CN103163638A - Microscope automatic focusing method based on sample determination surface plane equation - Google Patents
Microscope automatic focusing method based on sample determination surface plane equation Download PDFInfo
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- CN103163638A CN103163638A CN 201110407847 CN201110407847A CN103163638A CN 103163638 A CN103163638 A CN 103163638A CN 201110407847 CN201110407847 CN 201110407847 CN 201110407847 A CN201110407847 A CN 201110407847A CN 103163638 A CN103163638 A CN 103163638A
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Abstract
The invention discloses a method which confirms a determination point out-of-focus deviation amount according to a sample determination surface plane equation to achieve automatic focusing. The method comprises building a method of the sample determination surface plane equation, improving representativeness of the sample determination surface plane equation, removing a mechanical error in the process of automatic focusing, reducing measures like focusing times, adequately controlling a servo system and the like. The total focusing time does not exceed 2 minutes to achieve the aim that an automatic scanning determination sample still keeps an accurate focusing state.
Description
Technical field
Belong to the optical microscope field of automatically focusing, be applicable to sample and automatically measure process.
Background technology
the necessity of microscope focusing
Working sample imaging clearly in the accurate burnt scope of optical microphotograph endoscope objective lens is called accurate burnt.Regulating the microscope focal length makes sample be called focusing in accurate burnt process.After sample moves, generally need focusing accurate Jiao again.Microscope has focusing knob, can drive the lifting of microscope thing platform and realize focusing.Some high-grade microscope is separately with electric control gear, and operation the corresponding button is controlled the focusing of driven by motor focusing knob.The sample press top and bottom depth of parallelism is inadequate, and the plasticine resilience causes sample can not keep suppressing rear state, and microscopes optical axis and thing platform verticality all can cause not etc. that sample moves and retrodeviate from the burnt state of standard, be called out of focus.Accurate burnt scope is relevant with the micro objective enlargement factor.The low power objective wide ranges, still can keep accurate burnt among a small circle after mobile example.The high power objective narrow range, mobile example can exceed accurate burnt scope among a small circle, causes image blur.Keeping sample is the prerequisite that obtains correct measurement result in the burnt state of standard, and various automatic focusings (also referred to as microscope automatic focusing, microscope automatic focus etc.) technology is arisen at the historic moment.
existing microscope Autofocus Technology characteristics
Existing Autofocus Technology is all passed through to control servo control mechanism (as stepper motor) and is substituted artificial rotating microscope focusing knob lifting thing platform execution, and difference is to determine that defocus offset amount principle is different.The one, gather the gray scale difference XOR marginal sharpness judgement focal length state of image and calculate the defocus offset amount by analysis
[1-5].The 2nd, with various micrometers apart from technology direct-detection defocus offset amount.The former accounts for main flow, and the latter waits perfect.Common ground is that each focusing is independently carried out, irrelevant with sample position, to accurate burnt the getting final product of the image gathered, in the automatic mensuration that therefore can be used for realizing according to image recognition technology.Common shortcoming has:
(1) speed is slow.Every bit is focused all by comparison chart picture or micrometer value, nearly 10 seconds of focus process
[3-4], and experienced operator's focus process 1-2 second only.Therefore arrange that more focusing loses actual use value;
(2) can not utilize the relation between the focusing coordinate simplify focus process and reduce the focusing number of times;
(3) inapplicable point by point scanning automatic measuring technology field.When measuring point reaches up to ten thousandly when even hundreds thousand of, the independent focusing of each point is unrealistic.In working sample only, during a certain component data, for avoiding the interference of other components, often require the microscope ken in minimum state, can't gather complete image and image border.Measure a sample, artificial total general about 2-3 minute of focusing time.If automatically focus, T.T. surpasses this value, and meaning only is embodied in and reduces focusing troublesome operation aspect.The reasons such as these shortcomings and attachment device are expensive have restricted applying of this class technology of auto.Especially need the Autofocus Technology more fast and effectively of exploitation applicable to point by point scanning determination techniques field.
List of references
1, Jiang Haihua, the microscope automatic focusing technical research [J] based on Image Definition. optical technology, 2008, the 34 volume supplementary issues.
2, Zhang Hongjun, Ma Guanying, the design of Microscope Autocontrol System [J]. Linyi Normal University's journal, 2006, the 28 the 3rd phases of volume.
3, permitted to contain Li Jianwei, the advanced microscope system [J] based on intelligent automatic focusing. photoelectric project, 2000, the 27 the 1st phases of volume.
