Difference between revisions of "Electron Tomography"

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(→‎case of using mrcImageCorrelation)
(→‎take tilt-series images)
 
(31 intermediate revisions by the same user not shown)
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<tr>  
 
<tr>  
 
<td>[[File:Input-Tomogram1.png]]<br>
 
<td>[[File:Input-Tomogram1.png]]<br>
−
<p align="Center">1 Axis Tilt(Center is 0°'s image)<br></p>
+
<p align="Center">1 Axis Tilt(Center is untilted image)<br></p>
 
</td>  
 
</td>  
 
</tr>  
 
</tr>  
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<tr>  
 
<tr>  
 
<td>[[File:Input-Tomogram2.png]]<br>
 
<td>[[File:Input-Tomogram2.png]]<br>
−
<p align="Center">2 Axis Tilt(Center is 0°'s image)(as 90° rotation image for 1 Axis Tilt's image)<br></p>
+
<p align="Center">2 Axis Tilt(Center is untilted image)(as 90° rotation image for 1 Axis Tilt's image)<br></p>
 
</td>  
 
</td>  
 
</tr>  
 
</tr>  
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　Align roughly each tilt image by using the correlation among images around center.
 
　Align roughly each tilt image by using the correlation among images around center.
  
−
<div>[[Media:1WDC-Tom-2dSet.zip|Input file]](rotation at range: ±60°, interval: 2°, around Y-axis)<br>
+
<div>[[Media:1WDC-Tom-2dSet.zip|Input file]](tilt at range: ±60°, interval: 2°, around Y-axis)<br>
 
<table>  
 
<table>  
 
<tr>  
 
<tr>  
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== [[Fine Alignment]] ==
 
== [[Fine Alignment]] ==
−
<div>　傾斜画像の軸の位置,角度を出来る限り一致させます.</div>
+
<div>　Align the position and the angle of tilt axis as possible.</div>
 
<br>
 
<br>
  
−
===実行例1(傾斜軸の調整)===
+
===Example 1(Alignment of tilt axis)===
−
<div>　軸の傾きは[[mrcImageTiltAxisSearch]]で算出することができます.</div>
+
<div>　Calculate tilt of axis by using [[mrcImageTiltAxisSearch]].</div>
 
<br>
 
<br>
  
−
<div>[[Media:1WDC-Tom-Tilt-2dSet.zip|Input file]](傾斜軸を10°傾けた１軸傾斜のdata)<br>
+
<div>[[Media:1WDC-Tom-Tilt-2dSet.zip|Input file]](1 axis tilt data whose axis is tilted by 10 degree)<br>
 
<table>  
 
<table>  
 
<tr>  
 
<tr>  
 
<td><p align="Center">[[File:1WDC-Tom-Tilt-2dSet.png]]<br>
 
<td><p align="Center">[[File:1WDC-Tom-Tilt-2dSet.png]]<br>
−
10°刻みで表示<br></p>  
+
10° interval<br></p>  
 
</td>  
 
</td>  
 
</tr>  
 
</tr>  
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<br>
 
<br>
  
−
<div>[[Media:Makefile-Tomogram1.zip|使用Makefile]]のSetting</div>
+
<div>[[Media:Makefile-Tomogram1.zip|Used Makefile]]'s Setting</div>
 
<pre>
 
<pre>
 
# RotMode
 
# RotMode
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<br>
 
<br>
  
−
<div>コマンド</div>
+
<div>Command</div>
 
<pre>
 
<pre>
 
make TiltFit
 
make TiltFit
Line 172: Line 172:
 
<br>
 
<br>
  
−
<div>Outputした軸の傾き(.tiltinfofileに格納される)</div>
+
<div>Output of axis tilt(It is stored at .tiltinfofile)</div>
 
<pre>
 
<pre>
 
9.895
 
9.895
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<br>
 
<br>
  
−
<div>３次元再構成したときの違い</div>
+
<div>difference about 3D Reconstruction</div>
 
<table>  
 
<table>  
 
<tr>  
 
<tr>  
−
<td>補正前</td>  
+
<td>Before correction</td>  
 
</tr>  
 
</tr>  
 
<tr>  
 
<tr>  
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</tr>  
 
</tr>  
 
<tr>  
 
<tr>  
−
<td>補正後</td>  
+
<td>After correction</td>  
 
</tr>  
 
</tr>  
 
<tr>  
 
<tr>  
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<br>
 
<br>
  
−
===実行例2(傾斜軸の繰り返し調整)===
+
===Example2(Repeatedly Alignment of tilt axis)===
−
<div>Outputされた軸の傾きを中心として,繰り返し[[mrcImageTiltAxisSearch]].</div>
+
<div>Repeat to use [[mrcImageTiltAxisSearch]], referring tilt of output axis.</div>
 
<br>
 
<br>
  
−
<div>[[Media:1WDC-Tom-Tilt1-2dSet.zip|Input file]](傾斜軸を1°傾けた１軸傾斜のdata)<br>
+
<div>[[Media:1WDC-Tom-Tilt1-2dSet.zip|Input file]](1 axis tilt data whose axis is tilted by 1 degree)<br>
 
<table>  
 
<table>  
 
<tr>  
 
<tr>  
 
<td><p align="Center">[[File:1WDC-Tom-Tilt1-2dSet.png]]<br>
 
<td><p align="Center">[[File:1WDC-Tom-Tilt1-2dSet.png]]<br>
−
10°刻みで表示<br></p>  
+
10° interval<br></p>  
 
</td>  
 
</td>  
 
</tr>  
 
</tr>  
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<br>
 
<br>
  
−
<div>[[Media:Makefile-Tomogram2.zip|使用Makefile]]のSetting</div>
+
<div>[[Media:Makefile-Tomogram2.zip|Used Makefile]]'s Setting</div>
 
<pre>
 
<pre>
 
# For mrcImageTiltAxisSearch
 
# For mrcImageTiltAxisSearch
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<br>
 
<br>
  
−
<div>コマンド</div>
+
<div>Command</div>
 
<pre>
 
<pre>
 
make TiltFit
 
make TiltFit
Line 251: Line 251:
 
<br>
 
<br>
  
−
<div>軸の傾きの遷移</div>
+
<div>transition of tilt of axis</div>
 
<pre>
 
<pre>
 
0.084
 
0.084
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0.701
 
0.701
 
</pre>
 
</pre>
−
<div>傾斜軸の傾きが1付近に近づきます.</div>
+
<div>The tilt of axis approaches gradually 1.</div>
 
<br>
 
<br>
  
 
== [[3D Reconstruction]] ==
 
== [[3D Reconstruction]] ==
−
<div>　２次元画像のセットから３次元画像を再構成します.</div>
+
<div>　Reconstruct 3D image from 2D image set.</div>
 
<br>
 
<br>
  
−
<div>[[Media:1WDC-shift1.3d|元の3Ddata]]'s image</div>
+
<div>[[Media:1WDC-shift1.3d|Original 3D data]]'s image</div>
 
<table>  
 
<table>  
 
<tr>  
 
<tr>  
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===[[mrc2Dto3D]]===
 
===[[mrc2Dto3D]]===
−
====実行例1(1軸傾斜での再構成)====
+
====Example1 (Reconstruction of 1 axis tilt)====
−
<div>　連続傾斜像の撮影で軸にズレがない場合は角度刻みをそのままInput fileリストにSettingします.</div>
+
<div>　If the tilt axis don't deviate, set the angle information directly to input file list.</div>
 
<br>
 
<br>
  
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<tr>  
 
<tr>  
 
<td><p align="Center">[[File:1WDC-Tom-Fit-2d.png]]<br>
 
<td><p align="Center">[[File:1WDC-Tom-Fit-2d.png]]<br>
−
10°刻みで表示<br></p>  
+
10° interval<br></p>  
 
</td>  
 
</td>  
 
</tr>  
 
</tr>  
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<br>
 
<br>
  
−
<div>コマンド: [[mrc2Dto3D]] -i Input.3dlst -o Input.3d -m 1</div>
+
<div>Command: [[mrc2Dto3D]] -i Input.3dlst -o Input.3d -m 1</div>
 
