CN102818797B - Vector type scanning method of laser scanning confocal microscope - Google Patents
Vector type scanning method of laser scanning confocal microscope Download PDFInfo
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- CN102818797B CN102818797B CN201210264322.8A CN201210264322A CN102818797B CN 102818797 B CN102818797 B CN 102818797B CN 201210264322 A CN201210264322 A CN 201210264322A CN 102818797 B CN102818797 B CN 102818797B
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Abstract
The invention relates to a vector type scanning method of a laser scanning confocal microscope. The method comprises the following steps: pre-scanning a sample rapidly; carrying out ROI (region of interest) identification on the pre-scanned sample; carrying out vector type scanning path generation on the sample according to the ROI identification, wherein the generated scanning path covers the sample ROI; carrying out vector fine scanning on the sample ROI according to the vector type scanning path; and under a driving function of a controller, rotating a scanning galvanometer according to the vector type scanning path and obtaining a scanning image of the sample by the laser scanning confocal microscope.
Description
[technical field]
The present invention relates to a kind of scan imaging method of laser scanning confocal micro-scope, be specifically related to a kind of vector expression scan method of laser scanning confocal micro-scope.
[background technology]
Laser scanning confocal micro-scope (Laser Scanning Confocal Microscope, LSCM) utilize laser spots as the exciting light of fluorescence and by scanister, continuous sweep carried out to sample, and stopping a kind of microscope of focal plane light and imaging by spatial conjugation diaphragm (pin hole); Laser scanning confocal micro-scope is the effective technology means of research sub-micron fine structure, is the large-scale scientific research apparatus of the scientific worker's indispensability being engaged in biomedicine and material science research.
As shown in Figure 1, be two galvanometer X-Y scanning mechanisms of laser scanning confocal micro-scope, comprise controller and X-Y scanning galvanometer, described controller for driving, the motion of gated sweep galvanometer.Above-mentioned laser scanning confocal micro-scope adopts the imaging mode of spot scan, needs to enable analyzing spot travel through whole microscopic field of view in scanning process.Wherein, the scanning angle θ of galvanometer
x, θ
ythere are certain mapping relations with the point (x, y) on X-Y plane:
Specifically, the scanning process of laser scanning confocal micro-scope is divided into coarse scanning and close scanning, lines by line scan in above-mentioned coarse scanning process, and the number of scanning lines few (spacing of every line scanning is larger), roughly depicts image outline; In above-mentioned close scanning process, still adopt the mode of lining by line scan, but the spacing of every line scanning reduces, the number of scanning lines is increased sharply, and due in scanning process, scanning galvanometer needs back and forth movement, and is only going journey to enter line scanning.Therefore, for effectively reducing the galvanometer regulation time in scanning process, need a backhaul on a large scale fast, but scanning galvanometer is strenuous vibration in this process, accelerating the aging of galvanometer plated film, reduce the serviceable life of galvanometer system.
[summary of the invention]
The technical problem to be solved in the present invention is: for the above-mentioned defect of prior art, a kind of vector expression scan method of laser scanning confocal micro-scope is provided, galvanometer regulation time in the effective minimizing scanning galvanometer course of work, without the quick-speed large-scale return stroke of pure line scanning switching mode, be of value to and extend galvanometer system serviceable life.
For solving the problems of the technologies described above, the vector expression scan method that the invention provides a kind of laser scanning confocal micro-scope comprises:
Step one, quick prescan is carried out to sample;
Step 2, ROI identification is carried out to the sample after quick prescan;
Step 3, to identify based on described ROI, the generation of vector expression scanning pattern is carried out to sample, the scanning pattern Covering samples ROI of generation;
Step 4, carry out vector fine scanning according to described vector expression scanning pattern to sample ROI: under the driving of controller, rotate according to vector expression scanning pattern scanning galvanometer, laser scanning confocal micro-scope obtains the scan image of sample.
Preferably, described quick prescan is that scanning galvanometer carries out rough scanning in the mode of line scanning to sample, obtains the essential information of sample ROI.
Preferably, after execution step 3, before execution step 4, scanning pattern judgement is also comprised; Described scanning pattern judges, judges whether vector expression scanning pattern scope meets scanning demand, as met, then performs step 4; As do not met, then sample ROI being adjusted, performing the coordinates measurement that step 3 re-starts scanning.
Preferably, described vector expression scanning spiral is inside spin scanning or external spiral scanning.
Preferably, described spiral scan is square.
Preferably, described spiral scan is triangular in shape.
Preferably, described spiral scan is rounded.
Preferably, described spiral scan ovalize.
