CN104730702A - Micro-scanning platform, shooting method and work area flatness calibration method - Google Patents
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Abstract
The invention discloses a micro-scanning platform, a shooting method and a work area flatness calibration method. Continuous scanning on a low precision platform can be achieved. The method mainly comprises the steps that a scanning coordinate system is set up in a work area; through a plane formula A1X+B1Y+C1Z+D1=0 and a standard slide, the calibration values En of the flatnesses of all scanning points in the work area of an objective table are determined; then, in actual sample slide shooting, the actual focusing position of each scanning point is determined according to the corresponding En. In this way, all scanning points can be adjusted to be in place before a camera shoots all the scanning points. The continuous scanning on the low precision platform can be achieved. Moreover, the scanning precision is high, scanning efficiency is high, and shot photos are clear. Due to the fact that high-precision continuous scanning is achieved on the low precision platform, the manufacturing cost of a high-precision scanning device is remarkably reduced.
Description
Technical field
The present invention relates to microscope continuous sweep field, particularly relate to Microscanning platform, image pickup method and perform region flatness calibration steps.
Background technology
Microscope is the common tool observing microworld, is widely used at present in biological medicine diagnosis research and commercial production.The use of current micrometron is more and more extensive, i.e. the shooting of the movement of microslide on objective table, focusing between camera lens and sample, sample image has all been linked by the XY axle objective table of robotization, Z axis lens driving and digital camera.
Usual microscopic examination sample is all placed on (25mm x 75mm) on microslide, microslide is placed on the perform region of objective table, then passes through transillumination (biological specimen is in the majority) or is directly placed on objective table by falling to penetrating optical illumination (industrial in the majority).If the tolerance of sample plane is in the field depth of the focal plane of camera lens, in theory to the plane Z axis height that can calculate whole sample after three point focusing of sample, then link with XY axle objective table and can realize continuous rapid scanning.
Realize there is following prerequisite with single focussing plane continuous sweep:
1, Z axis stepping accuracy is less than the focal plane depth of field (the 100X oil mirror focal plane depth of field is 0.2um) of camera lens;
2, the flatness of sample plane is in the focal plane depth of field of camera lens;
The flatness of 3, XY axle objective table is in the focal plane depth of field of camera lens.
Prerequisite 1 can use piezoelectric ceramics lens driver, and the positioning precision of Z axis can reach 20nm.
Prerequisite 2, cell smear or histotomy thickness are 3-10um, and microslide flatness is less than 0.2um, after lens focus sample, can obtain sharply defined image in field depth.
Prerequisite 3, XY axle objective table shift motion is minimum is Y-direction 25mm, X-direction 75mm, is generally X 100mm, and Y 75mm, XY and sample contact thing are placed on the high-precision objective table of various principle more.But due to the factor such as stroke range, assembling, the difference in height (flatness) of whole movement is about about 2um, and exceeded the focal plane depth of field of camera lens, during continuous sweep, subregion there will be phenomenon out of focus.If use the objective table of more high precision guide rail, cost payout can rise by straight line, and hardware complexity also can increase (as used air supporting or magnetic suspended guide).
Summary of the invention
The technical matters that the present invention solves is to provide a kind of image pickup method being conducive to realizing the Microscanning platform of continuous sweep when guide precision is lower.
