CN102116930A - Tri-differential confocal microscope imaging method with high axial resolution and imaging device - Google Patents
Tri-differential confocal microscope imaging method with high axial resolution and imaging device Download PDFInfo
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- CN102116930A CN102116930A CN 201110049995 CN201110049995A CN102116930A CN 102116930 A CN102116930 A CN 102116930A CN 201110049995 CN201110049995 CN 201110049995 CN 201110049995 A CN201110049995 A CN 201110049995A CN 102116930 A CN102116930 A CN 102116930A
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Abstract
The invention provides a tri-differential confocal microscope imaging method with high axial resolution and an imaging device. The axial resolution of the original confocal microscope is improved by tri-differential detection. In the optical path of a system, the axial position of a pinhole at the optical detecting position of the confocal microscope can be changed by a pinhole axial micro-displacement device, thus realizing tri-differential detection of signals. The imaging method and the imaging device have the advantages of ensuring the stability of the displacement of the pinhole and simultaneously improving the resolving power, and being simple to realize.
Description
Technical field
The present invention relates to a kind of device and formation method that improves confocal microscope resolution, particularly a kind of have axial high-resolution three differential confocal microscope formation method and an imaging device, realizes that three differential detections of signal improve the imaging of confocal microscope axial resolution.
Background technology
Confocal microscope is to be proposed first in nineteen fifty-seven by American scholar M.Minsky, and obtains United States Patent (USP) in 1961, and the patent No. is US3013467.Confocal microscope places the conjugate position that corresponds to each other with pointolite, some thing and point probe three, and the resolution of system is significantly improved.General confocal microscope ultimate principle as shown in Figure 3, behind the light process object lens 21 and semi-transparent semi-reflecting lens 22 that pointolite 20 sends, in the irradiation sample 23 on the focal plane any surveyed and can be detected device 26 behind the reflected light of this some process semi-transparent semi-reflecting lens 22 and the light collecting lens 24 at detecting pinhole 25 places.By mobile example or utilize light path scanning, just can finish point by point scanning to the sample focal plane, obtain the confocal images of sample optics transversal section.Pointolite 20 is conjugation with detecting pinhole 25 with respect to focal plane of lens, and the point outside the focal plane can be in the imaging of detecting pinhole place, so it has improved the resolution of system greatly.
In order to improve the axial resolution of confocal microscope, Zhao Weiqian etc. have proposed three differential micro imaging methods, and this method is surveyed three road signals simultaneously, improve system axial resolution by handling three road signals.Specific implementation method is referring to the Chinese invention patent of " three differential confocal microscopic imaging method and device ", and application number is 200410073652.4.This method utilizes the space beam split to realize differential detection, to improve the axial resolution of confocal microscope.The efficiency of light energy utilization was low when but this method was measured at the low light level, and because this method adopts non-light channel structure altogether, therefore was difficult to guarantee the stability of pin hole displacement, and environment for use is had relatively high expectations.
Summary of the invention
The objective of the invention is for overcoming the deficiency of above-mentioned prior art, a kind of have axial high-resolution three differential confocal microscope formation method and imaging device are provided, same in the stability that guarantees the pin hole displacement improved resolution characteristic.