<br>
 
<br>
  
−
<div>[[Media:Makefile-Tomogram.zip|こちらのMakefile]]を使用すれば,以下のコマンドでも実行できます.</div>
+
<div>By using [[Media:Makefile-Tomogram.zip|This Makefile]], perform the following command for reconstruction.</div>
−
<div>Setting内容</div>
+
<div>Setting data</div>
 
<pre>
 
<pre>
 
# For Reconstruction
 
# For Reconstruction
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ROT3MAX=0
 
ROT3MAX=0
 
</pre>
 
</pre>
−
<div>コマンド</div>
+
<div>Command</div>
 
<pre>
 
<pre>
 
make 3DList
 
make 3DList
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<br>
 
<br>
  
−
====実行例2(2軸傾斜での再構成)====
+
====Example2 (Reconstruction of 2 axis tilt)====
−
<div>２軸で傾斜した画像にて３次元再構成を行います.</div>
+
<div>Perform 3D Reconstruction for images of 2 axis tilt.</div>
−
<div>[[Media:1WDC-Tom1-2dSet.zip|Input file]](X, Y-axis周りに±60°の範囲でそれぞれ10°刻みで傾斜)<br>
+
<div>[[Media:1WDC-Tom1-2dSet.zip|Input file]](tilt at range: each ±60°, interval: each 10°, around X, Y-axis))<br>
 
<table>  
 
<table>  
 
<tr>  
 
<tr>  
 
<td><p align="Center">[[File:1WDC-Tom1-2dSet.png]]<br>
 
<td><p align="Center">[[File:1WDC-Tom1-2dSet.png]]<br>
−
1行目がX-axis傾斜,２行目がY-axis傾斜<br></p>  
+
1st line is X-axis tilt, 2nd line is Y-axis tilt<br></p>  
 
</td>  
 
</td>  
 
</tr>  
 
</tr>  
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<br>
 
<br>
  
−
<div>[[Media:Makefile-Tomogram6.zip|こちらのMakefile]]を使って３次元再構成を行います.</div>
+
<div>By using [[Media:Makefile-Tomogram6.zip|This Makefile]], perform 3D Reconstruction.</div>
−
<div>コマンド</div>
+
<div>Command</div>
 
<pre>
 
<pre>
 
make all
 
make all
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<br>
 
<br>
  
−
<div>途中で使用する範囲を決めるために[[Display2]]が起動しますが,今回は全ての範囲を選択してROIInformationを保存します.<br>
+
<div>Halfway, X runs for determine essential part of image. This time, select whole field and save to ROI information.<br>
−
今回OutputしたInformationfile</div>
+
Output Information file</div>
 
<pre>
 
<pre>
 
DataA_006.mrcsmth-0000.roi Rect        0.000000      0.000000      63.000000      0.000000      63.000000      63.000000        0.000000      63.000000
 
DataA_006.mrcsmth-0000.roi Rect        0.000000      0.000000      63.000000      0.000000      63.000000      63.000000        0.000000      63.000000
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<br>
 
<br>
  
−
====実行例3(Doubleを使用した2軸傾斜での再構成)====
+
====Example3 (Reconstruction of 2 axis tilt with Double)====
−
<div>[[mrc2Dto3D]]のOption -Doubleを使用して３次元再構成を行います.</div>
+
<div>By using [[mrc2Dto3D]]'s Option -Double, perform 3D Reconstruction.</div>
 
<br>
 
<br>
  
−
<div>変更箇所</div>
+
<div>Changed part</div>
 
<pre>
 
<pre>
 
.roilst.mrc3d:
 
.roilst.mrc3d:
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<br>
 
<br>
  
−
===ラドン変換を使った３次元再構成===
+
===3D Reconstruction with Radon Transform)===
−
<div>　[[mrc2Dto3D]]を使用するほかにラドン変換から３次元像を作る方法があります.ラドン変換は以下の流れで行います.
+
<div>　Radon Transform can perform 3D reconstruction without using [[mrc2Dto3D]]. Perform as the Following.
 
<br>
 
<br>
  
−
位置合わせ済みの2Dリスト<br>
+
Aligned 2D image list<br>
 
↓[[mrcImageSinogramCreate]]<br>
 
↓[[mrcImageSinogramCreate]]<br>
−
Sinogramリスト<br>
+
Sinogram list<br>
 
↓[[mrcRadon2Dto3D]]<br>
 
↓[[mrcRadon2Dto3D]]<br>
−
3Dラドンfile<br>
+
3D Radon file<br>
 
↓[[mrcImageInverseRadonTransform]]<br>
 
↓[[mrcImageInverseRadonTransform]]<br>
−
3Dfile(完了)</div>
+
3D file (Finished)</div>
 
<br>
 
<br>
  
−
====実行例1====
+
====Example 1====
−
<div>[[Media:Makefile-Tomogram7.zip|こちらのMakefile]]を使い,[[Media:1WDC-Tom2-2dSet.zip|実行例2でCreateされた .roilstfile]]について実行します.</div>
+
<div>Use [[Media:Makefile-Tomogram7.zip|This Makefile]] and Input is [[Media:1WDC-Tom2-2dSet.zip|.roilstfile by Example2]].</div>
 
<br>
 
<br>
  
−
<div>コマンド</div>
+
<div>Command</div>
 
<pre>
 
<pre>
 
make Radon3D
 
make Radon3D
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<br>
 
<br>
  
−
====実行例2====
+
====Example2====
−
<div>　[[Media:Makefile-Tomogram7.zip|こちらのMakefile]]から逆ラドン変換のOptionを変更して実行します.</div>
+
<div>　Use [[Media:Makefile-Tomogram7.zip|This Makefile]] changing option of Inverse Radon Transform.</div>
 
<br>
 
<br>
  
−
<div>変更内容</div>
+
<div>Changed part</div>
 
<pre>
 
<pre>
 
### RadonTransform
 
### RadonTransform
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</pre>
 
</pre>
  
−
<div>コマンド</div>
+
<div>Command</div>
 
<pre>
 
<pre>
 
make Radon3D
 
make Radon3D
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== [[Problems of Electron Tomography image]] ==
 
== [[Problems of Electron Tomography image]] ==
−
===ミッシングエリア===
+
===Missing Area===
−
<div>　全方位からのProjection像を得られないために,ミッシングエリア(１軸の場合はウェッジ,２軸の場合はピラミッド)とよばれるInformationがない領域があります.そのため,方向依存でのボケを生じます.</div>
+
<div>　Reconstructed image don't have enough angle data, so it have the region that doesn't have information called "Missing Area" (1-Axis: Wedge, 2-Axis: Pyramid). Thus, it has a Blur depending on angle.</div>
 
<br>
 
<br>
  
−
<div>[[:Media:Makefile-Tomogram5.zip|使用MakeFile]].</div>
+
<div>[[:Media:Makefile-Tomogram5.zip|Used MakeFile]].</div>
−
<div>[[:Media:Input-ellipsoidal.3d|元の３次元像]](Target.ini3dにリネーム)</div>
+
<div>[[:Media:Input-ellipsoidal.3d|Original 3D image]](rename to Target.ini3d)</div>
 
<table>  
 
<table>  
 
<tr>  
 
<tr>  
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<br>
 
<br>
  
−
<div>１軸回転でProjectionして３次元再構成した場合(Y-axis: -60° ~ 60°: 10°刻み)</div>
+
<div>3D Reconstruction of Projection image of 1 Axis Tilt(Y-axis: -60° ~ 60°: 10° interval)</div>
−
<div>変更箇所</div>
+
<div>Changed part</div>
 
<pre>
 
<pre>
 
TILTAXISNUMBER=1 # Single: 1 Double: 2  
 
TILTAXISNUMBER=1 # Single: 1 Double: 2  
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<br>
 
<br>
  
−
<div>コマンド</div>
+
<div>Command</div>
 
<pre>
 
<pre>
 
make Target.ini2d
 
make Target.ini2d
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<br>
 
<br>
  
−
<div>２軸回転でProjectionして３次元再構成した場合(X, Y-axis: 各-60° ~ 60°: 10°刻み)</div>
+
<div>3D Reconstruction of Projection image of 2 Axis Tilt(X, Y-axis: -60° ~ 60°: 10° interval)</div>
−
<div>変更箇所</div>
+
<div>Changed part</div>
 