The technical solution adopted for the present invention to solve the technical problems is:
The image pickup method of Microscanning platform, comprising:
A, the perform region of objective table is divided into n analyzing spot along X-axis and Y-axis, forms scan coordinate system Sn (x, y), the area of the perform region that area/every photos is corresponding of n=perform region;
B, preparation standard slide, at scan coordinate system Sn (x, y) 3 points are selected in, described 3 points are respectively Si (x, y), Sj (x, y) with Sk (x, y), at Si (x, y), Sj (x, y) with Sk (x, y) place's focus criteria slide respectively, obtain 3 theoretical basic points: P1 (x, y, z), P2 (x, y, z) with P3 (x, y, z), wherein, P1 and Si is corresponding, P2 and Sj is corresponding, P3 and Sk is corresponding, the Z coordinate of each theoretical basic point is the actual focal position on Z axis, according to P1, P2 and P3 and plane equation A
1x+B
1y+C
1z+D
1=0 calculates parameter A
1, B
1, C
1and D
1,
C, respectively at each point place focus criteria slide of scan coordinate system Sn (x, y), obtain Zn, Zn is the actual focal position when the various point locations focus criteria slide of scan coordinate system Sn (x, y) on Z axis;
D, Sn (x, y) is substituted into A
1x+B
1y+C
1z+D
1=0, obtain Zpn, Zpn is the theoretical focal position when the various point locations focus criteria slide of scan coordinate system Sn (x, y) on Z axis, then calculate En=Zn-Zpn, En is the modified value of the flatness of each analyzing spot of the perform region of objective table;
E, prepare actual sample slide, at scan coordinate system Sn (x, y) 3 points are selected in, described 3 points are respectively Sm (x, y), Sq (x, y) with Sg (x, y), at Sm (x, y), Sq (x, y) with Sg (x, y) place focuses on actual sample slide respectively, obtain the zm of 3 some correspondences, zq and zg, wherein zm, actual focal position when zq and zg is focusing on Z axis, then calculate: zpm=zm-Em, zpq=zq-Eq, zpg=zg-Eg, wherein, Sm and zpm is corresponding, Sq and zpq is corresponding, Sg and zpg is corresponding, according to Sm, Sq, Sg, zpm, zpq, zpg and plane equation A
2x+B
2y+C
2z+D
2=0 calculates parameter A
2, B
2, C
2and D
2,
F, when actual sample slide is taken, determine the analyzing spot St (x, y) of the scan coordinate system Sn (x, y) needing the position of the actual sample slide of shooting corresponding, St (x, y) is substituted into A
2x+B
2y+C
2z+D
2=0 calculates Zpt, then calculates Zt=Et+Zpt, is taken by camera lens according to Zt after Z axis is adjusted to the right place.
By above-mentioned steps A to D, the present invention can determine that the perform region of objective table is at the Changing Pattern with objective table moving process midplane degree, this rule embodies by En value, also can be calibrated by En.Owing to precomputing En value, so can adjust fast camera lens targetedly in follow-up shooting process, to adapt to the change of perform region flatness.Namely step e and F can allow camera lens rapid adjustment put in place.This just makes continuous sweep be achieved, even if the precision of guide rail is not high, also can realize the fast continuous scan to sample.And because camera lens takes after rapid adjustment puts in place again, the photo therefore taken is all clearly, can not be fuzzy.
Further: the standard slide in step B is standing striation slide.
Further: 3 somes Si (x, y) in step B, Sj (x, y) and Sk (x, y) ensure that when selecting the triangle that the line of described 3 points is formed is the triangle that in the perform region of objective table, area is maximum.
Further: 3 somes Sm (x, y) in step e, Sq (x, y) and Sg (x, y) ensure that when selecting the triangle that the line of described 3 points is formed is the triangle that in the perform region of objective table, area is maximum.
Present invention also offers the perform region flatness calibration steps of the objective table of Microscanning platform, comprising:
A, the perform region of objective table is divided into n analyzing spot along X-axis and Y-axis, forms scan coordinate system Sn (x, y), the area of the perform region that area/every photos is corresponding of n=perform region;
B, preparation standard slide, at scan coordinate system Sn (x, y) 3 points are selected in, described 3 points are respectively Si (x, y), Sj (x, y) with Sk (x, y), at Si (x, y), Sj (x, y) with Sk (x, y) place's focus criteria slide respectively, obtain 3 theoretical basic points: P1 (x, y, z), P2 (x, y, z) with P3 (x, y, z), wherein, P1 and Si is corresponding, P2 and Sj is corresponding, P3 and Sk is corresponding, the Z coordinate of each theoretical basic point is the actual focal position on Z axis, according to P1, P2 and P3 and plane equation A
1x+B
1y+C
1z+D
1=0 calculates parameter A
1, B
1, C
1and D
1,
C, respectively at each point place focus criteria slide of scan coordinate system Sn (x, y), obtain Zn, Zn is the actual focal position when the various point locations focus criteria slide of scan coordinate system Sn (x, y) on Z axis;
D, Sn (x, y) is substituted into A
1x+B
1y+C
1z+D
1=0, obtain Zpn, Zpn is the theoretical focal position when the various point locations focus criteria slide of scan coordinate system Sn (x, y) on Z axis, then calculate En=Zn-Zpn, En is the modified value of the flatness of each analyzing spot of the perform region of objective table.
