CN101126834B - An in-plane scanning method and system for a point-scanning laser confocal microscope - Google Patents
An in-plane scanning method and system for a point-scanning laser confocal microscope Download PDFInfo
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Abstract
本发明公开了一种用于点扫描激光共焦显微镜的扫描方法和系统，该方法首先将样品台、计算机和扫描共焦显微镜测量物镜构成激光共焦扫描显微镜的三维扫描系统，实现自动控制扫描；然后在样品台上对被测样品定位；三维扫描系统使样品台依次进行旋转、一维横向移动，直至被测样品扫描完一层，接着复位进行轴向扫描被测样品下一层，直至被测样品测量完毕。该方法在于不改变光路的情况下，通过样品台的旋转移动完成扫描，克服了以往扫描台存在的机构复杂，扫描速度慢的缺点。主要应用于点扫描的激光共焦显微镜中，但也可用于类似的光点扫描测量系统中。
The invention discloses a scanning method and system for a point-scanning laser confocal microscope. In the method, a sample stage, a computer and a scanning confocal microscope measuring objective lens are firstly used to form a three-dimensional scanning system of a laser confocal scanning microscope to realize automatic control of scanning. ; Then position the sample to be tested on the sample stage; the three-dimensional scanning system makes the sample stage rotate and move one-dimensionally laterally until the sample to be tested scans one layer, and then resets to axially scan the next layer of the sample to be tested until The measured sample is measured. The method is to complete the scanning through the rotation and movement of the sample stage without changing the optical path, which overcomes the disadvantages of complex mechanism and slow scanning speed existing in the previous scanning stage. Mainly used in point-scanning laser confocal microscopes, but can also be used in similar spot-scanning measurement systems.
Description
Technical field
The invention belongs to the technical field of photoelectric detection that scientific instrument such as biology, medical treatment, minute manufacturing are used, relate to the scan method of confocal laser scanning microscope, particularly a kind of microscopical interscan method and system of point scanning laser confocal that be used for.
Background technology
Confocal laser scanning microscope is compared with simple microscope, has higher resolution and tomography ability, is widely used in the scientific instrument of biology, life, medical treatment and micro-nano manufacturing and material etc.(X, Y) scan method adopts the beam flying mode more in the face in the existing confocal laser scanning microscope, adopts device such as galvanometer to realize X, Y scanning usually.And adopt the scan mode of movement of objects to be bidimensional rectilinear scanning.The former benefit is to improve measuring speed greatly, but the system architecture complexity, to optical system quality requirements harshness; Latter's light path is stable, and system architecture is simple, simultaneously owing to only need to consider to eliminate axle and go up a some aberration and get final product, so the reduction of optical system cost, but sweep velocity is slow, is difficult to practicability.
Summary of the invention
Defective or deficiency at above-mentioned prior art is created the object of the present invention is to provide a kind of microscopical interscan method and system of point scanning laser confocal that can be used for, and it is simple that this method has mechanism simultaneously, and light path is stable, has high sweep velocity simultaneously.
In order to realize above-mentioned task, the present invention takes following technical solution:
A kind of microscopical interscan method of point scanning laser confocal that be used for is characterized in that this method comprises the following steps:
Step 4, the process of repeating step three finishes until the sample measurement.
Scanning sequency can be that rotation sweep and transversal scanning hocket, and also can be that synchronous coordination carries out.Transversal scanning refers to measure perpendicular to scanning confocal microscope any one direction (being made as directions X) of objective lens optical axis, and axial scan refers to the direction parallel with the aforementioned lights direction of principal axis (being made as the Z direction).Laterally moving can be by realizations such as straight-line motion mechanism such as stepper motor, servomotors, and rotation can be by realizations such as various rotating mechanisms such as electric rotating machines.Axial scan can be that sample stage moves, and also can be that scanning confocal microscope measurement object lens move, and can also be other axial scanning method.Described scanning mechanism links to each other with computing machine, realizes automatic gated sweep.This method is applicable to the confocal scan microscope of any form such as reflective, transmission-type etc.
Realize system for carrying out said process, this system comprises light-source system and measuring system, it is characterized in that:
Described light-source system includes light source, and first optical filter, convergent lens, first pin hole, collimation lens, neutral colour filter, spectroscope and observing system are arranged on the light path of light source;
Described measuring system includes microcobjective, and microcobjective is provided with second optical filter, light collecting lens, second pin hole, photodetector, amplifying circuit;
The microcobjective of measuring system and sample are aimed at;
Sample stage is used to place sample, and this sample stage mainly comprises rotating mechanism and X displacement platform; If axial scan adopts sample stage to move, this scan table also comprises z to scanning mechanism (as shown in Figure 1), and object lens move if axial scan adopts measurement, and this scan table does not comprise that Z is to scanning mechanism; Sample stage links to each other with a computing machine, and computing machine links to each other with the amplifying circuit of measuring system.
The present invention the rotation by sample stage, moves and can finish scanning under the situation that does not change light path, overcome mechanism's complexity that scan table in the past exists simultaneously, the shortcoming that sweep velocity is slow.The present invention is mainly used in the laser scanning confocal microscopy of spot scan, but also can be used in the similar spot scan measuring system.