<pre>
 
<pre>
 
TILTAXISNUMBER=2 # Single: 1 Double: 2  
 
TILTAXISNUMBER=2 # Single: 1 Double: 2  
 
</pre>
 
</pre>
−
<div>コマンド</div>
+
<div>Command</div>
 
<pre>
 
<pre>
 
make Target.ini2d1
 
make Target.ini2d1
Line 683: Line 683:
 
<br>
 
<br>
  
−
===画像の位置,フォーカスの問題===
+
===Problem of position and focus of image===
−
<div>　試料を傾けながら撮影するため回転軸からの距離に応じてレンズとの距離が変化していきます.これによって生じる問題をいかに補正するかも一つのポイントとなります.</div>
+
<div>　Because sample is taken being gradually tilted, the  distance between it and lens is change as well. Thus, how to correct is one of the point.</div>
 
<br>
 
<br>
  
−
====parallel transformの補正====
+
====Correction of parallel transform====
−
<div>　傾斜する角度が大きくなるほど,視野が広くなるため試料は傾斜軸の方へ近づいていきます.これを補正するために[[mrcImageCorrelation]]などを使ってどれだけparallel transformが必要かを計算します.また,試料を傾斜する毎に形も変わっていきますので比較はできるだけ近い画像同士で行います.</div>
+
<div>　As tilt angle is large, the photo field is wide, and the sample approaches tilt axis. For this correction, calculate the distance of parallel shift by using [[mrcImageCorrelation]]. In addition, compare between near images, because these shape change depending on tilt of sample</div>
 
<br>
 
<br>
  
−
====軸の決定====
+
====Determine axis====
−
<div>　撮影した画像は必ずしもSettingした軸を中心に傾斜しているとは限りません.これは電子顕微鏡自体の性能や試料の設置方法などによって多少のズレが生じてしまうからです.傾斜軸のズレは[[mrcImageTiltAxisSearch]]などを使って計算します.</div>
+
<div>　Tomogram does not always tilt around set axis. Because it has a little error depending on several factors as performance of electron microscopy or situation of sample. Speaking about error of tilt axis, [[mrcImageTiltAxisSearch]] can calculate it.</div>
 
<br>
 
<br>
  
−
== トモグラフに使われる画像処理 ==
+
== Image Process for Tomograph ==
−
<div>　再構成した３次元画像のAnalysisを行う前処理として以下のような処理を施します.</div>
+
<div>　Perform the following preprocesses for analysis of reconstructed 3D image.</div>
 
<br>
 
<br>
  
 
=== Smoothing ===
 
=== Smoothing ===
−
<div>　再構成した３次元像のNoiseを除去するためSmoothingを行います.</div>
+
<div>　Perform the smoothing for noise removing of reconstructed 3D image.</div>
 
<br>
 
<br>
  
−
====実行例1====
+
====Example1====
 
<div>[[Media:Input-Tomogram3D.mrc|Input file]]</div>
 
<div>[[Media:Input-Tomogram3D.mrc|Input file]]</div>
 
<table>  
 
<table>  
Line 732: Line 732:
 
<br>
 
<br>
  
−
<div>コマンド: [[mrcImageSmoothing]] -i Input-Tomogram3D.mrc -o Input-TomogramSmth.mrc -m 1</div>
+
<div>Command: [[mrcImageSmoothing]] -i Input-Tomogram3D.mrc -o Input-TomogramSmth.mrc -m 1</div>
 
<br>
 
<br>
  
Line 763: Line 763:
 
<br>
 
<br>
  
−
=== セグメンテーション ===
+
=== Segmentation ===
−
<div>　２次元画像や３次元画像を必要な部分ごとに切り出します.</div>
+
　Because tomogram contains a variety of structures, it requires tasks such as cutting out a segment of interest from the 3D image. Support software is required for it. There is cutting out from 2D image in some cases.<br>
 
<br>
 
<br>
  
−
====３次元画像の切り出し====
+
====Cut out 3D image====
−
<div>　再構成された３次元画像から粒子と考えられる部分などを切り出します.切り出したdataは３次元画像のAnalysisや平均化するときに使用することができます.</div>
+
<div>　Cut out parts that is determined as particle from reconstructed 3D image. Cut out data is used by 3D image analysis or average.</div>
 
<br>
 
<br>
  
−
=====実行例1=====
+
=====Example1=====
−
<div>切り出したい部分のcoordinates,Widthが分かっている場合は[[mrcImageCenterGet]]を使います.</div>
+
<div>If the width and the coordinates of the part to be cut has been found, you can use [[mrcImageCenterGet]].</div>
 
<br>
 
<br>
  
Line 803: Line 803:
 
<br>
 
<br>
  
−
<div>コマンド: [[mrcImageCenterGet]] -i Input-TomogramSmth.mrc -o Input-TomogramSub.mrc -Cx 67 -Cy 67 -Cz 67 -Nx 27 -Ny 27 -Nz 27</div>
+
<div>Command: [[mrcImageCenterGet]] -i Input-TomogramSmth.mrc -o Input-TomogramSub.mrc -Cx 67 -Cy 67 -Cz 67 -Nx 27 -Ny 27 -Nz 27</div>
 
<br>
 
<br>
  
Line 834: Line 834:
 
<br>
 
<br>
  
−
====２次元画像の切り出し====
+
====Cut out 2D image====
−
<div>　視野全体ではなく,一部だけ再構成するために傾斜画像から部分的に２次元画像を切り出す場合もあります.視野全体の再構成よりもコストが削減されるので,部分的なdataだけあれば十分な場合はこの手法を用います.試料を傾けながら撮影しているので,画像から切り出す位置や角度を画像によって変える必要があります.切り出すときには[[mrcImageCenterGet]]や[[mrcImageROI]],[[mrcImageROIs]]などを使用します.切り出し範囲を全て手動で決めるのはかなりの時間を要しますので,基準(0°)'s imageでの切り出し範囲を手動で決めると残りの切り出し位置を傾斜角度に応じて自動で計算して切り出しを行う方法をとります.</div>
+
<div>　If a target of reconstruction is not whole photo field but a part of field, there is a case to cut out partially 2D image from tilt image. It is low cost than reconstruction of whole. Therefore this method is used in case of not needing whole data. Because samples are taken being tilted gradually, coordinates and angles need to be changed for each image. Cut out by using [[mrcImageCenterGet]], [[mrcImageROI]], [[mrcImageROIs]] and so on. It takes significant time that all parts are cut out. Thus, it takes how to calculate and cut out automatically other tilted image by deciding manually cut out of range in the reference(untilted) image.</div>
−
操作動画: [[:Media:Tomography4-1.mov|(.mov)]] [[:Media:Tomography4-1.mp4|(.mp4)]]<br>
+
Operation movie: [[:Media:Tomography4-1.mov|(.mov)]] [[:Media:Tomography4-1.mp4|(.mp4)]]<br>
 
<br>
 
<br>
  
−
<div>以下の流れで３次元再構成まで行います.</div>
+
<div>Perform 3D Reconstruction as the following.</div>
 
<pre>
 
<pre>
−
傾斜画像(2D画像複数)(前処理済)
+
Tilted Image(Multiple 2D images)(Prepared)
  
−
↓ 切り出し範囲のSetting(Display2: 0°'s imageのみ)
+
↓ Set range of cutting out.(Display2: untilted image only)
  
−
0°の切り出しInformationfile(ROIInformation)
+
Information file for cutting out untilted image(ROIInformation)
  
−
↓ 各傾斜画像の切り出し範囲を計算する(現在はMakefile内で処理)
+
↓ Calculate the range of cutting out tilted images.(Currently, Makefile performs this process.)
  
−
各傾斜画像の切り出しInformation
+
Information file for cutting out each tilted image
  
−
↓ 画像の切り出し(mrcImageROIs)
+
↓ Cut out images(mrcImageROIs)
  
−
切り出された複数's imagefile(切り出し数 × 傾斜数分)
+
Multiple cut out image files(Number of cutting out × Number of tilted image)
  
−
↓ 位置合わせ(mrcImageCorrelarion + Makefileの処理)
+
↓ Alignment(mrcImageCorrelarion + Makefile's process)
  
−
位置合わせ済みの切り出しInformation(傾斜数分)
+
Aligned information file for cutting out (Number of tilted image)
  
−
↓ 再度画像の切り出し(mrcImageROIs) (※ 切り出し画像をシフトするのではなく,切り出し位置を再Settingして切り出します)
+
↓ Cut out images again.(mrcImageROIs) (* It resets the position and cut out them.)
  