The vector expression scan method of laser scanning confocal micro-scope of the present invention, the sample after quick prescan carries out ROI identification; And the generation of vector expression scanning pattern is carried out to sample ROI, according to scanning pattern, vector fine scanning is carried out to sample ROI.In vector expression scanning process, the coordinated movement of various economic factors of X-direction and Y-direction forms laser facula track while scan, without the quick-speed large-scale return stroke of existing pure line scanning switching mode, the effective galvanometer regulation time reduced in the scanning galvanometer course of work, improves the overall image taking speed of ROI; And owing to adopting spiral scan, avoid the aging problem causing galvanometer plated film in prior art due to strenuous vibration in line scanning galvanometer process, be of value to the serviceable life extending galvanometer.
[accompanying drawing explanation]
Fig. 1 illustrates the schematic diagram of two galvanometer X-Y scanning mechanisms of laser scanning confocal micro-scope in prior art.
Fig. 2 illustrates the theory diagram of laser scanning confocal micro-scope galvanometer scanning system of the present invention.
Fig. 3 illustrates the vector expression scan method process flow diagram of laser scanning confocal micro-scope of the present invention.
Fig. 4 illustrates the process flow diagram of the vector expression scan method of another embodiment of laser scanning confocal micro-scope of the present invention.
Fig. 5 illustrates the sample external spiral scanning schematic diagram of the vector expression scan method of laser scanning confocal micro-scope of the present invention.
Fig. 6 illustrates the sample inside spin spiral scan schematic diagram of the vector expression scan method of laser scanning confocal micro-scope of the present invention.
Fig. 7 illustrates the rectangle spiral scan schematic diagram of the vector expression scan method of laser scanning confocal micro-scope of the present invention.
Fig. 8 illustrates the square spiral scanning schematic diagram of the vector expression scan method of laser scanning confocal micro-scope of the present invention.
Fig. 9 illustrates the triangle spiral scan schematic diagram of the vector expression scan method of laser scanning confocal micro-scope of the present invention.
Figure 10 illustrates the round screw thread scanning schematic diagram of the vector expression scan method of laser scanning confocal micro-scope of the present invention.
Figure 11 illustrates the elliptical spiral scanning schematic diagram of the vector expression scan method of laser scanning confocal micro-scope of the present invention.
[embodiment]
Shown in figure 2, the invention provides a kind of vector expression scan method of laser scanning confocal micro-scope, for laser scanning confocal micro-scope, comprise: two scanning galvanometer 30, feedback units 40 of controller 10, the scanning galvanometer driver 20 connected with controller 10 and orthogonal setting, described controller 10 carries out rotation through scanning galvanometer driver 20 pairs of scanning galvanometers 30 and controls, and adjusts according to the rotation of scanning galvanometer positional information to scanning galvanometer 30 that feedback unit 40 detects.
Shown in figure 3, vector expression scan method of the present invention comprises:
S1, quick prescan is carried out to sample;
S2, the sample after quick prescan carried out to ROI (Region Of Interest, area-of-interest) and identify;
S3, to identify based on described ROI, the generation of vector expression scanning pattern is carried out to sample, the scanning pattern Covering samples ROI of generation;
Described quick prescan is that scanning galvanometer carries out rough scanning in the mode of line scanning to sample, obtains the essential information of sample ROI.
S4, according to described vector expression scanning pattern, vector fine scanning is carried out to sample ROI: under the driving of controller, scanning galvanometer is rotated according to vector expression scanning pattern, obtain the scan image of sample.
Described vector fine scanning is that the vector expression scanning pattern gated sweep galvanometer rotation obtained according to step 3 travels through sample ROI, obtains the scan image of sample.
For the point (x on the scanning pattern generated
i, y
i), utilize mapping relations
Calculate scanning galvanometer pendulum angle (θ
i x, θ
i y), will containing angle information (θ by the controller of laser scanning confocal micro-scope
i x, θ
i y) controlled quentity controlled variable of sequence is sent to scanning galvanometer driver in sample plane, realizes vector expression scanning, finally obtains the scan image generating sample.
Laser scanning confocal micro-scope has wide application at biological and medical fields such as tissue biological, cell biology, molecular biology, genetics, embryology, neurology, pathology, immunology, epidemiology, oncology, bacteriology, virology.In addition, laser scanning confocal micro-scope also has a wide range of applications in the field such as research and development and production testing of the materials such as pottery, metal, semiconductor, chip.