It is a kind of that to have an axial high-resolution three differential confocal microscope formation method performing step as follows:
(1) photodetection place pin hole is fixed on axial micro-displacement apparatus one end of pin hole, the axial micro-displacement apparatus other end of pin hole is fixed on the substrate; Send the axial micro-displacement driving device of signal controlling pin hole by the signal Synchronization device and send drive signal, drive the axial micro-displacement apparatus of pin hole and change photodetection place pin hole position, successively pin hole is placed light collecting lens focus place, defocused u
mPlace and burnt preceding u
mPlace, wherein u
mBe the normalization optical coordinate of pin hole apart from the light collecting lens focal length;
(2) the signal Synchronization device is realized sample sweep signal, the axial micro-displacement apparatus drive signal of pin hole, the data collector trigger pip synchronously, guarantee after photodetection place pin hole moves, again to tested sample point by point scanning, and acquired signal simultaneously;
(3) three positions of above-mentioned pin hole in step (1), i.e. photodetection place pin hole lays respectively at light collecting lens focus place, defocused u
mPlace and burnt preceding u
mDuring the place, images acquired I
1(v, u, 0), I
2(v, u ,-u
m) and I
3(v, u ,+u
m), wherein u is axial normalization optical coordinate, v is horizontal normalization optical coordinate;
(4) with I
1(v, u, 0) subtracts I
2(v, u ,-u
m) obtain the difference I of two width of cloth images
A(v, u), I
1(v, u, 0) subtracts I
3(v, u ,+u
m) obtain the difference I of two width of cloth images
B(v, u), I
2(v, u ,-u
m) subtract I
3(v, u ,+u
m) obtain the difference I of two width of cloth images
C(v, u), by I
A(v, u), I
B(v, u), I
C(v, u) be improved axial resolution image I (v, u):
A kind of have an axial high-resolution three differential confocal microscope imaging device, comprising: the optical system of confocal microscope, photodetection place pin hole, the axial micro-displacement apparatus of pin hole, signal Synchronization device, the axial micro-displacement driving device of pin hole, signal inspection device, data collector and data processing equipment; Photodetection place pin hole is fixed on axial micro-displacement apparatus one end of pin hole, the axial micro-displacement apparatus other end of pin hole is fixed on the substrate, send the axial micro-displacement driving device of signal controlling pin hole by the signal Synchronization device and send drive signal, drive the axial location that the axial micro-displacement apparatus of pin hole moves photodetection place pin hole, translation stage displacement signal, the axial micro-displacement apparatus drive signal of pin hole, data collector trigger pip are carried out synchronously by synchronizing circuit; When photodetection place pin hole move finish after, move the point by point scanning sample by translation stage, data collector is by the signal inspection device images acquired simultaneously, the axial micro-displacement apparatus of pin hole moves the axial location of photodetection place pin hole, makes photodetection place pin hole light collecting lens focus place, the defocused u in the optical system of confocal microscope respectively
mPlace and burnt preceding u
mPlace, images acquired I successively
1(v, u, 0), I
2(v, u ,-u
m) and I
3(v, u ,+u
m), wherein u is axial normalization optical coordinate, v is horizontal normalization optical coordinate, u
mBe the normalization optical coordinate of photodetection place pin hole apart from the light collecting lens focal length; The periodic drive signal f that the axial micro-displacement driving device of signal Synchronization device control pin hole produces
d(t) be:
Wherein T promptly gathers three width of cloth image required times at cycle of drive signal, and E is the amplitude of signal, and t is the sampling time, and when 0＜t≤T/3, photodetection place pin hole is in the defocused u of light collecting lens
mPlace, images acquired I at this moment
2(v, u ,-u
m); When T/3＜t≤2T/3, photodetection place pin hole is in light collecting lens focus place, this moment images acquired T
1(v, u, 0); When 2T/3＜t≤T, photodetection place pin hole is in u before light collecting lens Jiao
mPlace, images acquired I at this moment
3(v, u ,+u
m); The image of data collector collection is delivered to and is handled image in the data processing equipment, with I
1(v, u, 0) subtracts I
2(v, u ,-u
m) must I
A(v, u), I
1(v, u, 0) subtracts I
3(v, u ,+u
m) must I
B(v, u), I
2(v, u ,-u
m) subtract I
3(v, u ,+u
m) must I
C(v, u), then obtain the image I that axial resolution improved (v u) is:
The position that the present invention utilizes the axial micro-displacement apparatus of pin hole to move photodetection place pin hole makes it respectively at the focus place of light collecting lens, defocused u
mPlace and burnt preceding u
mThe place, images acquired successively is to realize three differential detections of image.Work as u
m=5.21, during v=0, I
1(0, u, 0), I
2(0, u ,-u
m) and I
3(0, u ,+u
m) as shown in Figure 4.(0, halfwidth u) is significantly less than I to I as we can see from the figure
1The halfwidth of (0, u, 0), promptly azimuthal resolution improves.