−
位置合わせ済み's image(ROI)file(切り出し数 × 傾斜数分)
+
Aligned image(ROI)file(Number of cutting out × Number of tilted image)
  
−
↓ 角度InformationfileのCreate(Makefile内の処理)
+
↓ Create Angle Information file.(Makefile performs this process.)
  
−
角度Informationfile(切り出し数分)
+
Angle Information file(Number of cutting out)
  
−
↓ ３次元再構成(mrc2Dto3D)
+
↓ 3D Reconstruction(mrc2Dto3D)
  
−
3次元画像(切り出し数分)
+
3D image(Number of cutting out)
 
</pre>
 
</pre>
  
−
=====実行例1(1軸傾斜のサブトモグラム)=====
+
=====Example1(Subtomogram of 1 axis tilt)=====
−
<div>[[Media:Input-Tomogram2DSet1.zip|Input file]]'s image(一部)(y-axis回転: -60° ~ 60°: 2°刻み)</div>
+
<div>[[Media:Input-Tomogram2DSet1.zip|Input file]]'s image(parts)(y-axis tilt: -60° ~ 60°: 2° interval)</div>
 
<table>  
 
<table>  
 
<tr>  
 
<tr>  
Line 889: Line 889:
 
<br>
 
<br>
  
−
<div>[[:Media:Makefile-Tomogram4.zip|こちらのMakefile]].</div>
+
<div>Use [[:Media:Makefile-Tomogram4.zip|This Makefile]].</div>
−
<div>コマンド</div>
+
<div>Command</div>
 
<pre>
 
<pre>
 
make all
 
make all
 
</pre>
 
</pre>
  
−
<div>コマンドをInput後に[[Display2]](2Dビューワー)が開きますので,切り出したい部分(複数可)を選択しInformationfile(.roiinfo)をCreateします.</div>
+
<div>After input the command, [[Display2]](2D viewer) is opened, then select ranges of cutting out, and create the Information file(.roiinfo).</div>
 
<table>  
 
<table>  
 
<tr>  
 
<tr>  
Line 902: Line 902:
 
</tr>  
 
</tr>  
 
<tr>  
 
<tr>  
−
<td><p align="Center">切り出し部分決めてEdit->OKで決定します.</p></td>  
+
<td><p align="Center">Select ranges of cutting out, and determine by Edit->OK.</p></td>  
−
<td><p align="Center">複数切り出したいときはROI->MultiROIを選択します.</p> </td>  
+
<td><p align="Center">If you wish to cut out multiple images, select ROI->MultiROI.</p> </td>  
 
</tr>  
 
</tr>  
 
</table>  
 
</table>  
Line 911: Line 911:
 
</tr>  
 
</tr>  
 
<tr>  
 
<tr>  
−
<td>今回はInformationだけCreateすれば,切り出しは自動で行われます.<br>
+
<td>This time, Extracting "Information" only is enough for automatically cutting out.<br>
−
ROI InformationウィンドウにてInfoFileName右側のsaveボタンを押すとfileがCreateされます.<br>
+
Push the save button that is right side of "InfoFileName" on "ROI Information" window, then the file is created.<br>
−
(またはExtractModeをInformationにしてExtractボタンを押します.)</td>  
+
(Or set "Information" for "ExtractMode", and push the "Extract" button.)</td>  
 
</tr>  
 
</tr>  
 
</table>  
 
</table>  
 
<br>
 
<br>
  
−
<div>今回OutputしたInformationfile</div>
+
<div>Output Information file in this time.</div>
 
<pre>
 
<pre>
 
p1_031-0000.roi Rect 20 30 60 30 60 70 20 70
 
p1_031-0000.roi Rect 20 30 60 30 60 70 20 70
Line 939: Line 939:
 
<br>
 
<br>
  
−
<div>1枚's imageについて[[Display2]]からInformationfile(.roiinfo)をCreateすると,残り's imageについては傾きから切り出す範囲を計算し,自動で切り出しが行われます.</div>
+
<div>When you create Information file(.roiinfo) for one image by [[Display2]], it calculates automatically other image's range, and cuts out them.</div>
 
<br>
 
<br>
−
<div>自動で切り出された画像(縦: 選択範囲毎, 横: file(角度)毎)(.pad)</div>
+
<div>cut out automatically images(columns: each selected range, rows: each file(angle))(.pad)</div>
 
<table>  
 
<table>  
 
<tr>  
 
<tr>  
Line 956: Line 956:
 
<br>
 
<br>
  
−
<div>切り出し処理を終えた後はそれぞれ's imageに対して３次元再構成が行われます.</div>
+
<div>After cutting out, it performs 3D Reconstruction for each image.</div>
 
<br>
 
<br>
  
−
<div>再構成されたそれぞれの３次元画像(.mrc3d)</div>
+
<div>Reconstructed 3D images(.mrc3d)</div>
 
<table>  
 
<table>  
 
<tr>  
 
<tr>  
Line 976: Line 976:
 
<br>
 
<br>
  
−
=====実行例2(2軸傾斜のサブトモグラム)=====
+
=====Example2(Subtomogram of 2 axis tilt)=====
−
<div>　２軸で傾斜した画像から切り出し及び再構成を行ってみます.</div>
+
<div>　Perform cutting out and reconstruction from 2 axis tilted images.</div>
 
<br>
 
<br>
  
−
<div>[[Media:Input-Tomogram2DSet2.zip|Input file]]'s image(一部)(x-axis, y-axis回転: -60° ~ 60°: 10°刻み)</div>
+
<div>[[Media:Input-Tomogram2DSet2.zip|Input file]]'s images(parts)(x-axis, y-axis tilt: -60° ~ 60°: 10° interval)</div>
 
<table>  
 
<table>  
 
<tr>  
 
<tr>  
−
<td>x-axis回転</td>
+
<td>x-axis tilt</td>
 
<td><p align="Center">[[File:Input-Tomogram2DSet2.png]]<br>
 
<td><p align="Center">[[File:Input-Tomogram2DSet2.png]]<br>
 
-60°<br></p>  
 
-60°<br></p>  
Line 995: Line 995:
 
</tr>  
 
</tr>  
 
<tr>  
 
<tr>  
−
<td>y-axis回転</td>
+
<td>y-axis tilt</td>
 
<td><p align="Center">[[File:Input3-Tomogram2DSet2.png]]<br>
 
<td><p align="Center">[[File:Input3-Tomogram2DSet2.png]]<br>
 
-60°<br></p>  
 
-60°<br></p>  
Line 1,009: Line 1,009:
 
<br>
 
<br>
  
−
<div>[[:Media:Makefile-Tomogram5.zip|こちらのMakefile]].</div>
+
<div>Use [[:Media:Makefile-Tomogram5.zip|This Makefile]].</div>
 
<br>
 
<br>
  
−
<div>コマンド</div>
+
<div>Command</div>
 
<pre>
 
<pre>
 
make all
 
make all
Line 1,018: Line 1,018:
 
<br>
 
<br>
  
−
<div>今回はこの領域でROIfileの切り出しを行います.</div>
+
<div>This time, cut out ROI files about these regions.</div>
 
<div>[[File:Tomogram2-Segmanetation.png]]</div>
 
<div>[[File:Tomogram2-Segmanetation.png]]</div>
−
<div>今回OutputしたInformationfile</div>
+
<div>Output Information files</div>
 
<pre>
 
<pre>
 
DataA_006-0000.roi Rect        0.722314      137.147614      33.000000      106.000000      49.053597      122.636041      16.775911      153.783654
 
DataA_006-0000.roi Rect        0.722314      137.147614      33.000000      106.000000      49.053597      122.636041      16.775911      153.783654
Line 1,042: Line 1,042:
 
<br>
 
<br>
  
−
<div>自動で切り出された画像(縦: 選択範囲毎, 横: file(角度)毎)(.pad)</div>
+
<div>cut out automatically images(columns: each selected range, rows: each file(angle))(.pad)</div>
 
<table>  
 
<table>  
 
<tr>  
 
<tr>  
 
<td><p align="Center">[[File:Tomogram2-Segmanetation1.png]]<br>
 
<td><p align="Center">[[File:Tomogram2-Segmanetation1.png]]<br>
−
DataA(x-axis回転)<br></p>  
+
DataA(x-axis tilt)<br></p>  
 
