CN106783496B - A kind of electron microscope tomograph imaging method and system - Google Patents
A kind of electron microscope tomograph imaging method and system Download PDFInfo
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- CN106783496B CN106783496B CN201611207099.8A CN201611207099A CN106783496B CN 106783496 B CN106783496 B CN 106783496B CN 201611207099 A CN201611207099 A CN 201611207099A CN 106783496 B CN106783496 B CN 106783496B
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- H—ELECTRICITY
- H01—ELECTRIC ELEMENTS
- H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
- H01J37/00—Discharge tubes with provision for introducing objects or material to be exposed to the discharge, e.g. for the purpose of examination or processing thereof
- H01J37/26—Electron or ion microscopes; Electron or ion diffraction tubes
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- H—ELECTRICITY
- H01—ELECTRIC ELEMENTS
- H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
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- H—ELECTRICITY
- H01—ELECTRIC ELEMENTS
- H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
- H01J37/00—Discharge tubes with provision for introducing objects or material to be exposed to the discharge, e.g. for the purpose of examination or processing thereof
- H01J37/02—Details
- H01J37/22—Optical or photographic arrangements associated with the tube
- H01J37/222—Image processing arrangements associated with the tube
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- H—ELECTRICITY
- H01—ELECTRIC ELEMENTS
- H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
- H01J37/00—Discharge tubes with provision for introducing objects or material to be exposed to the discharge, e.g. for the purpose of examination or processing thereof
- H01J37/26—Electron or ion microscopes; Electron or ion diffraction tubes
- H01J37/28—Electron or ion microscopes; Electron or ion diffraction tubes with scanning beams
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- H—ELECTRICITY
- H01—ELECTRIC ELEMENTS
- H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
- H01J37/00—Discharge tubes with provision for introducing objects or material to be exposed to the discharge, e.g. for the purpose of examination or processing thereof
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Abstract
The invention discloses a kind of electron microscope tomograph imaging method and system, the described method includes：Step 1, system reset；Step 2, acquired projections and its parameter；Step 3, the projection collected according to step 23 and its parameter reconstruct the tomographic map of sample to be tested.The present invention can choose projective parameter in areas imaging, i.e., accurately control sample to be tested is in different angle combinations (alphai,phij), and under the projective parameter state of selection automatic collection sample to be tested projection, therefore the full-automatic data acquisition for being tiltedly inclined to picture with cone to the uniclinal of sample to be tested can be realized simultaneously, and the tomographic map for quick obtaining sample to be tested provides advantage.
Description
Technical field
The present invention relates to electron microscopic technical field of imaging, more particularly to a kind of electron microscope tomograph imaging method and
System.
Background technology
Electron microscope tomography technology is to be shot using electron microscope with rebuilding three-dimensional cell or Molecular Graphs picture.Electricity
The micro- fault imaging of son generally includes two steps, and the first step is the technical parameter of projection and the projection of collecting sample, and second
Step is the technical parameter of the projection and projection collected using the first step, reconstructs the tomographic map of sample.It is common micro-
Mirror tomography technology mainly includes three kinds of image pickup methods：Uniclinal is oblique, double inclinations and cone tilt.It can be obtained using cone angled manner
The former spatial information obtained hands over other two methods more.
But the electron microscope fault imaging used at present mainly uses the acquisition mode for having expert to participate in judge, i.e.,
The control device that feedback-less is operated by operating personnel moves sample, and carries out projection acquisition operation.Such mode of operation
Due to there is the closed-loop control that expert participates in, not only taking more and limited to the measurement accuracy of projective parameter, the projection collected
Parameter is more rough, so as to also result in second step reconstruction process take it is more.And due to manually judging the process whether being aligned
In, it is necessary to be repeatedly imaged to sample in same angle, the deformation that sample can be caused more serious.
Thus, it is desirable to have a kind of technical solution is come at least one in the drawbacks described above that overcomes or at least mitigate the prior art
It is a.
The content of the invention
It is an object of the invention to provide a kind of electron microscope tomograph imaging methods and system to overcome or at least mitigate
It is at least one in the drawbacks described above of the prior art.
To achieve the above object, the present invention provides a kind of electron microscope tomograph imaging method, and the electron microscope breaks
Layer imaging method includes：Step 1, system reset, the step specifically include：Step 11, by the center of sample stage adjust to positioned at
On the primary optical axis of electron microscope；Step 12, the sample to be tested being placed on sample stage is adjusted to nought state；Step 2, acquisition is thrown
Shadow and its parameter, the step specifically include：Step 21, transmission image of the sample to be tested under nought state is gathered, and in the transmission
Coordinate value (u, v, w) of the center of area-of-interest in three-dimensional system of coordinate is chosen on image, the origin of the three-dimensional system of coordinate is
The direction of the central point of sample stage upper surface, the upper surface of the plane Duplicate Samples sample platform where (u, v), u and v are mutually perpendicular to, w's
Direction is the normal direction of sample stage；Step 22, according to acquisition in need projective parameter in angle combinations (alphai,
phij), calculate corresponding each angle combinations (alphai,phij) under area-of-interest center (u, v, w) compared with electronics
Offset (the Δ u, Δ v, Δ w) of microscopical primary optical axis；Step 23, sample stage is controlled according to each angle in step 22
Combine (alphai,phij) rotation and according to each angle combinations (alphai,phij) corresponding offset (Δ u,
Δ v, Δ w) compensate movement, so that the center (u, v, w) of area-of-interest is always positioned on the primary optical axis of electron microscope,
Gathering sample to be tested is in each state (alpha simultaneouslyi,phij, u, v, w) under projection；Step 3, gathered according to step 23
The projection arrived and its parameter reconstruct the tomographic map of sample to be tested.
