the test of its spectrum that can't be used for tiny area more and geometric position and analysis.

At present, the research develop rapidly of the improvement of the relevant microscopical performance of confocal Raman spectra in the world, methods such as ultra-short pulse laser method, oil immersion objective method, fiber-optic probe method, image improvement method have appearred, but above-mentioned research, mainly concentrate on aspects such as light-source system that the confocal Raman spectra microscopic system relates to, spectrum investigating system, focusing objective len system, spectral information processing, although its overall performance to the confocal Raman spectra microscopic system improves to some extent, aspect the improving of confocal Raman spectra microscopic system spatial resolving power, there are not remarkable break-throughs.

Differential confocal technology has highly sensitive, good linearity, and measurement has absolute tracking power and bipolarity tracking characteristics, can realize the characteristics such as absolute measurement of geometric position, and be widely used this year; And along with the development of pupil filtering technology, the compressibility depth of focus greatly that combines of differential confocal technology and pupil filtering technology improves and focuses sensitivity, improves the resolving power of system.As: " the Effect of an annularpupil filter on differential confocal microscopy " of " optics express delivery " reported the experiment of using the pupil filtering technology to improve the axial sensitivity of differential confocal microscope and expanding the linear response interval.And the report of the figure spectral imaging technology of nanoscale microcell geometry patterns that use differential confocal technology to combine with the confocal Raman spectra Detection Techniques to obtain sample then simultaneously and component information is not seen so far as yet.

Summary of the invention

Above shortcomings when the objective of the invention is to overcome prior art and being used for microcosmic three-dimensional appearance and material spectrum and measuring merge Raman spectrum Detection Techniques and differential confocal microtechnic, propose laser differential confocal spectrum microscopy tomography device.This sniffer can measuring samples nanoscale microcell spectral information, obtain the micro-geometrical structure and the material component information of sample simultaneously, and have high spatial resolution; Comprise the two-dimensional scan drive unit in apparatus of the present invention simultaneously, can drive sample makes device can carry out the scanning of two dimension to sample, thereby generate the figure spectrogram of sample, for front subjects such as biomedicine, material science and high-energy physics provide strong observation method.

The objective of the invention is to realize by following apparatus.

Laser differential confocal spectrum microscopy tomography device, comprise Raman spectrum probe portion (25), object lens (6), first spectroscope of placing successively along light path (7), be positioned at the measurement object lens (9) of first spectroscope (7) transmission direction and be used for driving and measure object lens (9) at axially movable axial drive means (8) and two-dimentional drive unit (28); Also comprise and be placed on the reciprocal differential confocal probe portion of first spectroscope (7) reflection direction; Also comprise a laser instrument (1) and a data fusion processing system (24); Wherein laser instrument (1) is connected with Raman spectrum analysis part (25) by single-mode polarization maintaining fiber, is used for providing the illumination and the excitation source of test usefulness to system; Data Fusion system (24) links to each other with differential confocal probe portion (26) with Raman spectrum probe portion (25), is used for two parts of fusion treatment and surveys spectral information and the positional information that receives, and obtains the microcell figure spectrum information of sample;

Raman spectrum probe portion (25) comprises that a focus is positioned at the fiber-optic output head that links to each other with laser instrument and is used for the light of optical fiber output is expanded the light source object lens (2) of bundle and the narrow band pass filter (3) that is arranged in order along light path, dichroic filter (4), be positioned at dichroic filter (4) transmission direction converge object lens (5), be positioned at the reflection direction catoptron (20) of dichroic filter, be placed on the optical filter (21) of the reflection direction of catoptron, at the coupled lens (22) of optical filter (21) transmission direction with gather the Raman spectrum detector (23) of the spectral signal at coupled lens (22) focus place by single-mode polarization maintaining fiber; Differential confocal system (26) comprising: second spectroscope (11) is placed on first condenser (12), first pin hole (13) of second spectroscopical transmission direction successively and presses close to first detector (18) of pin hole; Be placed on second condenser (16), second pin hole (17) of the reflection direction of second spectroscope (11) successively and press close to second detector (18) of pin hole.

Axial drive means in apparatus of the present invention (8) and measurement object lens (9) can also be positioned at the reflection direction of polarization spectroscope (7).

Apparatus of the present invention can also comprise that diffraction light shaping device (27) is used for the depth of focus that object lens (9) are measured in compression, improve to focus precision; Diffraction light shaping device (27) can be positioned at and expand between bundle object lens (6) and first spectroscope (7), also can be positioned between first spectroscope (7) and the measurement object lens (9) or between first spectroscope (7) and second spectroscope (11), can also use two diffraction light shaping devices, lay respectively at second spectroscope (11) and first and converge mirror (12) and second and converge between the mirror (16).