</td>  
 
</td>  
 
<td><p align="Center">[[File:Tomogram2-Segmanetation2.png]]<br>
 
<td><p align="Center">[[File:Tomogram2-Segmanetation2.png]]<br>
−
DataB(y-axis回転)<br></p>  
+
DataB(y-axis tilt)<br></p>  
 
</td>  
 
</td>  
 
</tr>  
 
</tr>  
 
</table>  
 
</table>  
−
<div>切り出しサイズがバラバラで分かりにくくなっていますが,いずれの傾斜軸でもROIfileが自動で切り出されています.</div>
+
<div>ROI files have been cut out automatically for each tilt axis. (It is not easy that each of parts is found, because each size are different...)</div>
 
<br>
 
<br>
  
−
<div>再構成されたそれぞれの３次元画像(.mrc3d)</div>
+
<div>Reconstructed 3D images(.mrc3d)</div>
 
<table>  
 
<table>  
 
<tr>  
 
<tr>  
Line 1,074: Line 1,074:
  
 
== [[Interpretation of 3D Reconstruction]] ==
 
== [[Interpretation of 3D Reconstruction]] ==
−
　また,多様な構造を含むため,３次元画像から興味あるセグメントを切り出すなどの作業が必要です.そのための支援ソフトウェアが必要です.
+
 
  
 
=== [[Averaging of Subtomograms]]===
 
=== [[Averaging of Subtomograms]]===
−
<div>　セグメンテーションによって分割された３次元data同士が別方向を向いている同じ粒子であるとき,それを平均化することでミッシングエリアを補間し,より精密な３次元像にすることができます.</div>
+
<div>　If each 3D data that is divided by segmentation is same particle and have different angle, these average image interpolates these "Missing Area", and makes more correct 3D image.</div>
 
<br>
 
<br>
  
−
<div>分割したdata1</div>
+
<div>Divided data1</div>
 
<table>  
 
<table>  
 
<tr>  
 
<tr>  
Line 1,088: Line 1,088:
 
</tr>  
 
</tr>  
 
</table>  
 
</table>  
−
<div>分割したdata2(90°向きが違う)</div>
+
<div>Divided data2(90° rotation)</div>
 
<table>  
 
<table>  
 
<tr>  
 
<tr>  
Line 1,096: Line 1,096:
 
</tr>  
 
</tr>  
 
</table>  
 
</table>  
−
<div>平均したdata</div>
+
<div>Average data</div>
 
<table>  
 
<table>  
 
<tr>  
 
<tr>  
Line 1,111: Line 1,111:
 
<br>
 
<br>
  
−
====実行例1====
+
====Example1====
 
<div>[[Media:Input-Mean-SubTomogram.zip|Input file]]</div>
 
<div>[[Media:Input-Mean-SubTomogram.zip|Input file]]</div>
 
<table>  
 
<table>  
Line 1,129: Line 1,129:
 
<br>
 
<br>
  
−
<div>３次元像を平均化する前に[[mrcImageAutoRotationCorrelation3D]]を使って,向きを合わせます.</div>
+
<div>align by using [[mrcImageAutoRotationCorrelation3D]] before averaging 3D image.</div>
 
<br>
 
<br>
  
−
<div>[[Media:Input-TomogramSub.mrc|Reference file]](Input fileの真ん中のdataを使用)</div>
+
<div>[[Media:Input-TomogramSub.mrc|Reference file]](Center data of Input file)</div>
 
<table>  
 
<table>  
 
<tr>  
 
<tr>  
Line 1,160: Line 1,160:
 
<br>
 
<br>
  
−
<div>コマンド: [[mrcImageAutoRotationCorrelation3D]] -i c1-1.mrc -r c2-2.mrc -fit f1-1.mrc -EA YOYS -Rot1 0 0 30 -Rot2 0 330 30 -Rot3 0 330 30 -Xrange 0 0 -Yrange 0 0  -Zrange 0 0  </div>
+
<div>Command: [[mrcImageAutoRotationCorrelation3D]] -i c1-1.mrc -r c2-2.mrc -fit f1-1.mrc -EA YOYS -Rot1 0 0 30 -Rot2 0 330 30 -Rot3 0 330 30 -Xrange 0 0 -Yrange 0 0  -Zrange 0 0  </div>
 
<br>
 
<br>
  
−
<div>[[Media:Input-Fit-SubTomogram.zip|補正後のdata]](-fitのOutput file)</div>
+
<div>[[Media:Input-Fit-SubTomogram.zip|corrected data]](-fit 's Output file)</div>
 
<table>  
 
<table>  
 
<tr>  
 
<tr>  
Line 1,180: Line 1,180:
 
<br>
 
<br>
  
−
<div>補正fileに対し,[[mrcImageAverage]]にて平均化を行います.</div>
+
<div>Perform Averaging of corrected files by [[mrcImageAverage]].</div>
 
<div>Output file</div>
 
<div>Output file</div>
 
<table>  
 
<table>  
Line 1,207: Line 1,207:
 
</tr>  
 
</tr>  
 
</table>  
 
</table>  
−
<div>平均化することにより３次元像の質(特にyz-plane)が良くなりました.</div>
+
<div>3D image's quality has been improved by averaging. (Particular, xy-plane)</div>
 
<br>
 
<br>
  
−
====実行例2====
+
====Example2====
 
<div>[[Media:Outdata-Tomogram.zip|Input file]]</div>
 
<div>[[Media:Outdata-Tomogram.zip|Input file]]</div>
 
<table>  
 
<table>  
Line 1,228: Line 1,228:
 
<br>
 
<br>
  
−
<div>[[Media:Input-TomogramSub1.mrc|Reference file]](Input fileの先頭のdataを使用)</div>
+
<div>[[Media:Input-TomogramSub1.mrc|Reference file]](Use top data of Input file)</div>
 
<table>  
 
<table>  
 
<tr>  
 
<tr>  
Line 1,256: Line 1,256:
 
<br>
 
<br>
  
−
<div>同様にまず向きを合わせます.</div>
+
<div>First, perform alignment as well.</div>
−
<div>コマンド: [[mrcImageAutoRotationCorrelation3D]] -i p1-0001.mrc3d -r Target.mrc3dref -fit p1-0001.3dfit -EA XEYR -Rot1 0 355 5 -Rot2 0 355 5 -Rot3 0 0 30 -M 18</div>
+
<div>Command: [[mrcImageAutoRotationCorrelation3D]] -i p1-0001.mrc3d -r Target.mrc3dref -fit p1-0001.3dfit -EA XEYR -Rot1 0 355 5 -Rot2 0 355 5 -Rot3 0 0 30 -M 18</div>
 
<br>
 
<br>
−
<div>[[Media:Input-Fit-SubTomogram1.zip|補正後のdata]](-fitのOutput file)</div>
+
<div>[[Media:Input-Fit-SubTomogram1.zip|Corrected data]](-fit 's Output file)</div>
 
<table>  
 
<table>  
 
<tr>  
 
<tr>  
Line 1,276: Line 1,276:
 
<br>
 
<br>
  
−
<div>Reference fileに向きを合っているものと合っていないものがあります.<br>
+
<div>Some fits the reference file, and some are otherwise.<br>
−
これはそれぞれのサブトモグラムの３次元像にてミッシングエリアが異なるので,どちらかにdataがない角度では相関値が大Widthに下がってしまうためです.</div>
+
Because each 3D subtomogram image has different "Missing Area". Thus the correlation value of the orientation that is included in not both of them is Considerably lower.</div>
 
<div>[[File:Fig-SubTomogram.png]]</div>
 
<div>[[File:Fig-SubTomogram.png]]</div>
 
<br>
 
<br>
  
−
<div>補正fileに対し,[[mrcImageAverage]]にて平均化を行います.</div>
+
<div>Perform averaging of Corrected files by using [[mrcImageAverage]].</div>
 
<div>Output file</div>
 
<div>Output file</div>
 
<table>  
 
<table>  

Latest revision as of 04:26, 29 September 2014

Electron Tomography is one of 3D Reconstruction using electron microscope. It is the method that reconstruct 3D image by electron micrographs which is projected the same photo field for several angle, then it creates Tomogram by using computer.