Further, in the case of step 21 is used for relatively thin sample to be tested, specifically include：Step 211, low resolution is utilized
Rate gathers image of the sample to be tested under nought state, which obtains a wide range of transmission plot of sample to be tested after pretreatment
Picture；Step 212, the u and v in the coordinate value at the center of area-of-interest are chosen on a wide range of transmission image of sample to be tested,
And using the corresponding height of the mid-depth of sample to be tested as the coordinate value at the center of area-of-interest in w, with determine sense it is emerging
The centre coordinate value (u, v, w) in interesting region；In the case of step 21 is used for thicker sample to be tested, specifically include：Step 211 ',
Utilize image of the low resolution acquisition sample to be tested under nought state；Step 212 ', step 211 ' the image collected process is in advance
After processing, a wide range of transmission image of sample to be tested is obtained, region of interest is chosen on a wide range of transmission image of sample to be tested
U and v in the coordinate value at the center in domain, and using the corresponding height of the mid-depth of sample to be tested as the center of area-of-interest
Coordinate value in w, to primarily determine that the centre coordinate value (u, v, w) of area-of-interest；Step 213 ', utilize step 212 ' really
Fixed centre coordinate value (u, v, w) and a default angle combinations (alphai,phij), calculate offset (Δ u, Δ v, Δ
w)；Each angle combinations (alpha in step 214 ', control sample stage according to step 213 'i,phij) rotation and according to
With each angle (alphai,phij) (Δ u, Δ v, Δ w) compensate movement to the corresponding offset of combination, so that sense is emerging
The center in interesting region is always positioned on the primary optical axis of electron microscope；Step 215 ', existed using high-resolution acquisition sample to be tested
Image under nought state, according to step 212 ' identical method, with finally determine the centre coordinate value of area-of-interest (u, v,
w)。
Further, in step 22 (Δ u, Δ v, the calculation formula of Δ w) are：
In formula, α alphai,For phij, i, j are natural number.
Further, step 23 specifically includes：Step 231, the alpha angles by the fixation of alpha axis within the specified range
Angle value；Step 232, driving phi axis rotates the phi angle values to specified range；Step 233, control sample stage according to
Step 231 and step 232 angle combinations (alphai,phij) (Δ u, Δ v, Δ w) compensate movement for corresponding offset；
Step 234, the projection of the sample to be tested under 233 state of acquisition step；Step 235, return to step 231 is to step 234, until institute
Some angle combinations (alphai,phij) projection acquisition of sample to be tested under state completes.
Further, step 11 specifically includes：Step 111, place calibration sample on sample stage, calibration sample by
Electron lucent material is made, and identical with the size of sample to be tested, and one layer is arranged in radiation in the mid-depth of calibration sample
The nano level metal filament of shape distribution, filament are intersected in the center of calibration sample；Step 112, sample stage is adjusted
Alpha is 0, between the center by the calibration sample in the center and the calibration sample image of calibration sample image
Deviation, the centre coordinate (u, v) at the center of calibration sample platform are in (0,0)；Step 113, the alpha of sample stage is adjusted as most
Greatly, by the deviation between the center of the calibration sample in the center and the calibration sample image of calibration sample image,
The centre coordinate w at the center of calibration sample platform is in 0.
Further, step 112 and step 113 are specially：Step 114, the image of calibration sample is gathered, calculates image
Center and image in calibration sample center between deviation, if there are deviations, 115 are entered step, if there is no inclined
Difference, then sample stage calibration finish；Step 115, according to the deviation in step 114, deviation is adjusted, while using than in step 114
The higher resolution acquisition calibration sample of resolution ratio image, and return to step 114.
The present invention also provides a kind of electron microscope computed tomography (SPECT) system, the electron microscope computed tomography (SPECT) system bag
It includes：Various dimensions loading sample table device, including sample stage, three axis translation members, alpha rotating members and phi rotating members,
Wherein：The sample stage is used to place sample to be tested, the three axis translation member, alpha rotating members and phi rotating member roots
The sample stage movement is driven according to the control instruction of the master control set, so that the center of the area-of-interest of sample to be tested is always
On the primary optical axis of electron microscope；Electron microscope is used for the image data of acquisition sample to be tested in real time；Master control fills
It puts, including system reset module, projection and its parameter collection module and rebuilds module, wherein：The system reset module is used
In by drive dynamic triaxial translation member, alpha rotating members and/or phi rotating members by the center of the sample stage adjust to
On the primary optical axis of the electron microscope and the sample to be tested that is placed on the sample stage is adjusted to nought state；Projection
And its parameter collection module, including：Area-of-interest determination sub-module is used to gathering sample to be tested saturating under nought state
Image is penetrated, and coordinate value (u, v, w) of the center of area-of-interest in three-dimensional system of coordinate is chosen on the transmission image, this three
The origin of dimension coordinate system is the central point of the sample stage upper surface, the upper table of the parallel sample stage of plane where (u, v)
The direction of face, u and v are mutually perpendicular to, and the direction of w is the normal direction of the sample stage；Offset computational submodule, is used for root
According to acquisition in need projective parameter in angle combinations (alphai,phij), calculate each corresponding angle combinations
(alphai,phij) under area-of-interest center (u, v, w) compared with the primary optical axis of electron microscope offset (Δ u,
Δv,Δw)；Projection acquisition submodule is used to control the alpha rotating members and phi rotating members according to the offset
Each angle combinations (alpha that gauge operator module providesi,phij) rotation and control the three axis translation member according to
With each angle combinations (alphai,phij) (Δ u, Δ v, Δ w) compensate movement, so that sense is emerging for corresponding offset
The center (u, v, w) in interesting region is always positioned on the primary optical axis of the electron microscope, while is gathered sample to be tested and be in each
A state (alphai,phij, u, v, w) under projection；Module is rebuild, is used for according to the projection and its parameter collection module
The projection collected and its parameter reconstruct the tomographic map of sample to be tested.