The present invention compared with prior art has following advantage:
(1) the present invention utilizes the three differential confocal microscopic imaging technology, improves the axial resolution of confocal microscope.
(2) the present invention adopts the time domain beam split, the position that utilizes the axial micro-displacement apparatus of pin hole to move photodetection place pin hole, to obtain the image of pin hole at three diverse locations, this compares with the space beam split that light is divided into three tunnel, more reasonably utilize luminous energy, and can guarantee the stability of displacement.
(3) the present invention is simple in structure, and environment for use is less demanding, is easy to realize.
Description of drawings
Fig. 1 method and apparatus pie graph of the present invention, wherein 1 is light source, 2 is pin hole, 3 is beam expander, and 4 is semi-transparent semi-reflecting lens, and 5 is object lens, 6 is translation stage, and 7 is sample, and 8 is light collecting lens, 9 is the axial micro-displacement apparatus of pin hole, 10 photodetection place pin holes, and 11 is signal inspection device, 12 is the signal Synchronization device, and 13 is data collector, and 14 is data processing equipment, 15 is the axial micro-displacement driving device of pin hole, and 16 is the optical system of confocal microscope;
The axial micro-displacement apparatus schematic diagram of Fig. 2 photodetection place pin hole, wherein 9 is the axial micro-displacement apparatus of pin hole, and 10 are photodetection place pin hole, and 17 is substrate;
Confocal microscope schematic diagram among Fig. 3 the present invention, wherein 20 is pointolite, and 21 is object lens, and 22 is semi-transparent semi-reflecting lens, and 23 is sample, and 24 is light collecting lens, and 25 are photodetection place pin hole, and 26 is detector;
Confocal microscope axial response simulation curve among Fig. 4 the present invention;
The periodic drive signal synoptic diagram that the axial micro-displacement driving device of signal Synchronization device control pin hole produces among Fig. 5 the present invention.
Specific embodiments
As shown in Figure 1, 2, the present invention is made up of the axial micro-displacement apparatus 9 of optical system 16, photodetection place pin hole 10, pin hole, signal Synchronization device 12, the axial micro-displacement driving device 15 of pin hole, signal inspection device 11, data collector 13 and the data processing equipment 14 of confocal microscope.Wherein, the optical system 16 of confocal microscope is made up of light source 1, pin hole 2, beam expander 3, semi-transparent semi-reflecting lens 4, object lens 5 and light collecting lens 8; The light that light source 1 sends is converged on the sample 7 a bit by behind pin hole 2, beam expander 3 and the semi-transparent semi-reflecting lens 4 by object lens 5, by the mobile point by point scanning of finishing sample 7 of signal Synchronization device 12 control translation stages 6.The light that sample 7 reflects returns by original optical path, after semi-transparent semi-reflecting lens 4 reflections, surveyed by signal inspection device 11 by light collecting lens 8 and photodetection place pin hole 10, the signal that data collector 13 collection is detected is handled it by data processing equipment 14 then.