　In the following, describe its method in order.

take tilt-series images

　When sample is taken by this method, image that is tilted at a regular interval around an axis is made. As tilt angle increases, the photo field become wider, thus objects in image come around to the center gradually.


Input-Tomogram1.png

1 Axis Tilt(Center is untilted image)


When you will take 2 Axis Tilt 's image, rotate the sample 90 degree, and take as well as 1 Axis Tilt.
Input-Tomogram2.png

2 Axis Tilt(Center is untilted image)(as 90° rotation image for 1 Axis Tilt's image)


Image Correction

　Tilt-series image's defocus is larger than normal electron photograph's one, because it is taken as whole field is underfocus. In this case, CTF Correction is not required, because outside data of 1st thon ring don't have almost information if you use electron gun as LaB6. But, in the case of using field-emission electron gun (FEG), note that you might get a incorrect data.

Rough Alignment

　Align roughly each tilt image by using the correlation among images around center.

Input file(tilt at range: ±60°, interval: 2°, around Y-axis)

1WDC-Tom-2dSet.png
10° interval


Preprocess(Windowing)

　By Windowing, remove noise of edge of image.


Windowing of this time is x: 80%, y: 90%
Command(In the case of 1st image): mrcImageWindowing -i Set1000.roi -o Set1000.mask -W 0.2 0.2 0.1 0.1


When you use This Makefile, it performs for all roi file.
Setting data
# For Windowing
WIN_X=0.2
WIN_X_MAX=0.2
WIN_Y=0.1
WIN_Y_MAX=0.1
WIN_MODE=18
Command
make Windowing


Output file(.mask)

1WDC-Tom-mask-2dSet.png
10° interval


Alignment

　Because objects move parallelly depending on tilt angle, align about it. Now, align among the images whose angle is close to each other so as not to be affected by tilt.

e.g. case of images whose interval is 2 degree
　Align 2° image to 0° image.
　Align 4° image to Aligned 2° image.
　Align 6° image to Aligned 4° image.
　.
　.

　(Align "-" as well as "+")


case of using mrcImageCorrelation

　Perform alignment by using This Makefile. Even if you don't use the Makefile, you perform command sequentially, and you can align. But, Input file list for mrc2Dto3D is required to be created.


Example 1
Input file

1WDC-Tom-mask-2dSet.png
10° interval


Command
make CorFit1


1WDC-Tom-Fit-2d.png
10° interval


Fine Alignment

　Align the position and the angle of tilt axis as possible.


Example 1(Alignment of tilt axis)

　Calculate tilt of axis by using mrcImageTiltAxisSearch.


Input file(1 axis tilt data whose axis is tilted by 10 degree)

1WDC-Tom-Tilt-2dSet.png
10° interval


Used Makefile's Setting
# RotMode
ROTMODE=ZOYS
# Rot1
ROT1MIN=10
ROT1MAX=10
ROT1D=10
# Rot2
ROT2MIN=-60
ROT2MAX=60
ROT2D=2
# Rot3
ROT3MIN=0
ROT3MAX=0
ROT3D=30
### For mrcImageMove
SHIFT2MAX=10
SHIFT3MAX=0
### For mrcImageTiltAxisSearch
TILTMIN=0
TILTMAX=20
TILTN=10
TILTITER=1
TILTSCALE=5


Command
make TiltFit


Output of axis tilt(It is stored at .tiltinfofile)
9.895


difference about 3D Reconstruction
Before correction
Outdata-Tomogram4.png Outdata1-Tomogram4.png Outdata2-Tomogram4.png
After correction
Outdata-Tomogram5.png Outdata1-Tomogram5.png Outdata2-Tomogram5.png
xy-plane yz-plane zx-plane


Example2(Repeatedly Alignment of tilt axis)

Repeat to use mrcImageTiltAxisSearch, referring tilt of output axis.


Input file(1 axis tilt data whose axis is tilted by 1 degree)

1WDC-Tom-Tilt1-2dSet.png
10° interval


Used Makefile's Setting
# For mrcImageTiltAxisSearch
IN_TILT_EXT=roi
# RotMode
ROTMODE=ZEYR
# Rot1
ROT1MIN=1
ROT1MAX=1
ROT1D=1
# Rot2
ROT2MIN=-60
ROT2MAX=60
ROT2D=10
# Rot3
ROT3MIN=0
ROT3MAX=0
ROT3D=10
### For mrcImageTiltAxisSearch
TILTMIN=-10
TILTMAX=10
TILTN=10
TILTITER=100
TILTSCALE=5


Command
make TiltFit


transition of tilt of axis
0.084
0.161
0.230
0.294
0.345

...

0.701
0.700
0.685
0.706
0.701
The tilt of axis approaches gradually 1.


3D Reconstruction

　Reconstruct 3D image from 2D image set.


1WDC-shift1-3d.png
xy-plane

1WDC-shift1-3d2.png
yz-plane

1WDC-shift1-3d3.png
zx-plane

Min

Max
Mean
SD

SE

0 (0, 0, 0)

4 (31, 26, 26)
0.0116844
0.147033

0.000287174


mrc2Dto3D

Example1 (Reconstruction of 1 axis tilt)

　If the tilt axis don't deviate, set the angle information directly to input file list.


Input file

1WDC-Tom-Fit-2d.png
10° interval


Command: mrc2Dto3D -i Input.3dlst -o Input.3d -m 1


By using This Makefile, perform the following command for reconstruction.
Setting data
# For Reconstruction
IN_3D_EXT=fit
# RotMode
ROTMODE=YOYS
# Rot1
ROT1MIN=-60
ROT1MAX=60
ROT1D=2
# Rot2
ROT2MIN=0
ROT2MAX=0
# Rot3
ROT3MIN=0
ROT3MAX=0
Command
make 3DList
make Input.3d


Output file(Input.3d)

Outdata-mrc2Dto3D.png
xy-plane

Outdata1-mrc2Dto3D.png
yz-plane

Outdata2-mrc2Dto3D.png
zx-plane

Min

Max
Mean
SD

SE

-0.00437076 (39, 34, 36)

0.00799233 (37, 34, 36)
3.45089e-06
0.000342836

6.69601e-07


Example2 (Reconstruction of 2 axis tilt)

Perform 3D Reconstruction for images of 2 axis tilt.
Input file(tilt at range: each ±60°, interval: each 10°, around X, Y-axis))

1WDC-Tom1-2dSet.png
1st line is X-axis tilt, 2nd line is Y-axis tilt


By using This Makefile, perform 3D Reconstruction.
Command
make all


Halfway, X runs for determine essential part of image. This time, select whole field and save to ROI information.
Output Information file
DataA_006.mrcsmth-0000.roi Rect        0.000000       0.000000       63.000000       0.000000       63.000000       63.000000        0.000000       63.000000


Output file

Outdata-Tomogram2.png
xy-plane

Outdata1-Tomogram2.png
yz-plane

Outdata2-Tomogram2.png
zx-plane

Min

Max
Mean
SD

SE

-9.32199 (29, 37, 34)

21.995 (40, 35, 32)
0.0280746
1.38771

0.00271036


Example3 (Reconstruction of 2 axis tilt with Double)

By using mrc2Dto3D's Option -Double, perform 3D Reconstruction.


Changed part
.roilst.mrc3d:
#	mrc2Dto3D -I $*.roilst -o $*.mrc3d -single 0 -InterpolationMode 2 -m 1 
	mrc2Dto3D -I $*.roilst -o $*.mrc3d -Double -InterpolationMode 2 -m 1


Output file

Outdata-Tomogram6.png
xy-plane

Outdata1-Tomogram6.png
yz-plane

Outdata2-Tomogram6.png
zx-plane

Min

Max
Mean
SD

SE

-10360.9 (41, 24, 0)

8132.05 (40, 35, 31)
86.0574
673.594

1.31561


3D Reconstruction with Radon Transform)

　Radon Transform can perform 3D reconstruction without using mrc2Dto3D. Perform as the Following.