4, permitted into Li Jianwei, the research of the intelligent automatic focusing system of advanced microscope and realization [J]. optical instrument, 2000, the 22 the 5th phases of volume.
5, Tang Chenghui, Zhu Senyuan, Wang Xuhui, the microscope automatic focusing technology [J] under computer control. war industry's robotization, 1999, the 4 phases.
Summary of the invention
general introduction
Sample determination face through grinding, after polishing is a plane (the sample determination face that the beginner grinds is even is several planes, and it is the requirement on a plane that the sample determination face that training can make to grind a little meets).During mobile example, it is relevant with sample displacement that a variety of causes causes that the defocus offset amount all shows as: displacement is larger, and the defocus offset amount is larger.After determining sample determination facial plane equation, can determine the defocus offset amount and eliminate by the sample coordinate position, realize that sample is accurate burnt.This instructions provides determines sample determination facial plane equation method, by sample position calculating defocus offset metering method, reduces the measure of focusing number of times in the mensuration process, control system technical requirement etc.
2 mathematical principles
Make true origin
f 0 (
x 0 , y 0 , z 0 )on the sample determination face,
zaxle is microscope light path center (being the object lens centers).Set 3 control points on the sample determination face:
f 1 (x1, y1, z1), F 2 (x2, y2, z2), F 3 (x3, y3, z3), see accompanying drawing 1.Sample facial plane equation is (1) formula:
Because sample plane and object lens spacing are not 0,
d≠ 0, order
a 0 =A/D, B 0 =B/D, C 0 =C/D
With these 3 control point coordinates, set up system of equations (2) formula:
Separate this system of equations and determine statement
a 0 , B 0 , C 0 after, sample facial plane equation is (3) formula
Elevation coordinate when on sample, any point standard is burnt
zican be by this position coordinates
(xi, yi)determine, see (4) formula:
z i = - (A 0 x i +B 0 y i +1
)/C 0 （4）
If this point needs focusing, theoretical out of focus bias
t=-z
i
Eliminate
tmake sample accurate burnt
,sample plane is only done or lower movement on the z direction, and vergence direction does not become.Mobile example extremely again
(x j , y j )(j ≠ i) afterwards, new out of focus bias can
scan be determined by (5) or (6) formula:
When the adjusting direction is identical with last adjusting direction
s=-z j -T(5)
When regulating direction with last adjusting opposite direction
s=-z j + T(6)
Can obtain thus the theoretical out of focus bias of sample optional position.
determine sample plane equation implementation process
Stepper motor is by can accurately locating and have locking moment.Can accurately calculate the sample Minimum sliding distance by the stepper motor minimum step angle that drives the sample attachable mechanical stage.Be multiplied by the execution step number, be actual displacement.In like manner, the execution step number by driving focusing of microscope knob stepper motor, can accurately calculate microscope thing platform heave amplitude.Program can be determined each point coordinate position on sample plane after recording each stepper motor execution step number.First sample is moved to microscope light path center accurate burnt, this point is defined as true origin
f 0 (
x 0 , y 0 , z 0 ).again by 1 control point of programmed control automated movement to the
f 1 (being also the sweep measuring starting point), complete its coordinate position of defocused acquisition
(x 1 , y 1 , z 1 ).again by
f 1 automated movement extremely
f 2 and focusing, obtain
f 2 coordinate (
x 2 , y 2 , z 2 ).Again by
f 2 automated movement extremely
f 3 and focusing, obtain
f 3 coordinate (
x 3 , y 3 , z 3 ).Obtain thus by formula (2) and determine needed three the control point coordinate datas of sample plane equation.The focus process of control point must complete by controlling servo control mechanism, records focus process by program and could obtain each control point elevation coordinate
z 1 , z 2 with
z 3 ,manually direct control focusing of microscope knob carries out.
improve the representative measure of sample determination facial plane equation
Between control point, distance is larger, and definite plane equation is better to sample determination face representativeness.Therefore by program per sample size with measure position, area definition control point.Principle be control point should be in sample edge the 1mm left and right, measure area and the control point spacing tried one's best in far away avoid again sample edge taking full advantage of.For example, for the circular sample of diameter 15mm, measure area should be in diameter 14mm circle maximum inscribe square, its length of side is 10mm.3 control points should be positioned at its summit.