Apparatus of the present invention can also comprise: first filter amplification circuit (15) and second filter amplification circuit (19), two amplifying circuits link to each other with second detector (18) with first detector (14) respectively, and the signal that is used for two detectors are detected carries out filtering and processing and amplifying.

Apparatus of the present invention can also comprise a data fusion processing system (24), data handling system (24) links to each other with first filter amplification circuit (15), second filter amplification circuit (19) and links to each other with Raman spectrum detector (23), be used for three's output signal is carried out integration processing, realize the robotization of measurement, obtain the microcell figure spectrum information of sample.

The present invention contrasts existing technique device and has following remarkable advantage:

1) can carry out the two-dimensional scan light spectrum image-forming to sample, obtain the geometry pattern information and the material component information of sample simultaneously, obtain the information of the collection of illustrative plates unification of sample;

2) microscopical microscopic spectrum of confocal Raman spectra and geometric position detectivity are significantly improved;

3) can realize microcell collection of illustrative plates tomography, three dimension scale tomography, three kinds of imaging patterns of spectrum test simultaneously;

4) have absolute tracking zero point and bipolarity tracking characteristics, can realize the geometric position absolute measurement;

5) utilize diffraction optical device shaping and compression focusing objective len hot spot, its effect that can play the compression hot spot can reduce the loss of excitation energy again, thereby it is unlikely too big etc. that raman spectrum strength is reduced.

Description of drawings

Fig. 1 is existing confocal Raman spectra microscope schematic diagram and imaging region synoptic diagram;

Fig. 2 is a nanoscale laser differential confocal spectrum microscopy tomography device schematic diagram of the present invention;

Fig. 3 installs differential response curve for this.

Wherein: the 1-laser instrument, 2-light source object lens, the 3-narrow band filter slice, the 4-dichroic filter, 5-converges object lens, 6-expands the bundle object lens, 7-first spectroscope, the 8-axial drive means, 9-measures object lens, the 10-sample, 11-second spectroscope, 12-first converges mirror, 13-first pin hole, 14-first detector, 15-first filter amplification circuit, 16-second converges mirror, 17-second pin hole, 18-second detector, 19-second filter amplification circuit, the 20-catoptron, the 21-optical filter, the 22-object lens that are coupled, 23-Raman spectrum detector, 24-Data Fusion system, 25-Raman spectrum analysis part, 26-differential confocal system, 27-diffraction light shaping device, 28 two-dimentional drive units.

Embodiment

The invention will be further described below in conjunction with drawings and Examples.

The technology of the present invention principle is: adopt the differential confocal microscopic imaging technology that the confocal microscope receiving light path is arranged as burnt preceding and defocused two-way and survey light path, differential the subtracting each other of detecting by the two-way detector of the two-way intensity response signal with coordination phase not reaches the purpose of improving azimuthal resolution and improving antijamming capability, simultaneity factor comprises the Raman spectrum analysis system, make system's material spectrum signal of the microcosmos area of measuring samples simultaneously, can be to the conscientious analysis of the material component of sample; In addition, introduce diffraction light shaping device, measuring beam is carried out limit compression system depth of focus, reached the purpose that improves system resolution; By sample is carried out scanning probe, the microscopic spectrum information of the sample that utilization records and pattern information reconstruct the collection of illustrative plates of response sample three-dimensional appearance and material component in conjunction with the two-dimensional coordinate of sample at last.

The present invention has the laser differential confocal spectrum microscopy tomography device structural drawing concurrently as shown in Figure 2, comprise: Raman spectrum probe portion 25, object lens 6, first spectroscope 7, the measurement object lens of placing successively along light path 9 that are positioned at first spectroscope, 7 transmission direction, be used to drive measure object lens in the axial drive means 8 of axially-movable, drive sample at the hang down two-dimentional drive unit 28 of all plane motions of two dimension; Also comprise and be placed on the reciprocal differential confocal probe portion of first spectroscope, 7 reflection directions; Also comprise a laser instrument 1 and a Data Fusion system 24 that links to each other with differential confocal probe portion 26 with Raman spectrum probe portion 25 that is connected with Raman spectrum analysis part 25 by single-mode polarization maintaining fiber.Wherein, Raman spectrum probe portion 25 comprise that a focus is positioned at that the fiber-optic output head that links to each other with laser instrument is used for that light with optical fiber output expands light source beam object lens 2 and the narrow band pass filter 3, the dichroic filter 4 that are arranged in order along light path, the transmission direction that is positioned at dichroic filter 4 converge object lens 5, be positioned at dichroic filter reflection direction catoptron 20, be placed on the optical filter 21 of the reflection direction of catoptron, at the coupled lens 22 of optical filter 21 transmission direction with gather the Raman spectrum detector 23 of the spectral signal at coupled lens 22 focus places by single-mode polarization maintaining fiber; Differential confocal system 26 comprises: second spectroscope 11 is placed on first condenser 12, first pin hole 13 of second spectroscopical transmission direction successively and presses close to first detector 18 of pin hole; Be placed on successively second spectroscope 11 reflection direction second condenser 16, second pin hole 17 and press close to second detector 18 of pin hole.