Description of drawings
Fig. 1 is the laser confocal scanning microscope system synoptic diagram.
Fig. 2 is sample worktable transversal scanning movement locus (face interscan method) synoptic diagram.
Label is wherein represented respectively: 1, light source, 2, first optical filter, 3, convergent lens, 4, first pin hole, 5, collimation lens, 6, neutral colour filter, 7, microcobjective, 8, sample, 9, spectroscope, 10, observing system, 11, second optical filter, 12, light collecting lens, 13, second pin hole, 14, photodetector, 15, amplifying circuit, 16, computing machine, 17, z is to scanning mechanism, and 18, rotating mechanism, 19, the X displacement platform.
The invention will be further described below in conjunction with accompanying drawing.
Embodiment
Referring to Fig. 1, the scanning confocal microscope that the present invention relates to is measured the laser confocal scanning microscope system that object lens constitute, and comprises light-source system and measuring system; Described light-source system includes light source 1, and first optical filter 2, convergent lens 3, first pin hole 4, collimation lens 5, neutral colour filter 6, spectroscope 9 and observing system 10 are arranged on the light path of light source 1;
Measuring system includes microcobjective 7, and microcobjective 7 is provided with second optical filter 11, light collecting lens 12, second pin hole 13, photodetector 14, amplifying circuit 15;
The microcobjective 7 of measuring system and test sample product 10 are aimed at;
Sample stage is used to place sample 10, and this sample stage comprises that mainly z is to scanning mechanism, rotating mechanism and X displacement platform; Sample stage links to each other with a computing machine 16, and computing machine 16 links to each other with the amplifying circuit 15 of measuring system.
Wherein, the X displacement platform is measured objective lens optical axis perpendicular to scanning confocal microscope, and z is parallel to scanning confocal microscope to scanning mechanism and measures objective lens optical axis.
Laterally move by stepper motor or servomotor and realize that rotation is realized by electric rotating machine.
Said confocal scan microscope can be reflective (as shown in Figure 1) also can make transmission-type.
Amplifying circuit 15 adopts the amplifying circuit of this area routine.
Referring to Fig. 2, Fig. 2 is sample worktable transversal scanning movement locus of the present invention (face interscan method) synoptic diagram.The light that light source 1 sends is selected the light of required wave band by first optical filter 2, this light converges to first pin hole 4 by convergent lens 3, filtered back then produces directional light by collimation lens 5, enters microcobjective 7 through neutral colour filter 6 and spectroscope 9, shines on the tested exemplar 8 again; Light by sample 8 scatterings turns back to lens 7, arrives observing system 10 through a spectroscope part again, and a part arrives photodetectors 14 by second optical filter 11, light collecting lens 12 and pin hole 13.If the measured point of the correspondence of first pin hole 4, second pin hole 13 and sample 8 is in conjugation, the light intensity value maximum that detects of detector then.Tested sampling point to sample 8 correspondences carries out axial scan by computing machine 16 control Z to scanning mechanism, can find its corresponding conjugate points position.But, just must carry out point by point scanning for whole exemplar is measured.Characteristics of the present invention just are its scanning new method.Transversal scanning all is the scanning of adopting mutually perpendicular bidimensional X, Y direction in the scan method in the past, and the present invention adopts the method for one dimension rotation and one dimension translation to realize transversal scanning.After as accompanying drawing 2,2 (a) expression rotating mechanism 18 sample 8 whenever being turned around, x direction displacement platform 19 makes sample 8 move a fixed range along the x direction, is scanned up to the face of whole sample 8 to finish.If the initial alignment point of sample 8 is its center A, then the x direction of scanning can just can be born, and locates up to scanning maximum magnitude (circumference at B point place); If initial alignment point is edge's (B point as shown in FIG.), then the x direction of scanning is for to move towards the center of sample 8, till the A point of sample center.Move a fixed range and the analyzing spot movement locus that obtains in the x direction when Fig. 2 (b) expression rotating mechanism 18 makes sample 8 rotate a week, with the same reason of Fig. 2 (a), starting point can be the A point, also can be the B point.After transversal scanning is finished, finish to scanning mechanism 17 and axial move the transversal scanning of following one deck by Z.Hocket successively axially and transversal scanning, until the scanning of finishing whole exemplar measurement range.
Below in conjunction with embodiment feasibility of the present invention is analyzed.
Suppose and use enlargement factor to be 40X, numerical aperture is 0.65 microcobjective, the about 0.43 μ m of its horizontal theoretical resolution.We set horizontal sampling interval is 0.5 μ m, and promptly the fixed range that at every turn moves of x direction is 0.5 μ m, is 0.5 μ m also in adjacent 2 circumference distance of each circumference up-sampling.Photodetector 14 in the system adopts photomultiplier, because the response time of photomultiplier transit light is 10
-9The s magnitude is so can guarantee the consistance of each sampled point time shutter by controlling of sampling.The cross measure scope is set at 250 μ m (the visual field size that is equivalent to the 40X microcobjective).So the x direction moves 250/2/0.5=250 (individual interval), mean that also corotation moving 250 changes.If rotating mechanism 18 makes that the rotating speed of sample 8 is 1000 revolutions per seconds, and the scan mode of employing Fig. 2 (a), the time of then finishing a whole transversal scanning is 250 * 1000
-1=0.25 (s), even 100 layers of axial scans like this, the needed time only is 25s.As seen this method not only can guarantee scanning accuracy also raising greatly of its sweep velocity simultaneously, satisfies the demand of actual measurement fully.