eliminate machine error and improve the measure of sample plane equation degree of accuracy
The sample that stepper motor drives moves with the execution unit of focusing all unavoidably with machine error, affects the degree of accuracy of each control point coordinate, and then affects the degree of accuracy of definite sample determination facial plane equation.Solution: obtain control point
f 3 after coordinate, the sample automatic opening-closing is to control point
f 2 ,first by the theoretical out of focus bias of being determined by the sample plane equation, carry out focusing.If can not be accurate burnt, auxiliary focusing makes it accurate burnt.The defocus offset amount that auxiliary focusing is eliminated (is designated as
w 2 ) be control point
f 2 because machine error and the sample accumulated of repeatedly focusing exists
ydirection moves (now
x 3 =x 2 ) accumulation the error synthesis exercising result.After completing, the sample automatic opening-closing is to control point
f 1 and auxiliary focusing, the defocus offset amount of elimination (is designated as
w 1 ) be control point
f 1 because of the machine error of focusing accumulation repeatedly with
xdirection moves (now
y 2 =y 1 ) accumulation machine error combined action result.With
w 2 revise
z 3 ,
w 1 revise z
2after the control point coordinate again solve (2) formula system of equations more accurate sample plane equation after the execution unit machine error that is eliminated.
avoid accurate burnt accumulation of error measure in auto-focus process
During focusing, on the thing platform or lower amount of movement be subject to stepper motor minimum step angle restriction and have minimum value.The actual regulated quantity of carrying out can only be its integral multiple, can not accurately carry out theoretical out of focus bias.During in the burnt scope of standard, even depart from accurate burnt position, can not cause image blur when sample yet.For example, when the executable thing platform of reality amount of movement minimum value is 10um, if the defocus offset amount of need eliminating be+during 15um, can only carry out lower motive objects platform 10um or 20um.When if the accurate burnt scope of object lens is positive and negative 10um, carry out lower motive objects platform 10um or 20um and all can make sample accurate burnt, also all can produce the burnt error of standard of 5um.Accurate burnt error outwardness and can't eliminating.But as long as accurate burnt error is accurate burnt unaffected in allowed band, as long as therefore automatic focusing practical implementation is adjusted to accurate burnt scope.When the burnt accumulation of error of standard, still can cause image blur.Avoid the accurate burnt error of accumulation in implementation, the one, consider the last burnt error symbol of standard that the time produces of regulating, the 2nd, accumulate and calculate the accurate burnt margin of error.For upper example, regulating the burnt error symbol of the standard produced if last is negative (focusing quantity not sufficient), and this should be carried out and regulate 20um, offsets the last minus deviation produced of regulating.After adjusting, must recalculate the burnt error symbol of standard and the margin of error of accumulation, for regulate and prepare next time.Implementation just can make sample all the time in the burnt scope of standard like this.
reduce the measure of focusing number of times
The supporting process that focusing is just measured after all automatically, reduce the focusing number of times and can accelerate finding speed.During sweep measuring, the some line space is all very little, and generally at 50-100um, some mensuration projects even require to reach 1um.The defocus offset amount that so little displacement causes can not exceed accurate burnt scope.When exceeding accurate burnt scope with the displacement increase, the defocus offset amount just needs to regulate.Therefore every scan line arranges that the minority focusing gets final product.When the sample plane degree of tilt is little or use the object lens multiple not high, every row arranges that 2-5 focusing gets final product, otherwise can arrange more.For example, when the some line space of sweep measuring is 100um, while measuring at 10,000, can 20 somes focusings of every mensuration once, every row arranges that 5 focusings get final product.Measuring a sample focuses 50 times at most.Under normal circumstances, distance 2mm only between each focusing, it is rare that the defocus offset amount that so little displacement causes exceeds accurate burnt scope situation.When the defocus offset amount of focusing does not exceed accurate burnt scope, can not carry out focusing, unenforced defocus offset amount is added in the defocus offset amount of focusing next time.Therefore actual focusing number of times is far fewer than 50 times.The operator is generally by measuring a sample, and the fixed factors such as microscope, press that can understand use cause the sample plane inclined degree, determine suitable every row focusing number of times before the next sample of sweep measuring.To defocused, the sample determination face rises or falls, and only focusing elevation coordinate changes.
to controlling the executive system requirement
The 50 times of oil immersion objectives of Leitz company polarizing microscope of take are example, and its accurate burnt scope is only positive and negative 300um.Stepper motor single step angle of rotation makes thing platform lifting minor increment must be less than 300um, carries out the defocused sample that just can make in the accurate burnt scope of these object lens.Stepper motor also must have enough startups and holding torque, otherwise it is not in place to carry out lifting thing platform.The general volume of the stepper motor that moment is large, weight, running current are all large, the power amplifier complexity.Also can cause the execution unit difficulty is installed, increase the problems such as microscope mechanical system wearing and tearing.The advantages such as it is small and exquisite that the magneto decelerating step motor has volume, and the single step angle of rotation is little, and startup is large with torque lock, and the monophasic pulses if electric current is little, and power amplifier is simple, meet above-mentioned requirements.In addition, step motor shaft must be consistent with focusing of microscope knob web member axle center, avoids the operational process shake.Stepper motor must be fixed on microscope by link, offsets the reacting force that axle rotates.