Axial drive means 8 in apparatus of the present invention and measurement object lens 9 can also be positioned at the reflection direction of first spectroscope 7.

Can also comprise in apparatus of the present invention that diffraction light shaping device 27 is used to compress the depth of focus of measuring object lens 9, improve to focus precision; Diffraction light shaping device 27 can expand between the bundle object lens 6 and first spectroscope 7, also can and measure between the object lens 9 or between first spectroscope 7 and second spectroscope 11 at first spectroscope 7, can also use two diffraction light shaping devices, lay respectively at second spectroscope 11 and first and converge mirror 12 and second and converge between the mirror 16.Can also comprise: first filter amplification circuit 15 and 19, two amplifying circuits of second filter amplification circuit link to each other with second detector 18 with first detector 14 respectively, and the signal that is used for two detectors are detected carries out filtering and processing and amplifying; Can also comprise a data disposal system 24, data handling system 24 links to each other and links to each other with Raman spectrum detector 23 with first filter amplification circuit 15, second filter amplification circuit 19, be used for three's output signal is carried out integration processing, realize the robotization of measurement, obtain the microcell figure spectrum information of sample.

The present invention has the laser differential confocal spectrum microscopy tomography device measuring principle concurrently as shown in Figure 2: open laser instrument 1, the laser that laser instrument 1 sends is transferred to the focus place of light source object lens through single-mode polarization maintaining fiber, converge the back by narrow band filter slice 3 filtering, the Raman spectrum composition that filtering laser inspires by optical fiber through light source object lens 2; By the process of the light beam behind the narrow band filter slice 3 binomial look optical filter 4, luminous energy almost all passes through, after through converging object lens 5 and expanding bundle object lens 6 backs by diffraction light apparatus for shaping 27, modulate the light beam into after the needed form through first spectroscope 7, measure object lens 9 backs and converge on the surface of sample 10 and be reflected after becoming a small light spot, comprise Rayleigh scattering light, fluorescence excitation and Raman spectrum in the reflected light, in this process, can realize focusing or controlling spot size in axially-movable by a use axial drive means 8 driving measurement object lens 9; Light path is returned on the former road of reflected light, through measuring object lens 9, back by the first spectroscope beam split, wherein one the tunnel be reflected and enter differential confocal sniffer 26, light beam enters differential confocal sniffer 26 backs second spectroscope 11 and is divided into two bundles, a branch of converged to enter after mirror 12 converges by first be positioned at first and converge first pin hole 13 that mirror 12 focus front distances are the position of M, received by first detector 14; Another bundle is converged to enter after mirror 16 converges by second and is positioned at second and converges that distance is second pin hole 17 of the position of M after mirror 16 focuses, is received by second detector 18.Another Reuter penetrate the back by diffraction light apparatus for shaping 27, expand bundle object lens 6, converge object lens 5 backs and be transmitted into catoptron 20 by the dichroism optical filter; Direct light by the sample reflected back in this process in the light will be directly by the binomial colo(u)r filter and by filtering, the light of reflection is once more through passing through optical filter 21 after the reflection of catoptron 20, eliminate the fluorescent component in the light beam, process coupled lens 22 enters the material component spectral information that 23 detections of Raman spectrum detector obtain sample after light is coupled into single-mode polarization maintaining fiber then.Raman spectrum detector 23, first filter amplification circuit 15 and second filter amplification circuit 19 are transferred to the signal that records the four-dimensional signal that obtains forgiving sample three-dimensional geometric information and component spectra information after Data Fusion system 24 handles.

In measuring process when the sample surface is in focal plane or out of focus, laser instrument 1, light source object lens 2, first spectroscope 7, measurement object lens 9, first converge mirror 12, first pin hole 13 and first detector 14 and constitute " accurate confocal microscope ", the intensity response I that first detector 14 detects 1(u 1) be:

I 1 ( u 1 ) = [ sin ( u 1 / 2 ) ( u 1 / 2 ) ] 2 I 0

U wherein 1Be axial normalization coordinate,, I 0Be incident intensity.

