Considerations for tilt series alignment in IMOD - Revision history

Stefano Scaramuzza: /* Residual fixing */

2019-03-27T10:57:49Z

Residual fixing

Stefano Scaramuzza at 17:19, 26 March 2019

2019-03-26T17:19:34Z

Stefano Scaramuzza at 17:06, 26 March 2019

2019-03-26T17:06:30Z

Stefano Scaramuzza at 14:03, 11 March 2019

2019-03-11T14:03:21Z

Stefano Scaramuzza: /* Residual fixing */

2019-02-26T13:01:32Z

Residual fixing

Stefano Scaramuzza at 14:30, 31 January 2019

2019-01-31T14:30:17Z

Stefano Scaramuzza at 14:12, 30 January 2019

2019-01-30T14:12:39Z

Stefano Scaramuzza at 14:05, 30 January 2019

2019-01-30T14:05:57Z

Stefano Scaramuzza: Created page with "When doing the tilt series alignment in [https://bio3d.colorado.edu/imod/ IMOD] there are a few things we strongly recommend to follow in order to reach the best possible resu..."

2019-01-30T13:48:42Z

Created page with "When doing the tilt series alignment in [https://bio3d.colorado.edu/imod/ IMOD] there are a few things we strongly recommend to follow in order to reach the best possible resu..."

New page

When doing the tilt series alignment in [https://bio3d.colorado.edu/imod/ IMOD] there are a few things we strongly recommend to follow in order to reach the best possible results.

=== Alignment parameters ===
To avoid overfitting, the tilt parameters should describe a rigid body transformation, i.e., we only solve for the parameter “one rotation”. The parameter selection should look as follows:

[[File:imod_alignment_parameters.png|thumb|center|400px| Alignment parameters in IMOD for rigid body transformation. We only solve for one rotation and keep everything else fixed]]

=== Residual fixing ===
In the “residual fixing” step in IMOD, the residuals should be fixed individually but only in a way that makes sense, i.e., the centers of the model (green circles) should ''always'' be in the center of the gold beads, even if the residual (red arrow) points somewhere else. This can be achieved by manually moving/correcting the green circles (right mouse click) if necessary.

=== Removing outlier beads ===
If there are outlier beads in your model that keep having large residuals no matter how well centered the model is, they can be removed from the model. However, this should only be done in extreme cases. Make sure you keep enough gold beads in your final model (minimum 5, ideally 10-15 or as many as behave well).

=== Alignment error ===
The alignment error should preferably be below 2 pixels and in the best cases below 1 pixel. However, this should not be forced. As said before, keep the points of the model on the center of the beads and keep enough gold beads in the model. It can be that some tomograms simply cannot be aligned better.

=== Alignment quality ===
Once you are satisfied with the alignment you can quickly check the alignment quality by confirming that the beads are moving perfectly horizontal throughout the tilts. Drawing a “red box” with the corresponding tool in 3dmod can be helpful as a visual guide.

=== Tomogram quality ===
Another way to check the alignment quality is by inspecting the beads in the final tomogram. A good alignment results in perfectly symmetric beads (fig. 3).

@@ Line 6: / Line 6: @@
 === Residual fixing ===
-In the “residual fixing” step in IMOD, the residuals should be fixed individually but only in a way that makes sense, i.e., the centers of the model (green circles) should ''always'' be in the center of the gold beads, even if the residual (red arrow) points somewhere else (see figure below). This can be achieved by manually moving/correcting the green circles (right mouse click) if necessary. Suggestion: The “threshold for residual report“ can be set to 3 in the beginning and, after correcting the residuals as described, it can be lowered to 2. After computing a new alignment and fixing the residuals again it can be lowered even further to 1 or 0.5.
+In the “residual fixing” step in IMOD, the residuals should be fixed individually but only in a way that makes sense, i.e., the centers of the model (green circles) should ''always'' be in the center of the gold beads, even if the residual (red arrow) points somewhere else (see figure below). This can be achieved by manually moving/correcting the green circles (right mouse click) if necessary. Suggestion: The “threshold for residual report“ can be set to 3 in the beginning and, after correcting the residuals as described, it can be lowered to 2. After computing a new alignment and fixing the residuals again it can be lowered further to 1 or even 0.5.
   [[File:imod_residuals.jpg|thumb|center|300px| Residual fixing in IMOD (open full size image to see details).]]

@@ Line 7: / Line 7: @@
 === Residual fixing ===
 In the “residual fixing” step in IMOD, the residuals should be fixed individually but only in a way that makes sense, i.e., the centers of the model (green circles) should ''always'' be in the center of the gold beads, even if the residual (red arrow) points somewhere else (see figure below). This can be achieved by manually moving/correcting the green circles (right mouse click) if necessary. Suggestion: The “threshold for residual report“ can be set to 3 in the beginning and, after correcting the residuals as described, it can be lowered to 2. After computing a new alignment and fixing the residuals again it can be lowered even further to 1 or 0.5.
-  [[File:imod_residuals.jpg|thumb|center|300px| Residual fixing in IMOD.]]
+  [[File:imod_residuals.jpg|thumb|center|300px| Residual fixing in IMOD (open full size image to see details).]]
 === Removing outlier beads ===