The accompanying drawing explanation
Fig. 1 is sample determination face tilt schematic diagram.In Fig. 1,1 is micro objective, and 2 is working sample, and 3 is plasticine, and 4 is microslide, F
0for true origin (sample center) F
1-F
3for focusing control point, F
1for the sweep measuring starting point
embodiment
Technology of auto based on sample determination face equation produces a desired effect after adopting in HD full microscope photometer.Due to the focusing of same mensuration row, direction is consistent, regulates continuously, once completes the adjusting repeatedly that may exist while not having independent focusing, only about half second consuming time at every turn.Can not accumulate the machine error that the drive disk assembly gear clearance causes during due to equidirectional continuous adjusting, degree of regulation is high.Also owing to significantly having reduced the focusing number of times, therefore measure the sample time of always the focusing much less of more independently focusing.Guaranteeing in sample imaging clearly situation, how many points of sweep measuring no matter, the time of always focusing all can be in 2 minutes, obtained when Sample Scan and measured and count when being respectively 10,000,20,000 and 100,000, total minute is controlled at respectively 10 minutes, 15 minutes with the actual effect of 25 minutes.
Implementation process, except the above-mentioned theory analysis is realized by programming, has also adopted the useful ancillary method on automatic focusing (without these ancillary methods, not affect the automatic focusing effect.）：
(1) measure the process monitoring window: in the microscope light path, by transformation, the double light path interface unit is installed.One of them installs high resolving power high-speed camera head, and opens up the video monitoring window, but Real Time Observation automatic focusing effect
.(2) the sample determination process can be placed on backstage and carries out: do not affect mensuration person and carry out data analysis, Edit Document simultaneously, online By consulting literatures etc., can monitor the mensuration process but retain this window simultaneously, in order to measured rear data, deposit, measure next sample etc.Also can take pictures to the mensuration position of needs by this window, record mensuration process etc.(3) provide and suspend and auxiliary focusing function: for finding in time the problem that autofocus system exists.Suspend automatically and can assist focusing after mensuration, after completing, continuation is measured.If occur, repeatedly need to suspend and auxiliary focusing, illustrative system is undesired, needs to repair.Mistake in software programming, unexpected situation, control circuit, stepper motor fault etc. all can affect the system normal operation.When as undesired as a certain road pulse signal when the control step motor, can cause stepper motor meeting step-out or reduce moment.This class fault is unlikely to make autofocus system to quit work, therefore often out in the cold.Owing to can not accurately carrying out on request focusing bias, can make focusing automatically fall flat.
the usefulness of bringing after enforcement
The troublesome operation of 1 alternative manual observation sample and focusing, solve because of viewing microscope fatigue for a long time can not a plurality of samples of METHOD FOR CONTINUOUS DETERMINATION etc. problem, and be applicable to all samples through grinding
[annotating 1-2];
2 can avoid image blur, make the automatic determination data distortion of sample reliable;
3 compare with other Atomatic focusing methods, except all must possess control with execution unit, without extra means, easy to implement, there is speed fast, mechanical deviation is little, the precision high always is no more than 2 minutes for automatic focusing time of a sample;
4 fluctuation long periods determining instrument short time are little, and finding speed is conducive to improve the mensuration precision soon;
5 can improve the statistics representativeness relevant with sample size.For example during the aobvious charred coal porosity of labor measurement, within 4 hours, only can measure 400 data, and while automatically measuring with HD full microscope photometer, within 25 minutes, can measure 100,000 data;
6 can make some results of study walk out academic palace obtains practical application: need the research conclusion of statistical study for the production of actual time, often need more multisample capacity adaptation complex situations.As the single coal vitrinite reflectance distribution plan that forms coal petrology theoretical foundation is normal distribution law and must be based upon on the abundant basis of determination data.And for example in labor measurement coal reflectivity process, can find a small amount of non-coking coal material that coal chemical enterprise is mixed with in carrying out coal, but, because measuring point is few, be difficult to embody in measurement result.Only have and measure the truth that the abundant automatic measurement result of counting could reflect working sample, obtain practical application.For the useful result that instructs enterprise to come the coal rational stacking to produce with the coal distribution graph of reflectivity, because labor measurement coal reflectivity is wasted time and energy, measuring point is few, and the representativeness of statistics and actual effect all are difficult to the satisfied need of production that instructs.Can meet and instruct the automatic mensuration of need of production that fast automatic focus function must be arranged.