Further: the standard slide in step B is standing striation slide.
Further: 3 somes Si (x, y) in step B, Sj (x, y) and Sk (x, y) ensure that when selecting the triangle that the line of described 3 points is formed is the triangle that in the perform region of objective table, area is maximum.
Present invention also offers Microscanning platform, comprise camera lens, also comprise the image pickup method of above-mentioned Microscanning platform, described camera lens is adjusted to the right place according to described image pickup method.
The invention has the beneficial effects as follows: continuous sweep can be realized at rough grade platform.And scanning accuracy is high, scan efficiency is high, the apparent of shooting.Due to high precision continuous sweep can be realized at rough grade platform, significantly reduce the manufacturing cost of high precision scanning device.
Accompanying drawing explanation
Fig. 1 is the schematic diagram of the perform region on objective table;
Fig. 2 is the schematic diagram that perform region is divided into n analyzing spot;
Fig. 3 is the schematic diagram of actual focal position Distance Theory focussing plane;
Fig. 4 is the schematic diagram theoretical focal position being adjusted to actual focal position;
Be labeled as in figure: objective table 101, perform region 102.
Embodiment
Below in conjunction with the drawings and specific embodiments, the present invention is further described.
Give the perform region 102 on objective table 101 and objective table 101 in Fig. 1, microslide is generally arranged on perform region.The below of perform region 102 is generally provided with light source, and the top of perform region 102 is generally provided with camera lens.Objective table 101 can along X-axis and the determined planar movement of Y-axis, and perform region 102 is movement accordingly also.X-axis and Y-axis are based upon on objective table 101, and Z axis is and XY plane orthogonal.
Introduce method of the present invention below:
A, the perform region 102 of objective table 101 is divided into n analyzing spot along X-axis and Y-axis, forms scan coordinate system Sn (x, y), as shown in Figure 2, each analyzing spot corresponding a coordinate, such as S1 (x1, y1), S2 (x2, y1) etc.Wherein, the area of the perform region that area/every photos is corresponding of n=perform region.The area of perform region i.e. the migration area of objective table.Such as X-axis displacement is 100mm, and Y-axis displacement is 75mm, then work area is 7500 square millimeters.The area of the perform region that every photos is corresponding can be determined by microscope camera lens enlargement ratio and camera imaging chip target surface, and such as the area of the perform region that every photos is corresponding is 1 square millimeter.Like this, just perform region can be divided into 7500 analyzing spots, the area of each analyzing spot is 1 square millimeter.
B, prepare standard slide, as long as standard slide here can focus on and integral planar degree is less than the slide of the lens focus depth of field.Such as can be standing striation slide.In scan coordinate system Sn (x, y), select 3 points, 3 points can be selected arbitrarily, described 3 points are respectively Si (x, y), Sj (x, y) and Sk (x, y), at Si (x, y), Sj (x, y) and Sk (x, y) place focus criteria slide respectively, standard slide is placed on relevant position by focus criteria slide exactly, is then focused on by camera lens.3 theoretical basic points can be obtained like this: P1 (x, y, z), P2 (x, y, z) with P3 (x, y, z), wherein, actual focal position when the Z coordinate of P1 is the focusing of Si place on Z axis, actual focal position when the Z coordinate of P2 is the focusing of Sj place on Z axis, the actual focal position when Z coordinate of P3 is the focusing of Sk place on Z axis, and each Z coordinate above-mentioned directly can obtain in focusing afterwards according to prior art.The x coordinate of P1 and y coordinate are exactly x coordinate and the y coordinate of Si, and the x coordinate of P2 and y coordinate are exactly x coordinate and the y coordinate of Sj, and the x coordinate of P3 and y coordinate are exactly x coordinate and the y coordinate of Sk.
