Considerations for tomogram positioning in IMOD - Revision history

Stefano Scaramuzza at 13:34, 9 April 2019

2019-04-09T13:34:44Z

Stefano Scaramuzza at 13:33, 9 April 2019

2019-04-09T13:33:57Z

Stefano Scaramuzza at 16:30, 8 April 2019

2019-04-08T16:30:25Z

Stefano Scaramuzza at 16:27, 8 April 2019

2019-04-08T16:27:18Z

Stefano Scaramuzza at 14:39, 8 April 2019

2019-04-08T14:39:22Z

Stefano Scaramuzza at 14:45, 31 January 2019

2019-01-31T14:45:22Z

Stefano Scaramuzza: Created page with "During tomogram reconstruction in [https://bio3d.colorado.edu/imod/ IMOD] at some point the dimensions of the final tomogram are calculated in the "tomogram positioning" step...."

2019-01-30T16:44:42Z

Created page with "During tomogram reconstruction in [https://bio3d.colorado.edu/imod/ IMOD] at some point the dimensions of the final tomogram are calculated in the "tomogram positioning" step...."

New page

During tomogram reconstruction in [https://bio3d.colorado.edu/imod/ IMOD] at some point the dimensions of the final tomogram are calculated in the "tomogram positioning" step. In this step, we recommend to keep the x-axis tilt at zero and to leave enough room above and below the sample in the tomogram. Keeping the x-axis tilt at zero facilitates the interpretation of the tomograms, since the z-axis corresponds to the direction of the electrons. It also saves us from adapting the [[Indicating_the_missing_wedge | missing wedge]] mask at later steps. Leaving enough space at the top and bottom of the sample in the tomogram is necessary to crop subvolumes from sample that is close to the ice surfaces:

[[File:tomogram_thickness.jpg|thumb|center|600px| Left: The tomogram is too thin and subvolumes at the upper/lower sample border cannot be cropped. Right: Enough space on top and bottom of the tomogram allows cropping of volumes anywhere in the sample.]]

To keep the x-axis tilt at zero and to keep the tomogram thick enough we follow the following steps. First we choose a fairly large thickness for the sample tomogram (the sample tomogram will just be used to set the boundaries, it has not the final tomogram size). In the example below, an initial thickness of 4000 pixels was chosen:

[[File:tomopitch_parameters.png|thumb|center|300px| Tomopitch parameters. The sample tomogram thickness is set to 4000 and the binning to 8. After running tomopitch make sure the x-axis tilt is 0.0. ]]

We also select a binning level of 4 or 8 to save computing time. After we create the sample tomogram we open it to set the boundaries. Here we do not follow the IMOD tutorial but just stay in the normal 3dmod view and set the lower and upper boundaries as shown below. This will prevent the introduction of any x-axis tilt. It is important that in this step we leave enough space above and below the sample. We should be very generous with that (as explained in the first figure). After saving the model and clicking on “compute Z shift & pitch angles” there should be displayed a new z-shift and final tomogram thickness but the x-axis tilt should be 0.0.

[[File:tomopitch_limits.jpg|thumb|center|600px| Using the sample tomogram, the boundaries are set in 3dmod. Move down in z-direction until you are enough below the sample and mark 3 contours with 2 points each as shown. Move up until you are enough above the sample and do the same. You should have now 6 contours with 2 points each. Save and close.]]

← Older revision		Revision as of 13:34, 9 April 2019
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	ReconstructionState.a.sample.AxisZShift=0.0</tt>		ReconstructionState.a.sample.AxisZShift=0.0</tt>

−	to zero. 6) Open the ''tomopitch.com'' file and set the two values to zero. 7) Reopen etomo and check that it worked.	+	to zero. 6) Open the ''tomopitch.com'' file and set the two corresponding values to zero. 7) Reopen etomo and check that it worked.

← Older revision		Revision as of 13:33, 9 April 2019
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−	Note: If you click the button “compute Z shift & pitch angles” again, the newly computed value of the z-shift is ''added'' to the previously computed one. Therefore, make sure to click this button only once. Also, if you have generated an offset angle by mistake, you can not simply set it to zero again. In the case where a z-shift needs to be recomputed or where an already computed offset angle needs to be removed, the following steps need to be done: 1) Go to the "Tomogram Positioning" step and set the "Angle offset" and "Z shift" to zero. 2) Go to the "Fine Alignment" step and set the two values to zero too. Do not go back to the "Tomogram Positioning" step. 3) Instead, in the top left corner click "File" and then "Save". 4) Close etomo. 5) Open the ''*edf'' file and set the two parameters	+	Note: If you click the button “compute Z shift & pitch angles” again, the newly computed value of the z-shift is ''added'' to the previously computed one. Therefore, make sure to click this button only once. Also, if you have generated an offset angle by mistake, you can not simply set it to zero again. In the case where a z-shift needs to be recomputed or where an already computed offset angle needs to be removed, the following steps need to be done: 1) Go to the "Tomogram Positioning" step and set the "Angle offset" and "Z shift" to zero. 2) Go to the "Fine Alignment" step and set the two values to zero too. Do ''not'' go back to the "Tomogram Positioning" step. 3) Instead, in the top left corner click "File" and then "Save". 4) Close etomo. 5) Open the ''*edf'' file and set the two parameters