Claims (1)
1. one kind is used for microscopical interscan method of point scanning laser confocal, it is characterized in that this method comprises the following steps:
Step 1, with light source (1), first optical filter (2), convergent lens (3), first pin hole (4), collimation lens (5), neutral colour filter (6), spectroscope (9) and observing system (10) are formed light-source system, with microcobjective (7), second optical filter (11), light collecting lens (12), second pin hole (13), photodetector (14) and amplifying circuit (15) are formed measuring system, sample stage is linked to each other with computing machine, computing machine links to each other with the amplifying circuit (15) of measuring system, realize automatic gated sweep, wherein sample stage is by z scanning direction mechanism, rotating mechanism and x direction displacement platform are formed in the xy plane;
Step 2, on sample stage, sample (8) is located, the light that light source (1) is sent is selected the light of required wave band by first optical filter (2), this light converges to first pin hole (4) by convergent lens (3), filtered back then produces directional light by collimation lens (5), pass through neutral colour filter (6) successively and spectroscope (9) enters microcobjective (7), shine sample again (on 8, light by sample (8) scattering turns back to microcobjective (7), arrive observing system (10) through a spectroscope part again, a part is successively by second optical filter (11), light collecting lens (12) and second pin hole (13) arrive photodetector (14), and photodetector (14) links to each other with computing machine (16) by amplifying circuit (15);
Step 3, by rotating mechanism and x direction displacement platform in computer control z scanning direction mechanism, the xy plane make sample stage carry out in the xy plane rotation successively, the x direction moves, scanned one deck until sample, then reset and carry out one deck under the z scanning direction sample, track while scan to every layer of sample is concentric circles line or helix, and wherein the optical axis direction of microcobjective is parallel to the z direction;
Step 4, the process of repeating step three finishes until the sample measurement.
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| CN102262151B (en) * | 2010-05-27 | 2013-12-04 | 麦克奥迪实业集团有限公司 | Method for quickly scanning slice by microscope |
| US8390926B2 (en) * | 2010-08-12 | 2013-03-05 | Photon Dynamics, Inc. | High speed acquisition vision system and method for selectively viewing object features |
| CN102101642B (en) * | 2011-01-28 | 2013-01-02 | 清华大学 | Nano manufacturing system |
| CN102566023B (en) * | 2012-01-11 | 2015-09-30 | 麦克奥迪实业集团有限公司 | A kind of digital slide real time scanning automatic focusing system and method thereof |
| CN102679917A (en) * | 2012-05-08 | 2012-09-19 | 首钢总公司 | Method for detecting continuous casting blank surface oscillation marks by laser |
| CN102984423A (en) * | 2012-11-26 | 2013-03-20 | 中国科学院长春光学精密机械与物理研究所 | Spot scanning imaging method for trigger exposure of encoder |
| US20160274042A1 (en) * | 2013-11-12 | 2016-09-22 | X-Ray Optical Systems, Inc. | Non-homogeneous sample scanning apparatus, and x-ray analyzer applications thereof |
| CN105044895B (en) * | 2015-06-01 | 2017-06-06 | 西安交通大学 | A kind of super-resolution confocal microscopic imaging apparatus and method |
| CN105596088B (en) * | 2015-12-22 | 2018-05-18 | 吉林亚泰中科医疗器械工程技术研究院股份有限公司 | Adjustable three imaging shaft mechanisms based on skin confocal system |
| CN109363767B (en) * | 2018-09-22 | 2024-03-29 | 上海市激光技术研究所有限公司 | Laser output device and adjustment method |
| CN109470666B (en) * | 2018-11-07 | 2023-11-24 | 上海萨迦生物科技有限公司 | Near infrared laser confocal biochip scanner |
| CN113758912A (en) * | 2021-09-03 | 2021-12-07 | 中国工程物理研究院激光聚变研究中心 | Full-surface analysis system for free-form surface sample |
| CN117130145A (en) * | 2023-10-31 | 2023-11-28 | 深圳市凯佳光学科技有限公司 | Multispectral digital light processing projection system and digital light illumination control method |
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|---|---|---|---|---|
| US5532874A (en) * | 1992-12-18 | 1996-07-02 | Morphometrix Inc. | Multiscanning confocal microscopy |
| CN1791822A (en) * | 2003-05-20 | 2006-06-21 | 卢西德有限公司 | Confocal microscope for imaging of selected locations of the body of a patient |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| US5532874A (en) * | 1992-12-18 | 1996-07-02 | Morphometrix Inc. | Multiscanning confocal microscopy |
| CN1791822A (en) * | 2003-05-20 | 2006-06-21 | 卢西德有限公司 | Confocal microscope for imaging of selected locations of the body of a patient |
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