The vector expression scan method of laser scanning confocal micro-scope of the present invention, the sample after quick prescan carries out ROI identification; And the generation of vector expression scanning pattern is carried out to sample ROI, according to scanning pattern, vector fine scanning is carried out to sample ROI, in vector expression scanning process, the coordinated movement of various economic factors of X-direction and Y-direction forms laser facula track while scan, without the quick-speed large-scale return stroke of existing pure line scanning switching mode, galvanometer regulation time in the effective minimizing scanning galvanometer course of work, improves the overall image taking speed of ROI; And owing to adopting spiral scan, avoid the aging problem causing galvanometer plated film in prior art due to strenuous vibration in line scanning galvanometer process, be of value to the serviceable life extending galvanometer.
Below in conjunction with the drawings and specific embodiments, the present invention is described in further detail.
Embodiment 1
Shown in figure 4, vector expression scan method of the present invention comprises:
S11, quick prescan is carried out to sample;
S21, ROI identification is carried out to the sample after quick prescan;
S31, to identify based on described ROI, the generation of vector expression scanning pattern is carried out to sample, the scanning pattern Covering samples ROI of generation;
S41, scanning pattern judgement is carried out to vector expression scanning pattern; Described scanning pattern judges, judges whether vector expression scanning pattern scope meets scanning demand, as met, performs step S61;
S51, as do not met, then sample ROI to be adjusted, performing step S31;
S61, according to described vector expression scanning pattern, vector fine scanning is carried out to sample ROI: under the driving of controller, scanning galvanometer is rotated according to vector expression scanning pattern, obtain the scan image of sample.
Described scanning demand is: described scanning pattern contains the Zone Full of sample ROI, sample image is scanned complete; The invalid scanning area of described scanning pattern is minimum, improves the overall image taking speed of ROI; Also can the follow up demand of user of described scanning pattern adjusts.
Embodiment 2
Shown in figure 5, vector expression scan method of the present invention comprises:
S1, sample (in figure shown in 5a) carried out to quick prescan (in figure shown in 5b);
S2, the sample after quick prescan carried out to ROI identification (in figure shown in 5c);
S3, to identify based on described ROI, external spiral scanning pattern generation (in figure shown in 5d) is carried out to sample, the scanning pattern Covering samples ROI of generation;
S4, according to described external spiral scanning pattern (in figure shown in 5d), vector fine scanning is carried out to sample ROI: under the driving of controller, scanning galvanometer is rotated according to vector expression scanning pattern, obtain the scan image of sample.
Above-mentioned scanning process also can comprise step S41 according to embodiment 1: scanning pattern judges, S51: sample ROI carry out adjusting, S61: vector fine scanning, that is vector expression scanning pattern is in embodiment 1 external spiral scanning pattern.
Wherein, in figure, 5a is the profile schematic diagram of sample, and described sample is that biological cell comprises cell membrane 101, organelle 102 organelle 103, and namely vector expression scan method of the present invention will scan the image information of sample.
Embodiment 3
Shown in figure 6, vector expression scan method of the present invention comprises:
S11, quick prescan (in figure shown in 6b) is carried out to sample (in figure 6a shown in);
S21, the sample after quick prescan carried out to ROI identification (in figure shown in 6c);
S31, to identify based on described ROI, inside spin scanning pattern generation (in figure shown in 6d) is carried out to sample, the scanning pattern Covering samples ROI of generation;
S41, internally helical scan path carry out scanning pattern judgement; Described scanning pattern judges, judges whether external spiral scanning pattern scope meets scanning demand, as met, performs step S61;
S51, as do not met, then sample ROI to be adjusted, performing step S31;
S61, according to described inside spin scanning pattern (in figure shown in 6d), vector fine scanning is carried out to sample ROI: under the driving of controller, scanning galvanometer is rotated according to inside spin scanning pattern, obtain the scan image of sample.
Wherein, in figure, 6a is the profile schematic diagram of sample, and described sample is that biological cell comprises cell membrane 101, organelle 102 organelle 103, and namely vector expression scan method of the present invention will scan the image information of sample.
Embodiment 4
Shown in figure 7,8, vector expression scan method of the present invention, is carrying out in the helical scan path of laser scanning confocal micro-scope according to embodiment 1,2 or 3, and described helical scan path is the scanning of square external spiral or the scanning of square inside spin.
Described square can be rectangle or square, that is described helical scan path is rectangle external spiral scanning (in figure shown in 7a), rectangle inside spin scanning (in figure shown in 7b), square external spiral scanning (in figure shown in 8a) or square inside spin scanning (in figure shown in 8b), generates scanning pattern.