According to P1, P2 and P3 and plane equation A
1x+B
1y+C
1z+D
1=0 calculates parameter A
1, B
1, C
1and D
1, so just can determine a theoretical focussing plane A
1x+B
1y+C
1z+D
1=0.
C, respectively at scan coordinate system Sn (x, y) each point place focus criteria slide, the diverse location namely standard slide being put into perform region, then by lens focus, obtains Zn, such as Z1, Z2, Z3 etc., these Zn and Sn each point one_to_one corresponding.Zn is the actual focal position when the various point locations focus criteria slide of scan coordinate system Sn (x, y) on Z axis.Zn can directly obtain its value by prior art after focusing on.
D, Sn (x, y) is substituted into A
1x+B
1y+C
1z+D
1=0, Z value can be obtained, namely obtain Zpn, such as Zp1, Zp2, Zp3 etc.Zpn and Sn is corresponding.Zpn is the theoretical focal position when the various point locations focus criteria slide of scan coordinate system Sn (x, y) on Z axis, and then calculate En=Zn-Zpn, En is the modified value of the flatness of each analyzing spot of the perform region of objective table.Such as En can be E1, E2, E3 etc.En and Sn one_to_one corresponding, the namely corresponding En value of each analyzing spot.En embodies the actual focal position of each analyzing spot of perform region that the factors such as guide rail cause and the deviation of theoretical focal position.Correcting action can be played to subsequent samples shooting by En, eliminate the impact of the factors such as guide rail on focal position with this.Such as shown in Fig. 3.In Fig. 3, horizontal line representation theory focussing plane, each point is actual focal position.
E, preparation actual sample slide, actual sample slide i.e. actual needs carry out the sample slide taken.In scan coordinate system Sn (x, y), select 3 points, 3 points can be selected arbitrarily.Described 3 points are respectively Sm (x, y), Sq (x, y) with Sg (x, y), at Sm (x, y), Sq (x, y) respectively actual sample slide is focused on Sg (x, y) place, obtain zm, zq and zg of 3 some correspondences, actual focal position when wherein zm, zq and zg are focusing on Z axis.Then calculate: zpm=zm-Em, zpq=zq-Eq, zpg=zg-Eg, because actual focal position is subject to the factor impacts such as guide rail here, therefore need these factors to remove just can obtain a theoretical focal position, above-mentioned Em, Eq and Eg are determined by above-mentioned En, the Em in the En that namely Sm point is corresponding, the such as corresponding E1 of S1 point.In like manner, the Eg in the corresponding En of the Eq in the corresponding En of Sq, Sg.Wherein, Sm and zpm is corresponding, and Sq and zpq is corresponding, and Sg and zpg is corresponding, according to Sm, Sq, Sg, zpm, zpq, zpg and plane equation A
2x+B
2y+C
2z+D
2=0 calculates parameter A
2, B
2, C
2and D
2, concrete namely the x coordinate of Sm and y coordinate and zpm are substituted into above-mentioned equation, the x coordinate of Sq and y coordinate and zpq are substituted into above-mentioned equation, the x coordinate of Sg and y coordinate and zpg substitute into above-mentioned equation.By above-mentioned calculating, just obtain a theoretical focussing plane A
2x+B
2y+C
2z+D
2=0.
F, when actual sample slide is taken, determine the analyzing spot St (x of the scan coordinate system Sn (x, y) needing the position of the actual sample slide of shooting corresponding, y), shooting S4 (x4, y4) is such as needed, just S4 (x4, y4) is substituted into A
2x+B
2y+C
2z+D
2=0 calculates Z value, and this Z value i.e. Zpt, that such as S4 is corresponding is Zp4.Then calculate Zt=Et+Zpt, Et determines according to above-mentioned En exactly.Namely Z4=E4+Zp4, adds that modified value E4 is because the factors such as guide rail can make theoretical focal position generation deviation here, just can obtain actual focal position after adding modified value.Therefore camera lens is adjusted along Z axis according to Z4 position, camera lens just can be made to adjust to actual focal position take, corresponding photograph also can be clear, can not be fuzzy, the adjustment mode of camera lens can have multiple, such as, camera lens can be moved along Z axis, also objective table can be moved along Z axis, camera lens all can be adjusted to the right place by various mode, namely shoots clear photograph.Such as, in Fig. 4 (x3, y1) point, the zpt of this some correspondence in theoretical focussing plane (horizontal line position), after correction, the actual focal position deviation theory focussing plane of this some correspondence.