@@ Line 6: / Line 6: @@
 === Residual fixing ===
-In the “residual fixing” step in IMOD, the residuals should be fixed individually but only in a way that makes sense, i.e., the centers of the model (green circles) should ''always'' be in the center of the gold beads, even if the residual (red arrow) points somewhere else. This can be achieved by manually moving/correcting the green circles (right mouse click) if necessary. Suggestion: The “threshold for residual report“ can be set to 3 in the beginning and, after correcting the residuals as described, it can be lowered to 2. After computing a new alignment and fixing the residuals again it can be lowered even further to 1 or 0.5.
+In the “residual fixing” step in IMOD, the residuals should be fixed individually but only in a way that makes sense, i.e., the centers of the model (green circles) should ''always'' be in the center of the gold beads, even if the residual (red arrow) points somewhere else (see figure below). This can be achieved by manually moving/correcting the green circles (right mouse click) if necessary. Suggestion: The “threshold for residual report“ can be set to 3 in the beginning and, after correcting the residuals as described, it can be lowered to 2. After computing a new alignment and fixing the residuals again it can be lowered even further to 1 or 0.5.
 === Removing outlier beads ===

@@ Line 1: / Line 1: @@
-When doing the tilt series alignment in [https://bio3d.colorado.edu/imod/ IMOD] there are a few things we strongly recommend to follow in order to reach the best possible results.
+When doing the tilt series alignment in [https://bio3d.colorado.edu/imod/ IMOD], we strongly recommend to consider the following points in order to reach the best possible results.
 === Alignment parameters ===
@@ Line 11: / Line 11: @@
 === Removing outlier beads ===
-If there are outlier beads in your model that keep having large residuals no matter how well centered the model is, they can be removed from the model. However, this should only be done in extreme cases. Make sure you keep enough gold beads in your final model (minimum 5, ideally 10-15 or as many as behave well).
+If there are outlier beads in your model that keep having large residuals, no matter how well centered the model is, they can be removed from the model. However, this should only be done in extreme cases. Make sure you keep enough gold beads in your final model (absolute minimum 5, ideally 10-15 or as many as behave well).
 === Alignment error ===
-The alignment error should preferably be below 2 pixels and in the best cases below 1 pixel. However, this should not be forced. As said before, keep the points of the model on the center of the beads and keep enough gold beads in the model. It can be that some tomograms simply cannot be aligned better.
+The alignment error should preferably be below 2 pixels and in the best cases below 1 pixel. However, this should not be forced. As said before, keep the points of the model on the center of the beads and keep enough gold beads in the model. It can very well be that some tomograms simply cannot be aligned better.
 === Alignment quality ===
-Once you are satisfied with the alignment you can quickly check the alignment quality visually by confirming that the beads are moving perfectly horizontal throughout the tilts. Drawing a “red box” with the corresponding tool in 3dmod can be helpful as a visual guide (as shown below):
+Once you are satisfied with the alignment you can quickly check the alignment quality visually by confirming that the beads are moving perfectly horizontal throughout the tilts. Drawing a red rectangle with the corresponding tool in 3dmod can be helpful as a visual guide:
-  [[File:imod_red_box.png|thumb|center|400px| Drawing a red box in 3dmod helps to asses if the beads move perfectly horizontal throughout the tilt series.]]
+  [[File:imod_red_box.png|thumb|center|400px| Drawing a red rectangle in 3dmod helps to asses if the beads move perfectly horizontal throughout the tilt series.]]
 === Tomogram quality ===
 Another way to check the alignment quality is by visually inspecting the beads in the final tomogram. This is described in more detail [[Tomogram_quality_ assessment | here]].

@@ Line 4: / Line 4: @@
 To avoid overfitting, the tilt parameters should describe a rigid body transformation, i.e., we only solve for the parameter “one rotation”. The parameter selection should look as follows:
-  [[File:imod_alignment_parameters.png|thumb|center|300px| Alignment parameters in IMOD for rigid body transformation. We only solve for one rotation and keep everything else fixed]]
+  [[File:imod_alignment_parameters.png|thumb|center|300px| Alignment parameters in IMOD for rigid body transformation. We only solve for one rotation and keep everything else fixed.]]
@@ Line 17: / Line 17: @@
 === Alignment quality ===
-Once you are satisfied with the alignment you can quickly check the alignment quality by confirming that the beads are moving perfectly horizontal throughout the tilts. Drawing a “red box” with the corresponding tool in 3dmod can be helpful as a visual guide.
+Once you are satisfied with the alignment you can quickly check the alignment quality visually by confirming that the beads are moving perfectly horizontal throughout the tilts. Drawing a “red box” with the corresponding tool in 3dmod can be helpful as a visual guide (as shown below):
+ [[File:imod_red_box.png|thumb|center|400px| Drawing a red box in 3dmod helps to asses if the beads move perfectly horizontal throughout the tilt series.]]
 === Tomogram quality ===
-Another way to check the alignment quality is by inspecting the beads in the final tomogram. A good alignment results in perfectly symmetric beads (fig. 3).
+Another way to check the alignment quality is by visually inspecting the beads in the final tomogram. This is described in more detail [[Tomogram_quality_ assessment | here]].

← Older revision		Revision as of 14:05, 30 January 2019
Line 4:		Line 4:
	To avoid overfitting, the tilt parameters should describe a rigid body transformation, i.e., we only solve for the parameter “one rotation”. The parameter selection should look as follows:		To avoid overfitting, the tilt parameters should describe a rigid body transformation, i.e., we only solve for the parameter “one rotation”. The parameter selection should look as follows:

−	[[File:imod_alignment_parameters.png\|thumb\|center\|~~400px~~\| Alignment parameters in IMOD for rigid body transformation. We only solve for one rotation and keep everything else fixed]]	+	[[File:imod_alignment_parameters.png\|thumb\|center\|300px\| Alignment parameters in IMOD for rigid body transformation. We only solve for one rotation and keep everything else fixed]]