Further, the area-of-interest determination sub-module determines the region of interest of sample to be tested in the following manner
Domain：
For relatively thin sample to be tested：First, the image using low resolution acquisition sample to be tested under nought state, the figure
A wide range of transmission image as obtaining sample to be tested after pretreatment；Then, on a wide range of transmission image of sample to be tested
The u and v in the coordinate value at the center of area-of-interest are chosen, and it is emerging using the corresponding height of the mid-depth of sample to be tested as sense
W in the coordinate value at the center in interesting region, to determine the centre coordinate value (u, v, w) of area-of-interest；
For thicker sample to be tested：Utilize image of the low resolution acquisition sample to be tested under nought state, image warp
The a wide range of transmission image of sample to be tested is obtained after crossing pretreatment；Then, chosen on a wide range of transmission image of sample to be tested
U and v in the coordinate value at the center of area-of-interest, and using the corresponding height of the mid-depth of sample to be tested as region of interest
W in the coordinate value at the center in domain, to primarily determine that the centre coordinate value (u, v, w) of area-of-interest；Recycling primarily determines that
Centre coordinate (u, v, w) and a default angle combinations (alphai,phij), calculate offset (Δ u, Δ v, Δ w)；
Furthermore the alpha rotating members and phi rotating members are controlled according to each angle combinations (alphai,phij) rotation and
Control the three axis translation member according to each angle (alphai,phij) combination corresponding offset (Δ u, Δ v, Δ
W) movement is compensated, so that the center of area-of-interest is always positioned on the primary optical axis of electron microscope；Finally, high score is utilized
Resolution gathers image of the sample to be tested under nought state, and area-of-interest is chosen on a wide range of transmission image of sample to be tested
U and v in the coordinate value at center, and using the corresponding height of the mid-depth of sample to be tested as the seat at the center of area-of-interest
W in scale value, to be ultimately determined to the centre coordinate value (u, v, w) of final area-of-interest.
Further, the projection acquisition submodule gathers sample to be tested and is in each state in the following manner
(alphai,phij, u, v, w) under projection：First, the alpha by alpha rotating members fixation within the specified range
Angle value；The phi rotating members is driven to rotate the phi angle values to specified range again；Then, three axis is controlled to put down
Component is moved according to angle combinations (alphai,phij) (Δ u, Δ v, Δ w) compensate movement, synchronously adopt for corresponding offset
Collect the projection of the sample to be tested under current state, until all angle combinations (alphai,phij) sample to be tested under state
Projection acquisition is completed.
Further, the electron microscope computed tomography (SPECT) system further includes：Calibration sample, by electron lucent material
Be made, and identical with the size of sample to be tested, the mid-depth of the calibration sample arrange one layer radially distribute receive
Meter level filament, filament are intersected in the center of the calibration sample；The sample stage and phi rotating members are all by electricity
Sub- transparent material is made.
The present invention can choose projective parameter in areas imaging, i.e., accurately control sample to be tested is different
Angle combinations (alphai,phij), and under the projective parameter state of selection automatic collection sample to be tested projection, therefore can be with
The full-automatic data acquisition for being tiltedly inclined to picture with cone to the uniclinal of sample to be tested is realized simultaneously, is quick obtaining sample to be tested
Tomographic map provide advantage.
Description of the drawings
Fig. 1 is the signal of the principle of a preferred embodiment of electron microscope computed tomography (SPECT) system provided by the present invention
Figure.
Fig. 2 is the schematic diagram of the principle of a preferred embodiment of the master control set in Fig. 1.
Fig. 3 is the schematic diagram of the principle of a preferred embodiment of the projection and its parameter collection module in Fig. 2.
Fig. 4 is the structure diagram of a preferred embodiment of the master control set in Fig. 1.
Fig. 5 is the operation principle schematic diagram of the alpha rotating members in Fig. 1.
Fig. 6 is the operation principle schematic diagram of the phi rotating members in Fig. 1.
Fig. 7 is the structure diagram of the calibration sample in the present invention.
Specific embodiment
In the accompanying drawings, represent same or similar element using same or similar label or have the function of same or like
Element.The embodiment of the present invention is described in detail below in conjunction with the accompanying drawings.
In the description of the present invention, term " " center ", " longitudinal direction ", " transverse direction ", "front", "rear", "left", "right", " vertical ",
The orientation or position relationship of the instructions such as " level ", " top ", " bottom " " interior ", " outer " are to be closed based on orientation shown in the drawings or position
System is for only for ease of the description present invention and simplifies description rather than instruction or imply that signified device or element must have
Specific orientation, with specific azimuth configuration and operation, therefore it is not intended that limiting the scope of the invention.
As shown in Figure 1 to Figure 3, the electron microscope tomograph imaging method that the present embodiment is provided includes：
Step 1, system reset, the step specifically include：
Step 11, the center of sample stage is adjusted on the primary optical axis of electron microscope.
Step 12, the sample to be tested being placed on sample stage is adjusted to nought state.Determine the parameter bag of sample plane position
U, v, w, alpha and phi are included, " nought state " refers to that u, v, w, alpha and phi are 0.
U, v, w are usually made by being determined with reference to three-dimensional system of coordinate with reference to three-dimensional system of coordinate with placing the sample stage of sample to be tested
For references object, that is, the central point for defining sample stage upper surface is the origin with reference to three-dimensional system of coordinate, and the plane where (u, v) is put down
The direction of the upper surface of row sample stage, u axis and v axis is mutually perpendicular to, and the direction of w axis is the normal direction of sample stage, and u is to treat test sample
Compared with distance value of the origin with reference to three-dimensional system of coordinate along u axis, v is the center of sample to be tested compared with reference to three at the center of product
Distance value of the origin of dimension coordinate system along v axis, w be sample to be tested center compared with the origin with reference to three-dimensional system of coordinate along w axis
Distance value.U, v, w can be adjusted by the three axis translation members 12 shown in Fig. 4.
The normal direction that the sample stage 11 come is illustrated in Fig. 4 is defined, i.e., the direction of vertical paper is the shape that alpha is 0
State.Alpha rotating members 13 are rotated by the rotation arrows of such as Fig. 5, thus it is possible to vary the normal direction of sample stage 11 changes sample
Angle between the normal direction of sample stage 11 is shown in the current normal direction of sample platform 11 and Fig. 4.