Embodiment 5
Shown in figure 9, vector expression scan method of the present invention, according to embodiment 1 or, 2 or 3 carry out in the helical scan path of laser scanning confocal micro-scope, described helical scan path is triangle external spiral scanning (in figure shown in 9a) or triangle inside spin scanning (in figure shown in 9b), generates scanning pattern.
Embodiment 6
With reference to shown in Figure 10, vector expression scan method of the present invention, according to embodiment 1 or, 2 or 3 carry out in the helical scan path of laser scanning confocal micro-scope, described helical scan path is circular external spiral scanning (in figure shown in 10a) or circular inside spin scanning (in figure shown in 10b), generates scanning pattern.
Embodiment 7
With reference to shown in Figure 11, vector expression scan method of the present invention, according to embodiment 1 or, 2 or 3 carry out in the helical scan path of laser scanning confocal micro-scope, described helical scan path is oval external spiral scanning (in figure shown in 11a) or oval inside spin scanning (in figure shown in 11a), generates scanning pattern.
The vector expression scan method of laser scanning confocal micro-scope of the present invention, the sample after quick prescan carries out ROI identification; And the generation of vector expression scanning pattern is carried out to sample ROI, according to scanning pattern, vector fine scanning is carried out to sample ROI, in vector expression scanning process, the coordinated movement of various economic factors of X-direction and Y-direction forms laser facula track while scan, without the quick-speed large-scale return stroke of existing pure line scanning switching mode, galvanometer regulation time in the effective minimizing scanning galvanometer course of work, improves the overall image taking speed of ROI; And owing to adopting spiral scan, avoid the aging problem causing galvanometer plated film in prior art due to strenuous vibration in line scanning galvanometer process, be of value to the serviceable life extending galvanometer.
The above the specific embodiment of the present invention, does not form limiting the scope of the present invention.Any various other done by technical conceive of the present invention change and distortion accordingly, all should be included in the protection domain of the claims in the present invention.
Claims (8)
1. a vector expression scan method for laser scanning confocal micro-scope, is characterized in that, described vector expression scan method comprises:
Step one, quick prescan is carried out to sample;
Step 2, ROI identification is carried out to the sample after quick prescan;
Step 3, to identify based on described ROI, the generation of vector expression scanning pattern is carried out to sample, the scanning pattern Covering samples ROI of generation;
Step 4, carry out vector fine scanning according to described vector expression scanning pattern to sample ROI: under the driving of controller, rotate according to vector expression scanning pattern scanning galvanometer, laser scanning confocal micro-scope obtains the scan image of sample.
2. vector expression scan method according to claim 1, is characterized in that, described quick prescan is that scanning galvanometer carries out rough scanning in the mode of line scanning to sample, obtains the essential information of sample ROI.
3. vector expression scan method according to claim 1, is characterized in that, also comprises after execution step 3, before execution step 4
Scanning pattern judges: judge whether vector expression scanning pattern scope meets scanning demand,
As met, then perform step 4; As do not met, then sample ROI being adjusted, performing the coordinates measurement that step 3 re-starts scanning.
4. vector expression scan method according to claim 1, is characterized in that, described vector expression scanning is inside spin scanning or external spiral scanning.
5. vector expression scan method according to claim 4, is characterized in that, described spiral scan is square.
6. vector expression scan method according to claim 4, is characterized in that, described spiral scan is triangular in shape.
7. vector expression scan method according to claim 4, is characterized in that, described spiral scan is rounded.
8. vector expression scan method according to claim 4, is characterized in that, described spiral scan ovalize.
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| CN103323940B (en) * | 2013-05-29 | 2015-04-15 | 哈尔滨工业大学 | Electric translation device based on two-photon microscope and two-dimensional spiral scanning method thereof |
| 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 |
| CN108398779B (en) * | 2018-02-12 | 2020-05-15 | 西安知微传感技术有限公司 | Helical line scanning laser projection method and system based on galvanometer |
| US11073603B2 (en) * | 2018-04-03 | 2021-07-27 | GM Global Technology Operations LLC | Controlled scan pattern transition in coherent lidar |
| CN111239170B (en) * | 2020-02-11 | 2023-06-13 | 常州广为仪器科技有限公司 | X-ray detection device and detection method for measuring trace elements |
| JP2022163844A (en) * | 2021-04-15 | 2022-10-27 | 国立研究開発法人産業技術総合研究所 | Spectral data acquisition method, cell classification method, and Raman spectroscopy system |
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| CN1766661A (en) * | 2005-09-30 | 2006-05-03 | 电子科技大学 | Method for detecting polarity distribution of ferroelectric material in micro-scale by non-conductive probe |
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