Annotate: 1 when with stereoscopic microscope observing or while measuring without the sample that grinds, because sample is uneven everywhere, can not be simultaneously accurate burnt, and low power dry objective observation that can only be large by accurate burnt scope.The defocus offset amount caused after sample moves generally all is not more than the accurate burnt scope of object lens, does not need automatically to focus burnt technology.The sample that 2 beginners grind occasionally is several planes.Training can solve this abnormal conditions a little, meets the requirement of this patent automatic focusing method to working sample.
Claims (4)
1. realize the microscope method of focusing automatically according to sample determination facial plane equation for one kind.
2. the method for sample determination facial plane equation is determined in the setting control point that claim 1 comprises.
3. the raising sample determination facial plane equation representativeness that claim 1 comprises and the method for degree of accuracy.
4. reduce the method for focusing number of times in the automatic working sample process that claim 1 comprises.
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103728304A (en) * | 2013-12-18 | 2014-04-16 | 宁波江丰生物信息技术有限公司 | Focusing method for pathological section scanner |
| CN104730702A (en) * | 2015-03-16 | 2015-06-24 | 苏州创继生物科技有限公司 | Micro-scanning platform, shooting method and work area flatness calibration method |
| CN108344741A (en) * | 2018-02-09 | 2018-07-31 | 辽宁翔舜科技有限公司 | A kind of focal length automatic identification and control technology applied to coal petrography automatic detection |
| CN109001902A (en) * | 2018-09-05 | 2018-12-14 | 哈尔滨理工大学 | Microscope focus method based on image co-registration |
| CN109239900A (en) * | 2018-11-07 | 2019-01-18 | 华东师范大学 | A kind of full-automatic quick focusing method for the big visual field acquisition of microscopic digital image |
| CN111665259A (en) * | 2019-03-08 | 2020-09-15 | 深圳中科飞测科技有限公司 | Detection device and detection method |
| WO2022041197A1 (en) * | 2020-08-31 | 2022-03-03 | 深圳迈瑞生物医疗电子股份有限公司 | Sample image photographing method and sample image photographing apparatus |
| CN114442296A (en) * | 2020-10-30 | 2022-05-06 | 深圳市瑞图生物技术有限公司 | Image acquisition method for microscope, medical detection device and storage medium |
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- 2011-12-09 CN CN 201110407847 patent/CN103163638A/en active Pending
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103728304A (en) * | 2013-12-18 | 2014-04-16 | 宁波江丰生物信息技术有限公司 | Focusing method for pathological section scanner |
| CN104730702A (en) * | 2015-03-16 | 2015-06-24 | 苏州创继生物科技有限公司 | Micro-scanning platform, shooting method and work area flatness calibration method |
| CN108344741A (en) * | 2018-02-09 | 2018-07-31 | 辽宁翔舜科技有限公司 | A kind of focal length automatic identification and control technology applied to coal petrography automatic detection |
| CN109001902A (en) * | 2018-09-05 | 2018-12-14 | 哈尔滨理工大学 | Microscope focus method based on image co-registration |
| CN109001902B (en) * | 2018-09-05 | 2021-07-16 | 哈尔滨理工大学 | Microscope focusing method based on image fusion |
| CN109239900A (en) * | 2018-11-07 | 2019-01-18 | 华东师范大学 | A kind of full-automatic quick focusing method for the big visual field acquisition of microscopic digital image |
| CN111665259A (en) * | 2019-03-08 | 2020-09-15 | 深圳中科飞测科技有限公司 | Detection device and detection method |
| WO2022041197A1 (en) * | 2020-08-31 | 2022-03-03 | 深圳迈瑞生物医疗电子股份有限公司 | Sample image photographing method and sample image photographing apparatus |
| CN114442296A (en) * | 2020-10-30 | 2022-05-06 | 深圳市瑞图生物技术有限公司 | Image acquisition method for microscope, medical detection device and storage medium |
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Application publication date: 20130619 |