Laser instrument 1, light source object lens 2, first spectroscope 7, measurement object lens 9, second converge mirror 16, second pin hole 17 and second detector 18 and constitute " accurate confocal microscope ", the intensity response I that second detector 18 detects 2(u 2) be:

I 2 ( u 2 ) = [ sin ( u 2 / 2 ) ( u 2 / 2 ) ] 2 I 0

With I 1(u 1) and I 2(u 2) do the difference after obtain: I Diff(u)=I 1(u 1)-I 2(u 2):

I diff ( u ) = I 1 ( u 1 ) - I 2 ( u 2 ) = { [ sin ( u 1 / 2 ) u 1 / 2 ] 2 - [ sin ( u 2 / 2 ) u 2 / 2 ] 2 } } I 0

Computing machine is according to I DiffHandle in real time and calculate, by I DiffTwo coordinates of intensity curve light intensity magnitude and the sample position that from two-dimentional drive unit (28), obtains (x, y), reconstruct sample microcell three-dimensional appearance information I (x, y, z)

Laser instrument 1, light source object lens 2, dichroic filter 4, measurement object lens 9, coupling object lens 22, Raman spectrum detector 23 constitute confocal Raman microscope, and the spectral response that Raman spectrum detector 23 detects is I (λ).

Raman spectrum detector 23, first detector 14 and second detector 18 with output signal be transferred to the four-dimensional information I that obtains sample 10 in the Data Fusion system 23 (x, y, z, λ)

Below in conjunction with the accompanying drawings the specific embodiment of the present invention is described; but these explanations can not be understood that to have limited scope of the present invention; protection scope of the present invention is limited by the claims of enclosing, and any change on claim of the present invention basis all is protection scope of the present invention.

Claims (3)

1. laser differential confocal spectrum microscopy tomography device, object lens (6), first spectroscope (7) that it is characterized in that comprising Raman spectrum probe portion (25), places successively along light path, be positioned at the measurement object lens (9) of first spectroscope (7) transmission direction and be used for driving and measure object lens (9) at axially movable axial drive means (8) and two-dimensional scan drive unit (28); Also comprise and be placed on the reciprocal differential confocal probe portion of first spectroscope (7) reflection direction; Also comprise a laser instrument (1) and a data fusion processing system (24); Wherein laser instrument (1) is connected with Raman spectrum probe portion (25) by single-mode polarization maintaining fiber, is used for providing the illumination and the excitation source of test usefulness to system; Data Fusion system (24) links to each other with differential confocal probe portion (26) with Raman spectrum probe portion (25), is used for two parts of fusion treatment and surveys spectral information and the positional information that receives, and obtains the microcell figure spectrum information of sample; Raman spectrum probe portion (25) comprises that a focus is positioned at the fiber-optic output head that links to each other with laser instrument and is used for the light of optical fiber output is expanded the light source object lens (2) of bundle and the narrow band pass filter (3) that is arranged in order along light path, dichroic filter (4), be positioned at dichroic filter (4) transmission direction converge object lens (5), be positioned at the catoptron (20) of dichroic filter reflection direction, be placed on the optical filter (21) of the reflection direction of catoptron, at the coupled lens (22) of optical filter (21) transmission direction with gather the Raman spectrum detector (23) of the spectral signal at coupled lens (22) focus place by single-mode polarization maintaining fiber; Differential confocal system (26) comprising: second spectroscope (11) is placed on first condenser (12), first pin hole (13) of second spectroscopical transmission direction successively and presses close to first detector (18) of pin hole; Be placed on second condenser (16), second pin hole (17) of the reflection direction of second spectroscope (11) successively and press close to second detector (18) of pin hole.
2. laser differential confocal spectrum microscopy tomography device according to claim 1 is characterized in that: axial drive means (8) and measurement object lens (9) place the reflection direction of polarization spectroscope (7) to substitute the described transmission direction of claim 1.
3. laser differential confocal spectrum microscopy tomography device according to claim 1, it is characterized in that comprising: first filter amplification circuit (15) and second filter amplification circuit (19), two amplifying circuits link to each other with second detector (18) with first detector (14) respectively, and the signal that is used for two detectors are detected carries out filtering and processing and amplifying.
CN2009100822486A 2009-04-21 2009-04-21 Laser differential confocal spectrum microscopy tomography device CN101526477B (en)

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