a Cryo-Sample with Patch Tracking
University of Colorado,
This tutorial will guide you through processing a cryo tilt series without
fiducials, using the patch tracking method of alignment. The tilt series
is from a vitrified section and was acquired by Cedric Bouchet-Marquis. It
is recommended that you do the basic cryo
tutorial before this one.
- Download the sample data set from our web site.
- Move the data set file "cryosectionData.tar.bz" to the directory where you want to
work on it. Its contents will unpack into a subdirectory named "cryosection".
- cd to the directory with the file
- Enter the command:
or, anywhere except on Windows without Cygwin, you can use
tar -xjf cryosectionData.tar.bz
- Enter the dataset directory with:
- Start Etomo by entering
and press Build Tomogram.
Press the file chooser icon on the Datatset name line and select the stack file.
For SystemTemplate, select
Press Scan Header
Enter 0 for Fiducial diameter. Some parameters for X-ray removal
are set based on the fiducial size to avoid erasing fiducials, so it is
important to enter 0 rather than an arbitrary size when there are no fiducials.
Select Parallel Processing to use multiple CPUs to compute the reconstruction,
or select Graphics card processingto use the GPU of an Nvidia card.
In the Axis A group box, select Series was bidirectional from and set the
angle to 0.
- Press Create Com Scripts
Press Create Fixed Stack.
Press Show Min/Max for Fixed Stack.
There is still a view with a maximum near 700.
You could lower the criteria a little, but simply iterating with the
current criteria will work in this case and is always worth trying first. Do this by pressing Use Fixed Stack then Create Fixed Stack again.
Press Use Fixed Stack.
Press Calculate Cross-Correlation
When done, press Generate Coarse Aligned Stack
Fiducial Model Generation:
In this step, patches of image will be tracked from one view to the next, and
the center positions of the patches will be placed into a model file. This model
can be used to solve for the tilt series alignment, much as a model of true
fiducial markers can.
Select Use patch tracking to make fiducial model.
Change the Size of patches(X, Y) to 680,680 (if it is not already
set to that); this will give 4x4
Press the Advanced button at the bottom and change the High frequency
cutoff radius to 0.125 (if it is not already set to that). The
parameters for patch tracking are mostly based on the ones for coarse alignment
since the same program is being run. However, the correlations for coarse alignment
are done on almost the full image and are generally binned down, so the high
frequency filter cutoff of 0.25 is adequate for filtering out noise. The patches being correlated
are smaller and will not be binned, so stronger filtering is needed. Press
Basic to make it easier to see the basic controls.
- Press Track Patches.
- When done, press Open Tracked Patches to see the tracked locations on
the image and in Model View. No specific feature has been tracked, just
the whole patch area. Notice that there are just 16 contours going all the
way through the tilt series.
For this data set, the fine alignment process will involve cycling back to the
patch tracking page.
Press Compute Alignment. The mean residual is fairly high, 1.5
pixels. Such a high residual occurs because some of the positions tracked
through the patches do not correspond to projections from single points in 3D.
A better fit to the points can be gotten by breaking the contours into pieces.
Return to the Fiducial Model Generation page and select Break contours
Press Recut or Restore Contours.
Return to the Fine Alignment page and press Compute Alignment.
The mean residual is much lower because much smaller errors build up over the
shorter tilt ranges in each contour.
Press View/Edit Fiducial Model. In the Model View window, rotate
the model so that you can see all the tracks well by pressing the middle mouse
button and moving the mouse at a steep diagonal. Each track consists of 7
overlapping segments, which you can see by right-clicking near a track to select
a contour; only a small segment is highlighted.
Bring up the align log file and switch to the Coordinates tab. The
"mean resid" column shows the mean residual in each contour or segment of the
track. The range is large, from 0.2 to 1.2. When patch tracking was
first developed, the approach
was simply to delete the contours with the highest residuals, and the Bead Fixer
has a Look at contours mode to assist this process. Not only is this somewhat
arbitrary, but it would lead to trouble in this case, since 10 of the highest 11
residuals occur for contours passing through zero degrees. The robust
fitting that is now available is a better approach.
Turn on Do robust fitting with tuning factor and then turn on Find
weights for contours, not points. Press Compute Alignment.
Open the align log again. Each contour has been given a weight between 0
and 1, where a weight of 0 means that the points in the contour are ignored in
the fitting. On the Robust tab, a summary line indicates 7 contours have
weights under 0.5. On the Coordinates tab, there is a new column with
the weights. The contours that have small weights are ones that have large
residuals relative to other comparable contours (i.e., ones at similar tilt
For your own data sets with patch tracking, you will probably want to select the
robust fitting whenever you break contours into pieces. If contours are
not broken into pieces, there are usually too few contours to allow robust
This sample can be positioned by viewing X/Z slices in the binned-down tomogram.
Set Sample tomogram thickness to 800.
Press Create Whole Tomogram to build a whole, binned-down tomogram.
Press Create Boundary Model to open the tomogram
Open a Slicer window with Image-Slicer. Set X-rotation to 90
and Thick: Img to 20.
- Draw 3 pairs of lines: in the middle of the tomogram (axis position ~380) and at axis positions near
115 and 555.
- Save the model with the S key.
- Press Compute Z Shift & Pitch Angles. Note the large angle
offset, which means that the tilt angles assumed during patch tracking were
significantly different from the actual angles, enough to affect the
correlations. Do the following steps to incorporate this knowledge.
- Return to the Fiducial Model Generation page and press Advanced.
- Enter the angle offset from the positioning page in the Tilt angle offset
- Press Track Patches.
- Go to the Fine Alignment page and do Compute Alignment. Note the
substantial improvement in the weighted mean residual, from ~0.41 to ~0.37. You
now have a final alignment that incorporates the positioning information, so you
can skip to creating the aligned stack.
Final Aligned Stack Creation:
Press Create Full Aligned Stack.
Make sure only one of the two choices Use the GPU and Parallel
processing is selected, and press Generate Tomogram.
Press View Tomogram in 3dmod when it is done.
Turn on the rubber band in the Zap window and select a
subarea area if desired.
Scroll to the first slice that you want to keep and press
Lo, then find the last
slice you want to keep and press Hi. You should be able to trim off
most of the crevasses on the bottom.
In Etomo, press Get XYZ Volume Range from 3dmod to import these limits.
In the Zap window, select a narrower range of slices with Lo and Hi
that will be used to determine scaling.
In Etomo, press Get XYZ Sub-Area from 3dmod in the Scaling section
to import these limits.
Press Trim Volume. When done, press 3dmod Trimmed Volume to
see the result.
Archive the original stack and clean up as usual.