Specific embodiment of the present invention is as follows: stick with glue agent photodetection place pin hole 10 is bonded on axial micro-displacement apparatus 9 one ends of pin hole, the axial micro-displacement apparatus 9 of pin hole adopts piezoelectric scanner in the present embodiment, and axial micro-displacement apparatus 9 other ends of pin hole are bonded on the substrate 17.Move vertically by the axial micro-displacement apparatus 9 control photodetection place pin holes 10 of pin hole.At first, signal Synchronization device 12 sends control signal, and the signal Synchronization device in the present embodiment is the synchronizing circuit plate, and the axial micro-displacement driving device 15 of control pin hole produces drive signal.The axial micro-displacement driving device 15 of pin hole is a high-voltage amplifier in the present embodiment, and high-voltage amplifier produces drive voltage signal, drives the axial location that the axial micro-displacement apparatus 9 of pin hole moves photodetection place pin hole 10.Undertaken synchronously by 12 pairs of translation stage 6 displacement signals of signal Synchronization device, axial micro-displacement apparatus 9 drive signals of pin hole, data collector 13 trigger pips, data collector 13 adopts image pick-up card in the present embodiment.When photodetection place pin hole 10 move finish after, move point by point scanning sample 7 by translation stage 6, simultaneously data collector 13 images acquired.The axial micro-displacement apparatus 9 of pin hole moves the axial location of photodetection place pin hole 10, makes photodetection place the pin hole 10 8 focus places of the light collecting lens in the optical system 16 of confocal microscope, defocused u respectively
mPlace and burnt preceding u
mPlace, images acquired I successively
1(v, u, 0), I
2(v, u ,-u
m) and I
3(v, u ,+u
m), wherein u is axial normalization optical coordinate, v is horizontal normalization optical coordinate, u
mBe the normalization optical coordinate of photodetection place pin hole 10 apart from light collecting lens 8 focal lengths; The cycle drive voltage signal f that signal Synchronization device 12 control high-voltage amplifiers produce
d(t) be:
Wherein T promptly gathers three width of cloth image required times at cycle of drive voltage signal, and E is the amplitude of signal.Drive voltage signal as shown in Figure 5.The displacement and the drive voltage signal of piezoelectric ceramics are linear, so u can be expressed as u (t)=af
d(t), a is a constant, the displacement of expression piezoelectric ceramics and the ratio of its drive voltage signal.When 0＜t≤T/3, photodetection place pin hole 10 is in light collecting lens 8 defocused u
mPlace, images acquired I at this moment
2(v, u ,-u
m); When T/3＜t≤2T/3, photodetection place pin hole 10 is in light collecting lens 8 focus places, this moment images acquired I
1(v, u, 0); When 2T/3＜t≤T, photodetection place pin hole 10 be in light collecting lens 8 burnt before u
mPlace, images acquired I at this moment
3(v, u ,+u
m).
At data processing equipment 14, promptly handle image in the computing machine, with I
1(v, u, 0) subtracts I
2(v, u ,-u
m) must I
A(v, u), I
1(v, u, 0) subtracts I
3(v, u ,+u
m) must I
B(v, u), I
2(v, u ,-u
m) subtract I
3(v, u ,+u
m) must I
C(v, u), then obtain the image I that axial resolution improved (v u) is:
In above-mentioned measuring process, need to optimize the normalization optical coordinate u of photodetection place pin hole 10 apart from light collecting lens 8 focal positions
mAccording to Chinese invention patent " three differential confocal microscopic imaging method and device " application number be 200410073652.4 described I (0, the u when u) axial resolution is maximum
mCan determine by following formula is optimum:
Work as u
m=± 5.21 o'clock, and sensitivity k (0,0, u
m) corresponding absolute value maximum, the axial resolution of this moment is the highest.Fig. 4 is u
m=± 5.21 o'clock I
1(0, u, 0), I
2(0, u ,-u
m) and I
3(0, u ,+u
m) response curve, (0, halfwidth u) is significantly less than I to I as we can see from the figure
1The halfwidth of (0, u, 0), promptly azimuthal resolution improves.
The content that is not described in detail in the instructions of the present invention belongs to this area professional and technical personnel's known prior art.