Known by said method, the difficult point of continuous sweep is actual when being exactly to each spot scan, ensure that camera lens energy rapid adjustment puts in place, could realize the continuous sweep of real meaning like this.And said method of the present invention just can ensure that, when to each analyzing spot shooting, camera lens can rapid adjustment put in place, because calculate actual focal position.
The present invention is verified practical by great many of experiments.Such as be carved with the microslide of the metal blackstreak of 1-5um, the Z axis lens driver of 20nm precision by using, use Olympus 60x Oil 1.35NA APO camera lens, the depth of field is about 0.1um, adopts said method of the present invention that objective table can be enable to adapt to continuous sweep and uses.
On the basis of the above, 3 somes Si (x, y) in step B, Sj (x, y) and Sk (x, y) ensure that when selecting the triangle that the line of described 3 points is formed is the triangle that in the perform region of objective table, area is maximum.Due to the some Si (x of 3 in step B, y), Sj (x, y) with Sk (x, y) be determine theoretical basic point P1, P2, with the prerequisite of P3, therefore in order to reduce error, ensure that when selecting the triangle that the line of described 3 points is formed is the triangle that in the perform region of objective table, area is maximum.Concrete edge or the vertex position that Si (x, y), Sj (x, y) and Sk (x, y) can be selected in perform region.Similar, 3 somes Sm (x, y) in step e, Sq (x, y) and Sg (x, y) ensure that when selecting the triangle that the line of described 3 points is formed is the triangle that in the perform region of objective table, area is maximum.So also be to reduce error.
Above-described specific embodiment; object of the present invention, technical scheme and beneficial effect are further described; be understood that; the foregoing is only specific embodiments of the invention; be not limited to the present invention; within the spirit and principles in the present invention all, any amendment made, equivalent replacement, improvement etc., all should be included within protection scope of the present invention.
Claims (8)
1. the image pickup method of Microscanning platform, is characterized in that:
A, the perform region of objective table is divided into n analyzing spot along X-axis and Y-axis, forms scan coordinate system Sn (x, y), the area of the perform region that area/every photos is corresponding of n=perform region;
B, preparation standard slide, at scan coordinate system Sn (x, y) 3 points are selected in, described 3 points are respectively Si (x, y), Sj (x, y) with Sk (x, y), at Si (x, y), Sj (x, y) with Sk (x, y) place's focus criteria slide respectively, obtain 3 theoretical basic points: P1 (x, y, z), P2 (x, y, z) with P3 (x, y, z), wherein, P1 and Si is corresponding, P2 and Sj is corresponding, P3 and Sk is corresponding, the Z coordinate of each theoretical basic point is the actual focal position on Z axis, according to P1, P2 and P3 and plane equation A
1x+B
1y+C
1z+D
1=0 calculates parameter A
1, B
1, C
1and D
1,
C, respectively at each point place focus criteria slide of scan coordinate system Sn (x, y), obtain Zn, Zn is the actual focal position when the various point locations focus criteria slide of scan coordinate system Sn (x, y) on Z axis;
D, Sn (x, y) is substituted into A
1x+B
1y+C
1z+D
1=0, obtain Zpn, Zpn is the theoretical focal position when the various point locations focus criteria slide of scan coordinate system Sn (x, y) on Z axis, then calculate En=Zn-Zpn, En is the modified value of the flatness of each analyzing spot of the perform region of objective table;
E, prepare actual sample slide, at scan coordinate system Sn (x, y) 3 points are selected in, described 3 points are respectively Sm (x, y), Sq (x, y) with Sg (x, y), at Sm (x, y), Sq (x, y) with Sg (x, y) place focuses on actual sample slide respectively, obtain the zm of 3 some correspondences, zq and zg, wherein zm, actual focal position when zq and zg is focusing on Z axis, then calculate: zpm=zm-Em, zpq=zq-Eq, zpg=zg-Eg, wherein, Sm and zpm is corresponding, Sq and zpq is corresponding, Sg and zpg is corresponding, according to Sm, Sq, Sg, zpm, zpq, zpg and plane equation A
2x+B
2y+C
2z+D
2=0 calculates parameter A
2, B
2, C
2and D
2,
F, when actual sample slide is taken, determine the analyzing spot St (x, y) of the scan coordinate system Sn (x, y) needing the position of the actual sample slide of shooting corresponding, St (x, y) is substituted into A
2x+B
2y+C
2z+D
2=0 calculates Zpt, then calculates Zt=Et+Zpt, is taken by camera lens according to Zt after Z axis is adjusted to the right place.