Aligned 2D image list
↓mrcImageSinogramCreate
Sinogram list
↓mrcRadon2Dto3D
3D Radon file
↓mrcImageInverseRadonTransform

3D file (Finished)


Example 1


Command
make Radon3D
Output file
xy-plane yz-plane zx-plane
Outdata-Tomogram3.png Outdata1-Tomogram3.png Outdata2-Tomogram3.png

Min

Max
Mean
SD

SE

1.84923e+06 (0, 59, 28)

1.17991e+07 (34, 31, 30)
3.63631e+06
1.64358e+06

3536.42


Example2

　Use This Makefile changing option of Inverse Radon Transform.


Changed part
### RadonTransform
RBP_MODE=4
RBP_OPTION=-Rmin 0.05 -Rmax 0.1
Command
make Radon3D
Output file
xy-plane yz-plane zx-plane
Outdata-Tomogram7.png Outdata1-Tomogram7.png Outdata2-Tomogram7.png

Min

Max
Mean
SD

SE

-344789 (25, 25, 0)

631546 (37, 34, 28)
4381.1
76358.4

164.297


Problems of Electron Tomography image

Missing Area

　Reconstructed image don't have enough angle data, so it have the region that doesn't have information called "Missing Area" (1-Axis: Wedge, 2-Axis: Pyramid). Thus, it has a Blur depending on angle.


Original 3D image(rename to Target.ini3d)

Input-ellipsoidal.png
xy-plane

Input1-ellipsoidal.png
yz-plane

Input2-ellipsoidal.png
zx-plane

Min

Max
Mean
SD

SE

0 (0, 0, 0)

2 (29, 29, 14)
0.108302
0.364429

0.000711774


3D Reconstruction of Projection image of 1 Axis Tilt(Y-axis: -60° ~ 60°: 10° interval)
Changed part
TILTAXISNUMBER=1	# Single: 1 Double: 2


Command
make Target.ini2d
make TestData2DSet
make all


Output-ellipsoidal.png
xy-plane

Output1-ellipsoidal.png
yz-plane

Output2-ellipsoidal.png
zx-plane

Min

Max
Mean
SD

SE

-0.00330818 (52, 36, 32)

0.00425516 (13, 37, 32)
2.08481e-05
0.000467848

9.13766e-07


3D Reconstruction of Projection image of 2 Axis Tilt(X, Y-axis: -60° ~ 60°: 10° interval)
Changed part
TILTAXISNUMBER=2	# Single: 1 Double: 2
Command
make Target.ini2d1
make TestData2DSetDouble
make all


Output3-ellipsoidal.png
xy-plane

Output4-ellipsoidal.png
yz-plane

Output5-ellipsoidal.png
zx-plane

Min

Max
Mean
SD

SE

-0.00248567 (12, 38, 32)

0.00344027 (13, 37, 32)
2.07529e-05
0.000401237

7.83666e-07


Problem of position and focus of image

　Because sample is taken being gradually tilted, the distance between it and lens is change as well. Thus, how to correct is one of the point.


Correction of parallel transform

　As tilt angle is large, the photo field is wide, and the sample approaches tilt axis. For this correction, calculate the distance of parallel shift by using mrcImageCorrelation. In addition, compare between near images, because these shape change depending on tilt of sample


Determine axis

　Tomogram does not always tilt around set axis. Because it has a little error depending on several factors as performance of electron microscopy or situation of sample. Speaking about error of tilt axis, mrcImageTiltAxisSearch can calculate it.


Image Process for Tomograph

　Perform the following preprocesses for analysis of reconstructed 3D image.


Smoothing

　Perform the smoothing for noise removing of reconstructed 3D image.


Example1

Input-Tomogram3D.png
xy-plane

Input1-Tomogram3D.png
yz-plane

Input2-Tomogram3D.png
zx-plane

Min

Max
Mean
SD

SE

-0.00268221 (76, 92, 66)

0.00642324 (89, 42, 71)
2.0386e-06
0.000254286

1.63933e-07


Command: mrcImageSmoothing -i Input-Tomogram3D.mrc -o Input-TomogramSmth.mrc -m 1


Input-TomogramSmth.png
xy-plane

Input1-TomogramSmth.png
yz-plane

Input2-TomogramSmth.png
zx-plane

Min

Max
Mean
SD

SE

-0.00136636 (75, 42, 133)

0.00292404 (94, 65, 68)
-9.48921e-07
0.00013513

8.71152e-08


Segmentation

　Because tomogram contains a variety of structures, it requires tasks such as cutting out a segment of interest from the 3D image. Support software is required for it. There is cutting out from 2D image in some cases.

Cut out 3D image

　Cut out parts that is determined as particle from reconstructed 3D image. Cut out data is used by 3D image analysis or average.


Example1
If the width and the coordinates of the part to be cut has been found, you can use mrcImageCenterGet.


Input-TomogramSmth.png
xy-plane

Input1-TomogramSmth.png
yz-plane

Input2-TomogramSmth.png
zx-plane

Min

Max
Mean
SD

SE

-0.00136636 (75, 42, 133)

0.00292404 (94, 65, 68)
-9.48921e-07
0.00013513

8.71152e-08


Command: mrcImageCenterGet -i Input-TomogramSmth.mrc -o Input-TomogramSub.mrc -Cx 67 -Cy 67 -Cz 67 -Nx 27 -Ny 27 -Nz 27


Input-TomogramSub.png
xy-plane

Input1-TomogramSub.png
yz-plane

Input2-TomogramSub.png
zx-plane

Min

Max
Mean
SD

SE

-0.0012017 (5, 9, 13)

0.00263957 (13, 6, 15)
2.22923e-05
0.000388094

2.76625e-06


Cut out 2D image

　If a target of reconstruction is not whole photo field but a part of field, there is a case to cut out partially 2D image from tilt image. It is low cost than reconstruction of whole. Therefore this method is used in case of not needing whole data. Because samples are taken being tilted gradually, coordinates and angles need to be changed for each image. Cut out by using mrcImageCenterGet, mrcImageROI, mrcImageROIs and so on. It takes significant time that all parts are cut out. Thus, it takes how to calculate and cut out automatically other tilted image by deciding manually cut out of range in the reference(untilted) image.

Operation movie: (.mov) (.mp4)

Perform 3D Reconstruction as the following.
Tilted Image(Multiple 2D images)(Prepared)

↓	Set range of cutting out.(Display2: untilted image only)

Information file for cutting out untilted image(ROIInformation)

↓	Calculate the range of cutting out tilted images.(Currently, Makefile performs this process.)

Information file for cutting out each tilted image

↓	Cut out images(mrcImageROIs)

Multiple cut out image files(Number of cutting out × Number of tilted image)

↓	Alignment(mrcImageCorrelarion + Makefile's process)

Aligned information file for cutting out (Number of tilted image)

↓	Cut out images again.(mrcImageROIs) (* It resets the position and cut out them.)

Aligned image(ROI)file(Number of cutting out × Number of tilted image)

↓	Create Angle Information file.(Makefile performs this process.)

Angle Information file(Number of cutting out)

↓	3D Reconstruction(mrc2Dto3D)

3D image(Number of cutting out)
Example1(Subtomogram of 1 axis tilt)
Input file's image(parts)(y-axis tilt: -60° ~ 60°: 2° interval)

Input-Tomogram2DSet1.png
-60°(p1_001.mrc)

Input1-Tomogram2DSet1.png
0°(p1_031.mrc)

Input2-Tomogram2DSet1.png
60°(p1_061.mrc)


Command
make all
After input the command, Display2(2D viewer) is opened, then select ranges of cutting out, and create the Information file(.roiinfo).

Tomogram1.png

Tomogram2.png

Select ranges of cutting out, and determine by Edit->OK.

If you wish to cut out multiple images, select ROI->MultiROI.

Tomogram3.png
This time, Extracting "Information" only is enough for automatically cutting out.

Push the save button that is right side of "InfoFileName" on "ROI Information" window, then the file is created.

(Or set "Information" for "ExtractMode", and push the "Extract" button.)