Claims (6)
1. one kind has axial high-resolution three differential confocal microscope formation method, it is characterized in that performing step is as follows:
(1) photodetection place pin hole is fixed on axial micro-displacement apparatus one end of pin hole, the axial micro-displacement apparatus other end of pin hole is fixed on the substrate; Send drive signal by the axial micro-displacement driving device of signal Synchronization device control pin hole, drive the axial micro-displacement apparatus of pin hole and change photodetection place pin hole position, successively pin hole is placed the optical system light collecting lens focus place of confocal microscope, defocused u
mPlace and burnt preceding u
mPlace, wherein u
mBe the normalization optical coordinate of pin hole apart from the light collecting lens focal length;
(2) the signal Synchronization device is realized sample sweep signal, the axial micro-displacement apparatus drive signal of pin hole, the data collector trigger pip synchronously, guarantee after photodetection place pin hole moves, again to tested sample point by point scanning, and acquired signal simultaneously;
(3) three positions of above-mentioned pin hole in step (1), i.e. photodetection place pin hole lays respectively at light collecting lens focus place, defocused u
mPlace and burnt preceding u
mDuring the place, by the periodic drive signal f of the axial micro-displacement driving device generation of signal Synchronization device control pin hole
d(t) be:
Wherein T promptly gathers three width of cloth image required times at cycle of drive signal, and E is the amplitude of signal, and t is the sampling time, and when 0＜t≤T/3, photodetection place pin hole is in the defocused u of light collecting lens
mPlace, images acquired I at this moment
2(v, u ,-u
m); When T/3＜t≤2T/3, photodetection place pin hole is in light collecting lens focus place, this moment images acquired I
1(v, u, 0); When 2T/3＜t≤T, photodetection place pin hole is in u before light collecting lens Jiao
mPlace, images acquired I at this moment
3(v, u ,+u
m); Wherein u is axial normalization optical coordinate, and v is horizontal normalization optical coordinate;
(4) with I
1(v, u, 0) subtracts I
2(v, u ,-u
m) obtain the difference I of two width of cloth images
A(v, u), I
1(v, u, 0) subtracts I
3(v, u ,+u
m) obtain the difference I of two width of cloth images
B(v, u), I
2(v, u ,-u
m) subtract I
3(v, u ,+u
m) obtain the difference I of two width of cloth images
C(v, u), by I
A(v, u), I
B(v, u), I
C(v, u) be improved axial resolution image I (v, u):
2. according to claim 1 have an axial high-resolution three differential confocal microscope formation method, and it is characterized in that: the optical system of described confocal microscope (16) comprises light source (1), pin hole (2), beam expander (3), semi-transparent semi-reflecting lens (4), object lens (5) and light collecting lens (8); The light that light source (1) sends is by behind pin hole (2), beam expander (3) and the semi-transparent semi-reflecting lens (4), converged on the sample (7) a bit by object lens (5), by the mobile point by point scanning of finishing sample (7) of translation stage (6); The light that sample (7) reflects returns by original optical path, after semi-transparent semi-reflecting lens (4) reflection, is surveyed by signal inspection device (11) by light collecting lens (8) and photodetection place pin hole (10).
3. according to claim 1 have an axial high-resolution three differential confocal microscope formation method, it is characterized in that: the axial micro-displacement apparatus of described pin hole (9) can be the displacement driver that piezoelectric scanner, ferro-electricity single crystal, voice coil motor, linear electric motors or rare-earth magnetostrictive element are made.
4. one kind has axial high-resolution three differential confocal microscope imaging device, it is characterized in that comprising: the optical system of confocal microscope (16), photodetection place pin hole (10), the axial micro-displacement apparatus of pin hole (9), signal Synchronization device (12), the axial micro-displacement driving device of pin hole (15), signal inspection device (11), data collector (13) and data processing equipment (14); Photodetection place pin hole (10) is fixed on axial micro-displacement apparatus (9) one ends of pin hole, the axial micro-displacement apparatus of pin hole (9) other end is fixed on the substrate (17), send the axial micro-displacement driving device of signal controlling pin hole (15) by signal Synchronization device (12) and send drive signal, drive the axial location of the axial micro-displacement apparatus of pin hole (9) mobile photodetection place pin holes (10), translation stage (6) displacement signal, the axial micro-displacement apparatus of pin hole (9) drive signal, data collector (13) trigger pip are carried out synchronously by signal Synchronization device (12); When photodetection place pin hole (10) move finish after, by the mobile point by point scanning sample of translation stage (6) (7), data collector (13) is gathered the picture signal that signal inspection device (11) detects simultaneously, the axial location of the mobile photodetection place pin hole of the axial micro-displacement apparatus of pin hole (9) (10) makes photodetection place pin hole (10) light collecting lens (8) focus place, the defocused u in the optical system (16) of confocal microscope respectively