2. the image pickup method of Microscanning platform as claimed in claim 1, is characterized in that: the standard slide in step B is standing striation slide.
3. the image pickup method of Microscanning platform as claimed in claim 1, it is characterized in that: 3 some Si (x in step B, y), Sj (x, y) ensure when selecting that with Sk (x, y) triangle that the line of described 3 points is formed is the triangle that in the perform region of objective table, area is maximum.
4. the image pickup method of Microscanning platform as claimed in claim 1, it is characterized in that: 3 some Sm (x in step e, y), Sq (x, y) ensure when selecting that with Sg (x, y) triangle that the line of described 3 points is formed is the triangle that in the perform region of objective table, area is maximum.
5. the perform region flatness calibration steps of the objective table of Microscanning platform, is characterized in that:
A, the perform region of objective table is divided into n analyzing spot along X-axis and Y-axis, forms scan coordinate system Sn (x, y), the area of the perform region that area/every photos is corresponding of n=perform region;
B, preparation standard slide, at scan coordinate system Sn (x, y) 3 points are selected in, described 3 points are respectively Si (x, y), Sj (x, y) with Sk (x, y), at Si (x, y), Sj (x, y) with Sk (x, y) place's focus criteria slide respectively, obtain 3 theoretical basic points: P1 (x, y, z), P2 (x, y, z) with P3 (x, y, z), wherein, P1 and Si is corresponding, P2 and Sj is corresponding, P3 and Sk is corresponding, the Z coordinate of each theoretical basic point is the actual focal position on Z axis, according to P1, P2 and P3 and plane equation A
1x+B
1y+C
1z+D
1=0 calculates parameter A
1, B
1, C
1and D
1,
C, respectively at each point place focus criteria slide of scan coordinate system Sn (x, y), obtain Zn, Zn is the actual focal position when the various point locations focus criteria slide of scan coordinate system Sn (x, y) on Z axis;
D, Sn (x, y) is substituted into A
1x+B
1y+C
1z+D
1=0, obtain Zpn, Zpn is the theoretical focal position when the various point locations focus criteria slide of scan coordinate system Sn (x, y) on Z axis, then calculate En=Zn-Zpn, En is the modified value of the flatness of each analyzing spot of the perform region of objective table.
6. the flatness calibration steps of the perform region of Microscanning platform as claimed in claim 1, is characterized in that: the standard slide in step B is standing striation slide.
7. the flatness calibration steps of the perform region of Microscanning platform as claimed in claim 1, it is characterized in that: 3 some Si (x in step B, y), Sj (x, y) ensure when selecting that with Sk (x, y) triangle that the line of described 3 points is formed is the triangle that in the perform region of objective table, area is maximum.
8. Microscanning platform, comprises camera lens, it is characterized in that: the image pickup method also comprising the Microscanning platform in Claims 1-4 described in any one, and camera lens is adjusted to the right place according to described image pickup method.
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| CN113989393A (en) * | 2021-12-29 | 2022-01-28 | 江苏美克医学技术有限公司 | Scanning device objective table flatness correction method based on image information processing |
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