A radius is chosen in sample stage 11, the R0 come as reference point, passes through phi rotating members as illustrated in Fig. 6
14 rotate normal of the sample stage 11 around sample stage 11, then can reference point be rotated to R1, then, between radius R1 and R0
Angle be phi, that is to say, that if R0, as reference point, the position phi of R0 is 0 state.
Such as Fig. 5 illustratively, alpha and phi is to be three-dimensional system of coordinate with reference to three-dimensional system of coordinate (u, v, w) in the state of 0
(x, y, z), that is, the two three-dimensional system of coordinates overlap, and u at this time is x, v y, w z.
Step 2, acquired projections and its parameter, the step specifically include：
Step 21, transmission image of the sample to be tested under nought state is gathered, and region of interest is chosen on the transmission image
Coordinate value (u, v, w) of the center in domain in reference to three-dimensional system of coordinate.
Step 22, return to step 1, according to acquisition in need projective parameter in angle combinations (alphai,phij),
Calculate corresponding each angle combinations (alphai,phij) under area-of-interest center (u, v, w) compared with electron microscope
Primary optical axis offset (Δ u, Δ v, Δ w)；Δ u is the center (u, v, w) of area-of-interest compared with alpha axis and phi
The offset of the intersection point of axis in the x direction, Δ v are the center (u, v, w) of area-of-interest compared with alpha axis and phi axis
The offset of intersection point in y-direction, Δ w are the center (u, v, w) of area-of-interest compared with alpha axis and the intersection point of phi axis
Offset in a z-direction.The computational methods of Δ u, Δ v and Δ w are as follows：
In formula, α alphai,For phij, i, j are natural number.
Step 23, sample stage is controlled according to each angle combinations (alpha in step 22i,phij) rotation and according to
With each angle combinations (alphai,phij) (Δ u, Δ v, Δ w) compensate movement, so that sense is emerging for corresponding offset
The center (u, v, w) in interesting region is always positioned on the primary optical axis of electron microscope, while is gathered sample to be tested and be in each shape
State (alphai,phij, u, v, w) under projection.
Step 3, the projection collected according to step 23 and its parameter reconstruct the tomographic map of sample to be tested.Step 3 carries
The method for reconstructing of confession is the prior art, and details are not described herein.
The present embodiment can choose projective parameter in areas imaging, i.e., accurately control sample to be tested is in difference
Angle combinations (alphai,phij), and under the projective parameter state of selection automatic collection sample to be tested projection, therefore can
To realize that the uniclinal to sample to be tested is tiltedly inclined to the full-automatic data acquisition of picture with cone simultaneously, test sample is treated for quick obtaining
The tomographic map of product provides advantage.
In one embodiment, due to sample to be tested compared to can reconstruction regions it is larger, and before being shot, experiment
Person can not know specific area-of-interest, therefore need once to be shot to determine interest region, the step in a big way
21 provide the following two kinds pattern.
The first pattern, single determine method：For relatively thin sample to be tested, relatively thin sample to be tested in the present invention can be with
Be interpreted as when to rebuild area thickness direction position accuracy demand it is not high when sample to be tested.The step 21 specifically includes：
Step 211, using image of the low resolution acquisition sample to be tested under nought state, the image after pretreatment,
Obtain a wide range of transmission image of sample to be tested.In this step, electron accelerating voltage is far smaller than electron microscope shooting when institute
It is known as " low resolution " with accelerating potential." pretreatment " includes selectivity and carries out the processing such as noise reduction, dyeing and grayscale adjusting.
Step 212, the u in the coordinate value at the center of area-of-interest is chosen on a wide range of transmission image of sample to be tested
And v, and using the corresponding height of the mid-depth of sample to be tested as the coordinate value at the center of area-of-interest in w, with determine
The centre coordinate value (u, v, w) of area-of-interest.
The method that the present embodiment is provided is for relatively thin sample, and since its thickness is smaller, carrying out one-time positioning can essence
Really determine the specific scope of area-of-interest, thus it is easy to operate.
Second of pattern determines method twice：For thicker sample to be tested, thicker sample to be tested in the present invention can be with
Be interpreted as when to rebuild area thickness direction position accuracy demand it is high when sample to be tested.The step 21 specifically includes：
Step 211 ', utilize image of the low resolution acquisition sample to be tested under nought state；In this step, electronics accelerates electricity
Accelerating potential used is known as " low resolution " when pressure is far smaller than electron microscope shooting.
Step 212 ', step 211 ' the image collected after pretreatment, obtains a wide range of transmission plot of sample to be tested
Picture, u and v on a wide range of transmission image of sample to be tested in the coordinate value at the center of selection area-of-interest, and with to be measured
The corresponding height of mid-depth of sample is as the w in the coordinate value at the center of area-of-interest, to primarily determine that region of interest
The centre coordinate (u, v, w) in domain." pretreatment " includes selectivity and carries out the processing such as noise reduction, dyeing and grayscale adjusting.
The definite centre coordinate (u, v, w) of step 213 ', utilize step 212 ' and a default angle combinations
(alphai,phij), calculate offset (Δ u, Δ v, Δ w).
Step 214 ', control sample stage alpha axis and phi axis according to step 213 ' in each angle combinations
(alphai,phij) rotation and according to each angle (alphai,phij) the corresponding offset of combination (Δ u, Δ v,
Δ w) compensates movement, so that the center of area-of-interest is always positioned on the primary optical axis of electron microscope.
Step 215 ', using image of the high-resolution acquisition sample to be tested under nought state, according to step 212 ' identical
Method, finally to determine the centre coordinate (u, v, w) of area-of-interest.In this step, electron accelerating voltage and electron microscope
It is known as " high-resolution " when accelerating potential used is not much different during shooting.