Output Information file in this time.
p1_031-0000.roi	Rect	20	30	60	30	60	70	20	70
p1_031-0001.roi	Rect	15	75	55	75	55	115	15	115
p1_031-0002.roi	Rect	25	110	65	110	65	150	25	150
p1_031-0003.roi	Rect	45	0	85	0	85	40	45	40
p1_031-0004.roi	Rect	60	50	100	50	100	90	60	90
p1_031-0005.roi	Rect	55	80	95	80	95	120	55	120
p1_031-0006.roi	Rect	55	120	95	120	95	160	55	160
p1_031-0007.roi	Rect	85	15	125	15	125	55	85	55
p1_031-0008.roi	Rect	85	51	125	51	125	91	85	91
p1_031-0009.roi	Rect	85	91	125	91	125	131	85	131
p1_031-0010.roi	Rect	93	130	133	130	133	170	93	170
p1_031-0011.roi	Rect	115	25	155	25	155	65	115	65
p1_031-0012.roi	Rect	120	60	160	60	160	100	120	100
p1_031-0013.roi	Rect	123	100	163	100	163	140	123	140
p1_031-0014.roi	Rect	115	140	155	140	155	180	115	180
p1_031-0015.roi	Rect	152	152	192	152	192	192	152	192


When you create Information file(.roiinfo) for one image by Display2, it calculates automatically other image's range, and cuts out them.


cut out automatically images(columns: each selected range, rows: each file(angle))(.pad)
Tomogram-Segmentation.png
-60°

0°

 60°


After cutting out, it performs 3D Reconstruction for each image.


Reconstructed 3D images(.mrc3d)
xy-plane Outdata-Tomogram.png
yz-plane Outdata1-Tomogram.png
zx-plane Outdata2-Tomogram.png


Example2(Subtomogram of 2 axis tilt)
　Perform cutting out and reconstruction from 2 axis tilted images.


Input file's images(parts)(x-axis, y-axis tilt: -60° ~ 60°: 10° interval)
x-axis tilt

Input-Tomogram2DSet2.png
-60°

Input1-Tomogram2DSet2.png
0°

Input2-Tomogram2DSet2.png
60°
y-axis tilt

Input3-Tomogram2DSet2.png
-60°

Input4-Tomogram2DSet2.png
0°

Input5-Tomogram2DSet2.png
60°



Command
make all


This time, cut out ROI files about these regions.
Tomogram2-Segmanetation.png
Output Information files
DataA_006-0000.roi Rect        0.722314      137.147614       33.000000      106.000000       49.053597      122.636041       16.775911      153.783654
DataA_006-0001.roi Rect       15.000000       79.000000       42.000000       79.000000       42.000000      107.000000       15.000000      107.000000
DataA_006-0002.roi Rect       13.000000       39.000000       58.000000       39.000000       58.000000       64.000000       13.000000       64.000000
DataA_006-0003.roi Rect       41.000000      129.000000       84.000000      129.000000       84.000000      154.000000       41.000000      154.000000
DataA_006-0004.roi Rect       51.000000       85.000000       82.000000       85.000000       82.000000      119.000000       51.000000      119.000000
DataA_006-0005.roi Rect       47.269722       69.494676       78.455043       40.316766       95.000000       58.000000       63.814679       87.177909
DataA_006-0006.roi Rect       66.000000        4.000000       92.000000        4.000000       92.000000       44.000000       66.000000       44.000000
DataA_006-0007.roi Rect       77.597890      150.922358       95.484138      132.052220      120.886248      156.129861      103.000000      175.000000
DataA_006-0008.roi Rect       93.000000       92.000000      115.000000       92.000000      115.000000      132.000000       93.000000      132.000000
DataA_006-0009.roi Rect      100.000000       51.000000      121.000000       51.000000      121.000000       93.000000      100.000000       93.000000
DataA_006-0010.roi Rect       98.589377       19.087800      119.760822       10.100593      135.000000       46.000000      113.828555       54.987208
DataA_006-0011.roi Rect      122.000000      141.000000      146.000000      141.000000      146.000000      184.000000      122.000000      184.000000
DataA_006-0012.roi Rect      136.883039       95.213292      160.000000      104.000000      144.116961      145.786708      121.000000      137.000000
DataA_006-0013.roi Rect      127.000000       73.000000      146.061183       60.635990      169.000000       96.000000      149.938817      108.364010
DataA_006-0014.roi Rect      140.000000       22.000000      177.361289       43.286947      166.470292       62.402025      129.109004       41.115078
DataA_006-0015.roi Rect      163.000000      152.000000      183.000000      152.000000      183.000000      190.000000      163.000000      190.000000


cut out automatically images(columns: each selected range, rows: each file(angle))(.pad)

Tomogram2-Segmanetation1.png
DataA(x-axis tilt)

Tomogram2-Segmanetation2.png
DataB(y-axis tilt)
ROI files have been cut out automatically for each tilt axis. (It is not easy that each of parts is found, because each size are different...)


Reconstructed 3D images(.mrc3d)
xy-plane Outdata-Tomogram1.png
yz-plane Outdata1-Tomogram1.png
zx-plane Outdata2-Tomogram1.png


Interpretation of 3D Reconstruction

Averaging of Subtomograms

　If each 3D data that is divided by segmentation is same particle and have different angle, these average image interpolates these "Missing Area", and makes more correct 3D image.


Divided data1
Input-SubTomogram.png Input1-SubTomogram.png Input2-SubTomogram.png
Divided data2(90° rotation)
Input-SubTomogram1.png Input1-SubTomogram2.png Input2-SubTomogram3.png
Average data
Output-SubTomogram1.png Output1-SubTomogram2.png Output2-SubTomogram3.png
xy-plane yz-plane zx-plane


Example1

xy-plane Input-Mean-SubTomogram.png
yz-plane Input1-Mean-SubTomogram.png
zx-plane Input2-Mean-SubTomogram.png


align by using mrcImageAutoRotationCorrelation3D before averaging 3D image.


Reference file(Center data of Input file)

Input-TomogramSub.png
xy-plane

Input1-TomogramSub.png
yz-plane

Input2-TomogramSub.png
zx-plane

Min

Max
Mean
SD

SE

-0.0012017 (5, 9, 13)

0.00263957 (13, 6, 15)
2.22923e-05
0.000388094

2.76625e-06


Command: mrcImageAutoRotationCorrelation3D -i c1-1.mrc -r c2-2.mrc -fit f1-1.mrc -EA YOYS -Rot1 0 0 30 -Rot2 0 330 30 -Rot3 0 330 30 -Xrange 0 0 -Yrange 0 0 -Zrange 0 0


corrected data(-fit 's Output file)
xy-plane Input-Fit-SubTomogram.png
yz-plane Input1-Fit-SubTomogram.png
zx-plane Input2-Fit-SubTomogram.png


Perform Averaging of corrected files by mrcImageAverage.
Output file

Outdata-TomogramSub.png
xy-plane

Outdata1-TomogramSub.png
yz-plane

Outdata2-TomogramSub.png
zx-plane

Min

Max
Mean
SD

SE

-0.000536652 (6, 13, 12)

0.00177258 (13, 17, 13)
3.22171e-05
0.000228739

1.6304e-06
3D image's quality has been improved by averaging. (Particular, xy-plane)


Example2

xy-plane Outdata-Tomogram.png
yz-plane Outdata1-Tomogram.png
zx-plane Outdata2-Tomogram.png


Reference file(Use top data of Input file)

Input-TomogramSub1.png
xy-plane

Input1-TomogramSub1.png
yz-plane

Input2-TomogramSub1.png
zx-plane

Min

Max
Mean
SD

SE

-5.47817 (34, 19, 20)

16.0715 (31, 22, 19)
0.142234
1.22547

0.0048441


First, perform alignment as well.
Command: mrcImageAutoRotationCorrelation3D -i p1-0001.mrc3d -r Target.mrc3dref -fit p1-0001.3dfit -EA XEYR -Rot1 0 355 5 -Rot2 0 355 5 -Rot3 0 0 30 -M 18


Corrected data(-fit 's Output file)
xy-plane Input-Fit-SubTomogram1.png
yz-plane Input1-Fit-SubTomogram1.png
zx-plane Input2-Fit-SubTomogram1.png


Some fits the reference file, and some are otherwise.
Because each 3D subtomogram image has different "Missing Area". Thus the correlation value of the orientation that is included in not both of them is Considerably lower.
Fig-SubTomogram.png


Perform averaging of Corrected files by using mrcImageAverage.
Output file

Outdata-TomogramSub1.png
xy-plane

Outdata1-TomogramSub1.png
yz-plane

Outdata2-TomogramSub1.png
zx-plane

Min

Max
Mean
SD

SE

-2.32263 (22, 29, 20)

5.20816 (19, 24, 22)
0.0844992
0.49404

0.00195286

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