mPlace and burnt preceding u
mPlace, images acquired I successively
1(v, u, 0), I
2(v, u ,-u
m) and I
3(v, u ,+u
m), wherein u is axial normalization optical coordinate, v is horizontal normalization optical coordinate, u
mBe the normalization optical coordinate of photodetection place pin hole (10) apart from light collecting lens (8) focal length; The periodic drive signal f that signal Synchronization device (12) the control axial micro-displacement driving device of pin hole (15) produces
d(t) be:
Wherein T promptly gathers three width of cloth image required times at cycle of drive signal, and E is the amplitude of signal, and t is the sampling time, and when 0＜t≤T/3, photodetection place pin hole (10) is in the defocused u of light collecting lens (8)
mPlace, images acquired I at this moment
2(v, u ,-u
m); When T/3＜t≤2T/3, photodetection place pin hole (10) is in light collecting lens (8) focus place, this moment images acquired I
1(v, u, 0); When 2T/3＜t≤T, photodetection place pin hole (10) be in light collecting lens (8) burnt before u
mPlace, images acquired I at this moment
3(v, u ,+u
m); The image that data collector (13) is gathered is delivered to and is handled image in the data processing equipment (14), with I
1(v, u, 0) subtracts I
2(v, u ,-u
m) must I
A(v, u), I
1(v, u, 0) subtracts I
3(v, u ,+u
m) must I
B(v, u), I
2(v, u ,-u
m) subtract I
3(v, u ,+u
m) must I
C(v, u), then obtain the image I that axial resolution improved (v u) is:
5. according to claim 4 have an axial high-resolution three differential confocal microscope imaging device, and it is characterized in that: the optical system of described confocal microscope (16) comprises light source (1), pin hole (2), beam expander (3), semi-transparent semi-reflecting lens (4), object lens (5) and light collecting lens (8); The light that light source (1) sends is by behind pin hole (2), beam expander (3) and the semi-transparent semi-reflecting lens (4), converged on the sample (7) a bit by object lens (5), by the mobile point by point scanning of finishing sample (7) of translation stage (6); The light that sample (7) reflects returns by original optical path, after semi-transparent semi-reflecting lens (4) reflection, is surveyed by signal inspection device (11) by light collecting lens (8) and photodetection place pin hole (10).
6. according to claim 4 have an axial high-resolution three differential confocal microscope imaging device, it is characterized in that: the axial micro-displacement apparatus of described pin hole (9) can be the displacement driver that piezoelectric scanner, ferro-electricity single crystal, voice coil motor, linear electric motors or rare-earth magnetostrictive element are made.
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| CN103190889A (en) * | 2013-04-17 | 2013-07-10 | 北京大学 | Real-time tunable confocal microscopic imaging system |
| CN103674926A (en) * | 2012-08-29 | 2014-03-26 | 日立视听媒体股份有限公司 | Optical device |
| CN104635757A (en) * | 2014-12-09 | 2015-05-20 | 中国科学院苏州生物医学工程技术研究所 | Confocal microscopy pinhole positioning control method |
| CN109357623A (en) * | 2018-11-16 | 2019-02-19 | 中国科学院光电技术研究所 | A kind of method and apparatus with confocal microscope system measurement mobile phone faceplate thickness |
| CN112197714A (en) * | 2020-09-28 | 2021-01-08 | 东北电力大学 | Free-form surface measuring device and detection method based on differential color confocal technology |
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|---|---|---|---|---|
| CN103674926A (en) * | 2012-08-29 | 2014-03-26 | 日立视听媒体股份有限公司 | Optical device |
| CN103674926B (en) * | 2012-08-29 | 2016-01-20 | 日立乐金光科技株式会社 | Optical devices |
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| CN103190889A (en) * | 2013-04-17 | 2013-07-10 | 北京大学 | Real-time tunable confocal microscopic imaging system |
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| CN104635757A (en) * | 2014-12-09 | 2015-05-20 | 中国科学院苏州生物医学工程技术研究所 | Confocal microscopy pinhole positioning control method |
| CN109357623A (en) * | 2018-11-16 | 2019-02-19 | 中国科学院光电技术研究所 | A kind of method and apparatus with confocal microscope system measurement mobile phone faceplate thickness |
| CN112197714A (en) * | 2020-09-28 | 2021-01-08 | 东北电力大学 | Free-form surface measuring device and detection method based on differential color confocal technology |
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