The method that the present embodiment is provided is directed to the relatively large sample of thickness, takes and positions corresponding straight line side twice
Journey intersection point is the central point of area-of-interest, it is thus possible to improve the accuracy of the positioning to area-of-interest.
In one embodiment, step 23 specifically includes：
Step 231, alpha axis is fixed into alpha angle values within the specified range, the value range of alpha for (-
60 °, 60 °).
Step 232, driving phi axis rotates the phi angle values to specified range, the value range of phi for (0 °,
360°)。
Step 233, control sample stage according to step 231 and step 232 angle combinations (alphai,phij) corresponding
(Δ u, Δ v, Δ w) compensate movement to offset.
Step 234, the projection of the sample to be tested under 233 state of acquisition step.
Step 235, return to step 231 is to step 234, such as：Alpha axis is fixed within the specified range another
Alpha angle values, driving phi axis rotates the phi angle values to specified range, until all angle combinations (alphai,
phij) projection acquisition of sample to be tested under state completes.
The method that the present embodiment is provided all can automatically move sample to be tested before every time to the shooting of projection
It is dynamic, area-of-interest is made to be located on the primary optical axis of electron microscope, it is achieved thereby that automatic shooting process without human intervention,
The operating time is so not only greatly reduced, and since each compensation rate is directly given by controller, is reduced to sample
The exposure frequency of product, image error caused by so as to greatly reduce sample deformation.
This embodiment achieves the arbitrary selections of projective parameter state.
As shown in fig. 7, in one embodiment, step 11 specifically includes：
Step 111, calibration sample is placed on sample stage, calibration sample is made of electron lucent material, and with treating
The size of sample is identical, and one layer of nano level metal filament to radially distribute is arranged in the mid-depth of calibration sample,
Filament is intersected in the center of calibration sample.
Step 112, the alpha for adjusting sample stage is 0, passes through center and the calibration sample figure of calibration sample image
Deviation between the center of calibration sample as in, the centre coordinate (u, v) at the center of calibration sample platform are in (0,0)；
Step 113, the alpha of sample stage is adjusted as maximum, passes through center and the calibration sample of calibration sample image
Deviation between the center of calibration sample in product image, the centre coordinate w at the center of calibration sample platform are in 0.
By step 11, it can realize that the center of sample stage is adjusted on the primary optical axis of electron microscope.
Present embodiments provide a kind of calibration method to sample stage, advance calibration help to improve acquired projections and its
The accuracy of parameter, and then provide advantage more truly to reconstruct the tomographic map of sample to be tested.
In one embodiment, step 112 and step 113 are specially：
Step 114, the image of calibration sample is gathered, calculates the center of the calibration sample in the center and image of image
Between deviation, if there are deviations, enter step 115, if there is no deviation, sample stage calibration finishes；
Step 115, according to the deviation in step 114, deviation is adjusted, the method for the adjustment deviation is the prior art, herein
It repeats no more.Simultaneously using the image of resolution acquisition calibration sample more higher than resolution ratio in step 114, and return to step
Rapid 114.
As shown in Figure 1, the present invention also provides a kind of electron microscope computed tomography (SPECT) system, electron microscope fault imaging systems
System includes：Various dimensions loading sample table device 1, electron microscope 2 and master control set 3, wherein：
Various dimensions loading sample table device 1 includes sample stage 11, three axis translation members 12, alpha rotating members 13 and phi
Rotating member 14, wherein：
Sample stage 11 is used to place sample to be tested, three axis translation members 12, alpha rotating members 13 and phi rotating members
14 drive sample stage 11 to move according to the control instruction of master control set 3, so that the center of the area-of-interest of sample to be tested is always
On the primary optical axis of electron microscope 1.
Electron microscope 2 is for the real-time image data for gathering sample to be tested.
As shown in Fig. 2, master control set 3 includes system reset module 31, projection and its parameter collection module 32 and rebuilds mould
Block 33, wherein：
System reset module 31 is used to rotate by driving dynamic triaxial translation member 12, alpha rotating members 13 and/or phi
Component 14 controls the center of sample stage 11 to be located on the primary optical axis of electron microscope 2 and be placed on sample stage 11 and treats test sample
Product are adjusted to nought state.
Determining the parameter of sample plane position includes u, v, w, alpha and phi, and " nought state " refers to u, v, w, alpha
All it is 0 with phi.
U, v, w are usually made by being determined with reference to three-dimensional system of coordinate with reference to three-dimensional system of coordinate with placing the sample stage of sample to be tested
For references object, that is, the central point for defining sample stage upper surface is the origin with reference to three-dimensional system of coordinate, and the plane where (u, v) is put down
The direction of the upper surface of row sample stage, u and v are mutually perpendicular to, and the direction of w is the normal direction of sample stage.U, v, w can pass through Fig. 4
In the three axis translation members 12 that show be adjusted.
The normal direction that the sample stage 11 come is illustrated in Fig. 4 is defined, i.e., the direction of vertical paper is the shape that alpha is 0
State.Alpha rotating members 13 are rotated by the rotation arrows of such as Fig. 5, thus it is possible to vary the normal direction of sample stage 11 changes sample
Angle between the normal direction of sample stage 11 is shown in the current normal direction of sample platform 11 and Fig. 4.
A radius is chosen in sample stage 11, the R0 come as reference point, passes through phi rotating members as illustrated in Fig. 6
14 rotate normal of the sample stage 11 around sample stage 11, then can reference point be rotated to R1, then, between radius R1 and R0
Angle be phi, that is to say, that when R1 be located at R0 position phi be 0 state.
As shown in figure 3, projection and its parameter collection module 32 include area-of-interest determination sub-module 321, offset gauge
Operator module 322 and projection acquisition submodule 323, wherein：
Area-of-interest determination sub-module 321 is used to gather image of the sample to be tested under nought state, and on this image
Choose the centre coordinate (u, v, w) of area-of-interest.