	<tt> TotalReconstructionState.a.sample.AngleOffset=0.0		<tt> TotalReconstructionState.a.sample.AngleOffset=0.0

← Older revision		Revision as of 16:30, 8 April 2019
Line 17:		Line 17:


−	Note: If you click the button “compute Z shift & pitch angles” again, the newly computed value of the z-shift is ''added'' to the previously computed one. Therefore, make sure to click this button only once. Also, if you have generated an offset angle by mistake, you can not simply set it to zero again. In the case where a z-shift needs to be recomputed or where an already computed offset angle needs to be removed, the following steps need to be done: 1) Go to the "Tomogram Positioning" step and set the "Angle offset" and "Z shift" to zero. 2) Go to the "Fine Alignment" step and set the two values to zero too. Do not go back to the "Tomogram Positioning" step. 3) Instead, in the top left corner click "File" and then "Save". 4) Close etomo. 5) Open the ''*edf'' file and set the two parameters ''TotalReconstructionState.a.sample.AngleOffset=0.0~~'' and ''~~ReconstructionState.a.sample.AxisZShift=0.0'' to zero. 6) Open the ''tomopitch.com'' file and set the two values to zero. 7) Reopen etomo and check that it worked.	+	Note: If you click the button “compute Z shift & pitch angles” again, the newly computed value of the z-shift is ''added'' to the previously computed one. Therefore, make sure to click this button only once. Also, if you have generated an offset angle by mistake, you can not simply set it to zero again. In the case where a z-shift needs to be recomputed or where an already computed offset angle needs to be removed, the following steps need to be done: 1) Go to the "Tomogram Positioning" step and set the "Angle offset" and "Z shift" to zero. 2) Go to the "Fine Alignment" step and set the two values to zero too. Do not go back to the "Tomogram Positioning" step. 3) Instead, in the top left corner click "File" and then "Save". 4) Close etomo. 5) Open the ''*edf'' file and set the two parameters
		+
		+	<tt> TotalReconstructionState.a.sample.AngleOffset=0.0
		+	ReconstructionState.a.sample.AxisZShift=0.0</tt>
		+
		+	to zero. 6) Open the ''tomopitch.com'' file and set the two values to zero. 7) Reopen etomo and check that it worked.

← Older revision		Revision as of 16:27, 8 April 2019
Line 11:		Line 11:


−	After we create the sample tomogram, we open it to set the boundaries. Here we do not follow the IMOD tutorial but just stay in the normal 3dmod view and set the lower and upper boundaries as shown in the figure below. This will prevent the introduction of any x-axis tilt and angle offset. It is important that in this step we leave enough space above and below the sample. We should be very generous with that (as explained in the first figure). After saving the model and clicking on “compute Z shift & pitch angles”, there should be displayed a new z-shift and final tomogram thickness, but the x-axis tilt and angle offset should still be 0.0. Note that if “compute Z shift & pitch angles” is clicked again, the newly computed value of the z-shift is ''added'' to the previously computed one. Therefore, make sure to compute this value only once.	+	After we create the sample tomogram, we open it to set the boundaries. Here we do not follow the IMOD tutorial but just stay in the normal 3dmod view and set the lower and upper boundaries as shown in the figure below. This will prevent the introduction of any x-axis tilt and angle offset. It is important that in this step we leave enough space above and below the sample. We should be very generous with that (as explained in the first figure). After saving the model and clicking on “compute Z shift & pitch angles”, there should be displayed a new z-shift and final tomogram thickness, but the x-axis tilt and angle offset should still be 0.0.


	[[File:tomopitch_limits.jpg\|thumb\|center\|600px\| Using the sample tomogram, the boundaries are set in 3dmod. Left: Move down in z-direction until you are enough below the sample and mark 3 contours with 2 points each as shown. Right: Move up until you are enough above the sample and do the same. You should have now 6 contours with 2 points each. Save and close.]]		[[File:tomopitch_limits.jpg\|thumb\|center\|600px\| Using the sample tomogram, the boundaries are set in 3dmod. Left: Move down in z-direction until you are enough below the sample and mark 3 contours with 2 points each as shown. Right: Move up until you are enough above the sample and do the same. You should have now 6 contours with 2 points each. Save and close.]]
		+
		+
		+	Note: If you click the button “compute Z shift & pitch angles” again, the newly computed value of the z-shift is ''added'' to the previously computed one. Therefore, make sure to click this button only once. Also, if you have generated an offset angle by mistake, you can not simply set it to zero again. In the case where a z-shift needs to be recomputed or where an already computed offset angle needs to be removed, the following steps need to be done: 1) Go to the "Tomogram Positioning" step and set the "Angle offset" and "Z shift" to zero. 2) Go to the "Fine Alignment" step and set the two values to zero too. Do not go back to the "Tomogram Positioning" step. 3) Instead, in the top left corner click "File" and then "Save". 4) Close etomo. 5) Open the ''*edf'' file and set the two parameters ''TotalReconstructionState.a.sample.AngleOffset=0.0'' and ''ReconstructionState.a.sample.AxisZShift=0.0'' to zero. 6) Open the ''tomopitch.com'' file and set the two values to zero. 7) Reopen etomo and check that it worked.

@@ Line 1: / Line 1: @@
-During tomogram reconstruction in [https://bio3d.colorado.edu/imod/ IMOD], the dimensions of the final tomogram need to be calculated in the "tomogram positioning" step. In this step, we recommend to keep the x-axis tilt at zero and to leave enough room above and below the sample in the tomogram. Keeping the x-axis tilt at zero facilitates the interpretation of the tomograms, because only then the z-axis corresponds to the direction of the electrons. It also saves us from adapting the [[Indicating_the_missing_wedge | missing wedge]] mask at later steps. Leaving enough space at the top and bottom of the sample in the tomogram is necessary because it allows Dynamo to crop subvolumes from sample that is close to the ice surfaces, as illustrated in the figure below:
+During tomogram reconstruction in [https://bio3d.colorado.edu/imod/ IMOD], the dimensions of the final tomogram need to be calculated in the "tomogram positioning" step. In this step, we recommend to keep the x-axis tilt and angle offset at zero and to leave enough room above and below the sample in the tomogram. Keeping the x-axis tilt and angle offset at zero facilitates the interpretation of the tomograms, because only then the z-axis corresponds to the direction of the electrons. It also saves us from adapting the [[Indicating_the_missing_wedge | missing wedge]] mask at later steps. Leaving enough space at the top and bottom of the sample in the tomogram is necessary because it allows Dynamo to crop subvolumes from sample that is close to the ice surfaces, as illustrated in the figure below:
@@ Line 5: / Line 5: @@
-To keep the x-axis tilt at zero and to keep the tomogram thick enough we need to proceed as follows. First we choose a fairly large thickness for the sample tomogram (the sample tomogram will just be used to set the boundaries, it has not the final tomogram size). In the example below, an initial thickness of 4000 pixels is chosen. We also select a binning level of 4 or 8 to save computing time.
+To keep the x-axis tilt and angle offset at zero and to keep the tomogram thick enough we need to proceed as follows. First we choose a fairly large thickness for the sample tomogram (the sample tomogram will just be used to set the boundaries, it has not the final tomogram size). In the example below, an initial thickness of 4000 pixels is chosen. We also select a binning level of 4 or 8 to save computing time.
-  [[File:tomopitch_parameters.png|thumb|center|300px| Tomopitch parameters. The sample tomogram thickness is set to 4000 and the binning to 8. After running tomopitch (“compute Z shift & pitch angles”), make sure that the x-axis tilt is still 0.0. ]]
+  [[File:tomopitch_parameters.png|thumb|center|300px| Tomopitch parameters. The sample tomogram thickness is set to 4000 and the binning to 8. After running tomopitch (“compute Z shift & pitch angles”), make sure that the x-axis tilt and angle offset is still 0.0. ]]
-After we create the sample tomogram we open it to set the boundaries. Here we do not follow the IMOD tutorial but just stay in the normal 3dmod view and set the lower and upper boundaries as shown in the figure below. This will prevent the introduction of any x-axis tilt. It is important that in this step we leave enough space above and below the sample. We should be very generous with that (as explained in the first figure). After saving the model and clicking on “compute Z shift & pitch angles”, there should be displayed a new z-shift and final tomogram thickness, but the x-axis tilt should still be 0.0.
+After we create the sample tomogram, we open it to set the boundaries. Here we do not follow the IMOD tutorial but just stay in the normal 3dmod view and set the lower and upper boundaries as shown in the figure below. This will prevent the introduction of any x-axis tilt and angle offset. It is important that in this step we leave enough space above and below the sample. We should be very generous with that (as explained in the first figure). After saving the model and clicking on “compute Z shift & pitch angles”, there should be displayed a new z-shift and final tomogram thickness, but the x-axis tilt and angle offset should still be 0.0. Note that if “compute Z shift & pitch angles” is clicked again, the newly computed value of the z-shift is ''added'' to the previously computed one. Therefore, make sure to compute this value only once.
   [[File:tomopitch_limits.jpg|thumb|center|600px| Using the sample tomogram, the boundaries are set in 3dmod. Left: Move down in z-direction until you are enough below the sample and mark 3 contours with 2 points each as shown. Right: Move up until you are enough above the sample and do the same. You should have now 6 contours with 2 points each. Save and close.]]