Offset computational submodule 322 be used for according to acquisition in need angle combinations (alphai,phij), calculate it
In each angle combinations it is corresponding (Δ u, Δ v, Δ w), Δ u be area-of-interest center (u, v, w) compared with alpha axis
With the offset of the intersection point of phi axis in the x direction, Δ v is the center (u, v, w) of area-of-interest compared with alpha axis and phi
The offset of the intersection point of axis in y-direction, Δ w are the center (u, v, w) of area-of-interest compared with alpha axis and phi axis
The offset of intersection point in a z-direction.The computational methods of Δ u, Δ v and Δ w are as follows：
In formula, α alphai,For phij, i, j are natural number.
Projection acquisition submodule 323 be used for control sample stage alpha axis and phi axis according to offset computational submodule
322 each angle combinations (alpha providedi,phij) rotation and according to each angle combinations (alphai,phij)
(Δ u, Δ v, Δ w) compensate movement to corresponding offset, so that the center of area-of-interest is always positioned at electron microscopic
On the primary optical axis of mirror, acquisition sample stage is in each state (alphai,phij, u, v, w) under sample to be tested image data.
Module 33 is rebuild to be used to be in each state according to the sample stage that projection and its parameter collection module 32 collect
(alphai,phij, u, v, w) under sample to be tested image data, reconstruct the tomographic map of sample to be tested.Module 33 is rebuild to carry
The method for reconstructing of confession is the prior art, and details are not described herein.
The present embodiment can choose projective parameter in areas imaging, i.e., accurately control sample to be tested is in difference
Angle combinations (alphai,phij), and under the projective parameter state of selection automatic collection sample to be tested projection, therefore can
To realize that the uniclinal to sample to be tested is tiltedly inclined to the full-automatic data acquisition of picture with cone simultaneously, test sample is treated for quick obtaining
The tomographic map of product provides advantage.
In one embodiment, due to sample to be tested compared to can reconstruction regions it is larger, and before being shot, experiment
Person can not know specific area-of-interest, therefore need once to be shot to determine interest region, region of interest in a big way
Domain determination sub-module 321 provides the following two kinds pattern.
The first pattern, single determine method：For relatively thin sample to be tested, relatively thin sample to be tested in the present invention can be with
Be interpreted as when to rebuild area thickness direction position accuracy demand it is not high when sample to be tested.Single determines that method is specially：
First, the image using low resolution acquisition sample to be tested under nought state, the image obtain after pretreatment
The a wide range of transmission image of sample to be tested；Then, chosen on a wide range of transmission image of sample to be tested in area-of-interest
U and v in the coordinate value of the heart, and using the corresponding height of the mid-depth of sample to be tested as the coordinate at the center of area-of-interest
W in value.Accelerating potential used is known as " low resolution " when electron accelerating voltage is far smaller than electron microscope shooting." pre- place
Reason " includes selectivity and carries out the processing such as noise reduction, dyeing and grayscale adjusting.
Second of pattern determines method twice：It in the present invention can be with for the thicker sample to be tested of thicker sample to be tested
Be interpreted as when to rebuild area thickness direction position accuracy demand it is high when sample to be tested.Determine that method is specially twice：
Using image of the low resolution acquisition sample to be tested under nought state, which obtains treating test sample after pretreatment
The a wide range of transmission image of product；Then, the seat at the center of area-of-interest is chosen on a wide range of transmission image of sample to be tested
U and v in scale value, and using the corresponding height of the mid-depth of sample to be tested as the coordinate value at the center of area-of-interest in
W, to primarily determine that the centre coordinate of area-of-interest (u, v, w)；Recycle the centre coordinate (u, v, w) and one primarily determined that
A default angle combinations (alphai,phij), calculate offset (Δ u, Δ v, Δ w)；Furthermore control the alpha axis of sample stage
With phi axis according to each angle combinations (alphai,phij) rotation and according to each angle (alphai,phij) group
Closing corresponding offset, (Δ u, Δ v, Δ w) compensate movement, so that the center of area-of-interest is always positioned at electronic display
On the primary optical axis of micro mirror；Finally, the image using high-resolution acquisition sample to be tested under nought state, in the big model of sample to be tested
The u and v in the coordinate value at the center that area-of-interest is chosen on transmission image are enclosed, and it is corresponding with the mid-depth of sample to be tested
Highly as the w in the coordinate value at the center of area-of-interest, to be ultimately determined to the centre coordinate of final area-of-interest
(u,v,w)。
Accelerating potential used is known as " low resolution " when electron accelerating voltage is far smaller than electron microscope shooting.Electronics adds
It is known as " high-resolution " when accelerating potential used is not much different when fast voltage is shot with electron microscope." pretreatment " includes selection
Property carry out noise reduction, the processing such as dyeing and grayscale are adjusted.
In one embodiment, the projection acquisition submodule 323 gathers sample stage and is in each in the following manner
State (alphai,phij, u, v, w) under sample to be tested projection：
First, alpha axis is fixed into alpha angle values within the specified range, the value range of alpha for (- 60 °,
60°).Phi axis is driven to rotate the phi angle values to specified range again, the value range of phi is (0 °, 360 °).Then,
Sample stage is controlled according to angle combinations (alphai,phij) (Δ u, Δ v, Δ w) compensate movement, together for corresponding offset
The image data of sample to be tested under step acquisition current state；Finally, it is another alpha angle values by alpha axial adjustment, until
All angle combinations (alphai,phij) projection acquisition of sample to be tested under state completes.
As shown in fig. 7, the electron microscope computed tomography (SPECT) system further includes calibration sample 4, calibration sample 4 is by electricity
Sub- transparent material is made, and identical with the size of sample to be tested, and the mid-depth of calibration sample 4 arranges one layer radial point
The nano level metal filament of cloth, filament are intersected in the center of calibration sample 4.Sample stage 11 and phi rotating members 14 are all
It is made of electron lucent material.The calibration method of sample stage 11 is already mentioned above, herein not reinflated description.
It is last it is to be noted that：The above embodiments are merely illustrative of the technical solutions of the present invention, rather than its limitations.This
The those of ordinary skill in field should be understood：It can modify to the technical solution recorded in foregoing embodiments or right
Which part technical characteristic carries out equivalent substitution；These modifications are replaced, and the essence of appropriate technical solution is not made to depart from this
Invent the spirit and scope of each embodiment technical solution.
Claims (10)
1. a kind of electron microscope tomograph imaging method, which is characterized in that including：
Step 1, system reset, the step specifically include：
Step 11, the center of sample stage is adjusted on the primary optical axis of electron microscope；
Step 12, the sample to be tested being placed on sample stage is adjusted to nought state；
Step 2, acquired projections and its parameter, the step specifically include：
Step 21, transmission image of the sample to be tested under nought state is gathered, and area-of-interest is chosen on the transmission image
Coordinate value (u, v, w) of the center in three-dimensional system of coordinate, the origin of the three-dimensional system of coordinate is the central point of sample stage upper surface,
The direction of the upper surface of plane Duplicate Samples sample platform where (u, v), u and v are mutually perpendicular to, and the direction of w is the normal side of sample stage
To；
Step 22, according to acquisition in need projective parameter in angle combinations (alphai,phij), it calculates and corresponds to each
Angle combinations (alphai,phij) under area-of-interest center (u, v, w) compared with the primary optical axis of electron microscope offset
Measure (Δ u, Δ v, Δ w)；
Step 23, sample stage is controlled according to each angle combinations (alpha in step 22i,phij) rotation and according to it is every
One angle combinations (alphai,phij) (Δ u, Δ v, Δ w) compensates movement, so that region of interest for corresponding offset
The center (u, v, w) in domain is always positioned on the primary optical axis of electron microscope, while is gathered sample to be tested and be in each state
(alphai,phij, u, v, w) under projection；
Step 3, the projection collected according to step 23 and its parameter reconstruct the tomographic map of sample to be tested.
2. electron microscope tomograph imaging method as described in claim 1, which is characterized in that step 21 is for relatively thin to be measured
In the case of sample, specifically include：
Step 211, the image using low resolution acquisition sample to be tested under nought state, the image obtain after pretreatment
The a wide range of transmission image of sample to be tested；
Step 212, the u and v in the coordinate value at the center of area-of-interest are chosen on a wide range of transmission image of sample to be tested,
And using the corresponding height of the mid-depth of sample to be tested as the coordinate value at the center of area-of-interest in w, with determine sense it is emerging
The centre coordinate value (u, v, w) in interesting region；In the case of step 21 is used for thicker sample to be tested, specifically include：
Step 211 ', utilize image of the low resolution acquisition sample to be tested under nought state；
Step 212 ', step 211 ' the image collected after pretreatment, obtains a wide range of transmission image of sample to be tested,
The u and v in the coordinate value at the center of area-of-interest are chosen on a wide range of transmission image of sample to be tested, and with sample to be tested
The corresponding height of mid-depth is as the w in the coordinate value at the center of area-of-interest, to primarily determine that in area-of-interest
Heart coordinate value (u, v, w)；
The definite centre coordinate value (u, v, w) of step 213 ', utilize step 212 ' and a default angle combinations (alphai,
phij), calculate offset (Δ u, Δ v, Δ w)；
Each angle combinations (alpha in step 214 ', control sample stage according to step 213 'i,phij) rotation and according to
With each angle (alphai,phij) (Δ u, Δ v, Δ w) compensate movement to the corresponding offset of combination, so that sense is emerging
The center in interesting region is always positioned on the primary optical axis of electron microscope；
The identical method of step 215 ', using image of the high-resolution acquisition sample to be tested under nought state, according to step 212 ',
Finally to determine the centre coordinate value (u, v, w) of area-of-interest.
3. electron microscope tomograph imaging method as claimed in claim 1 or 2, which is characterized in that (Δ u, Δ in step 22
V, the calculation formula of Δ w) are：
In formula, α alphai, φ phij, i, j are natural number.
4. electron microscope tomograph imaging method as claimed in claim 3, which is characterized in that step 23 specifically includes：
Step 231, the alpha angle values by the fixation of alpha axis within the specified range；
Step 232, driving phi axis rotates the phi angle values to specified range；
Step 233, control sample stage according to step 231 and step 232 angle combinations (alphai,phij) corresponding offset
(Δ u, Δ v, Δ w) compensate movement to amount；
Step 234, the projection of the sample to be tested under 233 state of acquisition step；
Step 235, return to step 231 is to step 234, until all angle combinations (alphai,phij) test sample is treated under state
The projection acquisition of product is completed.
5. electron microscope tomograph imaging method as claimed in claim 4, which is characterized in that step 11 specifically includes：
Step 111, calibration sample is placed on sample stage, calibration sample is made of electron lucent material, and with treating test sample
The size of product is identical, and one layer of nano level metal filament to radially distribute, metal are arranged in the mid-depth of calibration sample
Filament is intersected in the center of calibration sample；
Step 112, the alpha for adjusting sample stage is 0, by the center of calibration sample image and the calibration sample image
Calibration sample center between deviation, the centre coordinate (u, v) at the center of calibration sample platform is in (0,0)；
Step 113, the alpha of sample stage is adjusted as maximum, passes through center and the calibration sample figure of calibration sample image
Deviation between the center of calibration sample as in, the centre coordinate w at the center of calibration sample platform are in 0.
6. electron microscope tomograph imaging method as claimed in claim 5, which is characterized in that step 112 and step 113 are specific
For：
Step 114, the image of calibration sample is gathered, between the center for calculating the calibration sample in the center and image of image
Deviation, if there are deviations, enter step 115, if there is no deviation, sample stage calibration finishes；
Step 115, according to the deviation in step 114, deviation is adjusted, while using than higher point of resolution ratio in step 114
Resolution gathers the image of calibration sample, and return to step 114.
7. a kind of electron microscope computed tomography (SPECT) system, which is characterized in that including：
Various dimensions loading sample table device (1), including sample stage (11), three axis translation members (12), alpha rotating members
(13) and phi rotating members (14), wherein：
The sample stage (11) for placing sample to be tested, the three axis translation member (12), alpha rotating members (13) and
Phi rotating members (14) drive the sample stage (11) to move according to the control instruction of master control set (3), so that sample to be tested
The center of area-of-interest is always positioned on the primary optical axis of electron microscope (1)；
Electron microscope (2) is used for the image data of acquisition sample to be tested in real time；
Master control set (3) including system reset module (31), projection and its parameter collection module (32) and rebuilds module
(33), wherein：
The system reset module (31) be used for by drive dynamic triaxial translation member (12), alpha rotating members (13) and/or
Phi rotating members (14) by the center of the sample stage (11) adjust on the primary optical axis positioned at the electron microscope (2) with
And the sample to be tested being placed on the sample stage (11) is adjusted to nought state；
Projection and its parameter collection module (32), including：
Area-of-interest determination sub-module (321) is used to gather transmission image of the sample to be tested under nought state, and at this thoroughly
It penetrates on image and chooses coordinate value (u, v, w) of the center of area-of-interest in three-dimensional system of coordinate, the origin of the three-dimensional system of coordinate
It is the central point of the sample stage (11) upper surface, the upper surface of the parallel sample stage (11) of the plane where (u, v), u and v
Direction be mutually perpendicular to, the direction of w is the normal direction of the sample stage (11)；
Offset computational submodule (322), be used for according to acquisition in need projective parameter in angle combinations (alphai,
phij), calculate corresponding each angle combinations (alphai,phij) under area-of-interest center (u, v, w) compared with electronics
Offset (the Δ u, Δ v, Δ w) of microscopical primary optical axis；
Projection acquisition submodule (323), be used to controlling the alpha rotating members (13) and phi rotating members (14) according to
Each angle combinations (alpha that the offset computational submodule (322) providesi,phij) rotate and control three axis
Translation member (12) according to each angle combinations (alphai,phij) (Δ u, Δ v, Δ w) are mended for corresponding offset
Movement is repaid, so that the center (u, v, w) of area-of-interest is always positioned on the primary optical axis of the electron microscope (2), is adopted simultaneously
Collection sample to be tested is in each state (alphai,phij, u, v, w) under projection；
Module (33) is rebuild, is used for the projection collected according to the projection and its parameter collection module (32) and its parameter,
Reconstruct the tomographic map of sample to be tested.
8. electron microscope computed tomography (SPECT) system as claimed in claim 7, which is characterized in that the area-of-interest determines son
Module (321) determines the area-of-interest of sample to be tested in the following manner：
For relatively thin sample to be tested：First, the image using low resolution acquisition sample to be tested under nought state, image warp
The a wide range of transmission image of sample to be tested is obtained after crossing pretreatment；Then, chosen on a wide range of transmission image of sample to be tested
U and v in the coordinate value at the center of area-of-interest, and using the corresponding height of the mid-depth of sample to be tested as region of interest
W in the coordinate value at the center in domain, to determine the centre coordinate value (u, v, w) of area-of-interest；
For thicker sample to be tested：Using image of the low resolution acquisition sample to be tested under nought state, the image is by pre-
The a wide range of transmission image of sample to be tested is obtained after processing；Then, it is emerging that sense is chosen on a wide range of transmission image of sample to be tested
U and v in the coordinate value at the center in interesting region, and using the corresponding height of the mid-depth of sample to be tested as area-of-interest
W in the coordinate value at center, to primarily determine that the centre coordinate value (u, v, w) of area-of-interest；During recycling primarily determines that
Heart coordinate (u, v, w) and a default angle combinations (alphai,phij), calculate offset (Δ u, Δ v, Δ w)；Furthermore
The alpha rotating members (13) and phi rotating members (14) are controlled according to each angle combinations (alphai,phij) rotation
And the control three axis translation member (12) according to each angle (alphai,phij) the corresponding offset (Δ of combination
U, Δ v, Δ w) compensate movement, so that the center of area-of-interest is always positioned on the primary optical axis of electron microscope；Finally,
Using image of the high-resolution acquisition sample to be tested under nought state, it is emerging that sense is chosen on a wide range of transmission image of sample to be tested
U and v in the coordinate value at the center in interesting region, and using the corresponding height of the mid-depth of sample to be tested as area-of-interest
W in the coordinate value at center, to be ultimately determined to the centre coordinate value (u, v, w) of final area-of-interest.
9. electron microscope computed tomography (SPECT) system as claimed in claim 8, which is characterized in that the projection acquisition submodule
(323) acquisition sample to be tested is in each state (alpha in the following manneri,phij, u, v, w) under projection：
First, by the fixed alpha angle values within the specified range of the alpha rotating members (13)；The phi is driven again
Rotating member (14) is rotated to the phi angle values in specified range；Then, the three axis translation member (12) is controlled according to angle
Degree combination (alphai,phij) (Δ u, Δ v, Δ w) compensate movement, under synchronous acquisition current state for corresponding offset
Sample to be tested projection, until all angle combinations (alphai,phij) sample to be tested under state projection acquisition it is complete
Into.
10. the electron microscope computed tomography (SPECT) system as any one of claim 7 to 9, which is characterized in that further include：
Calibration sample (4), is made of electron lucent material, and identical with the size of sample to be tested, the calibration sample
(4) mid-depth arranges one layer of nano level metal filament to radially distribute, and filament is intersected in the calibration sample
The center of product (4)；
The sample stage (11) and phi rotating members (14) are all made of electron lucent material.
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| JP5331780B2 (en) * | 2010-11-30 | 2013-10-30 | 株式会社日立ハイテクノロジーズ | Electron microscope, image reconstruction system for electron microscope, and image reconstruction method for electron microscope |
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