Tutorial - Tomographic Reconstructions in Priism (2/2)
This is part 2 of the tutorial for performing reconstructions of
tomographic datasets with Priism; part 1 of the tutorial is at
5. Use EMTAR to Determine the Tilt Axis Orientation
If you already know this from other reconstructions at the same
magnification or, for data collected with UCSF_TOMO, you believe the
calibrated value, you can skip this step. The accuracy of this step
is critically dependent upon the tilt offset being correct (the zero
degree tilt is actually zero degrees). The mass-normalization step
will usually do a good job of this, but you can do a better job when you
view the backprojection later. Iterating through the whole process will
therefore give you the best possible answer.
To determine the tilt axis orientation:
- In the EMTAR dialog, if you have not done so already, select
the input tilt series with the "TiltSeries" button or the adjacent
field. Also select the resolution of interest using the "Resolution"
field (using the highest resolution possible (zero) might provide
a better answer but will mean more computation time).
- Turn on the "Determine tilt axis" step in the EMTAR dialog.
- In the tilt axis determination parameters:
- Make sure the "Input alignment" field refers to the alignment
file you generated in the previous steps. If it doesn't and you
used the default name for the alignment parameters
in the previous steps, all you need to do is run on the toggle button
at the end of the line that begins with "Input alignment".
- By default, the generated alignment parameters will overwrite the
input parameters. That is normally okay, but if you want to keep
a copy of the parameters with whatever tilt axis orientation had
been set so far (usually the calibrated value from UCSF_TOMO), then
change the output file name.
- The default search range of five degrees is usually fine.
If after one run, you can play with this if the results don't make
sense. For the example dataset, the calibrated value for the tilt
axis from the microscope (-70.94 degrees) is far enough from the
probable actual value that we'll use a larger search range of 15
degrees to avoid multiple passes.
- For cryo conditions, like the sample dataset, the default values
for the first tilt pair are likely fine. For negative stain, you
might use 40 degrees and 10 degrees instead.
- The tilt range and smoothing parameters, and, in the special
parameters dialog, the Wiener coefficient and phase weighting,
are parameters that I leave as is during the first pass and only
adjust them to refine the solution.
- Press the "Do It" button at the bottom of the EMTAR's dialog to
generate the alignment parameters which include the calculated value
for the tilt axis orientation. For the sample dataset, it estimates
the tilt axis to be at an orientation of -79.94 degrees.
6. Use EMTAR to Compute the Alignment Shifts and Align the Tilt Series
We'll use three stages in this step: one to compute the shifts for the
alignment using cross-correlations, another to apply the alignment parameters
to generate an aligned tilt series, and a final step to reconstruct a
central slice of the 3D volume. To set things up, use the following:
- If you adjusted the resolution level for the tilt axis determination
calculation, set the value back to two in the main EMTAR dialog so the
subsequent sections use the second lower resolution (500 x 500) in the
input tilt series. Turn off the tilt axis determination step.
- In the EMTAR dialog, turn on the "Alignment" step. Stick with the
default parameters for the first run: those defaults will use the tilt
offset and tilt axis determined in the previous steps and will overwrite
the input alignment parameters. When you have problems with the alignment,
the option in the alignment step's special parameters to generate images
of the cross-correlations can be useful. By changing the Wiener parameter
for the phase weighting and the 2nd peak threshold level, you may be
able to overcome some alignment problems. The stretching, filtering,
and phase weighting options may also help in a few cases. Below are
example cross-correlation results for the sample data set. The image on
the right is the cross-correlation between the 7.99 degree and 9.99 degree
projection (section 36 in the image file) and the image on the left is the
cross-correlation between the 43.98 degree and 45.98 degree projection
(section 54 in the image file).
- Turn on the "Apply parameters" stage in the EMTAR dialog. That stage
uses the alignment and mass-normalization parameters to generate a new
tilt series where the projections have been interpolated to an aligned
coordinate system and the intensities have been normalized. In the
parameters for that stage, change the name of the output, in the
"AlignedSeries" field, so the last part of the file name is
"Cent_ufix_001_b4.MnAln" rather than "Cent_ufix_001.MnAln" to distinguish
that the aligned series will be binned down by a factor of 4 compared
to the input tilt series. Also, change the menu next to the
"Image formation" button to read "scaled linear". That is an appropriate
choice for the sample dataset. For other datasets, the logarithmic option
may be a better choice. The linear option does not normalize the
intensities at all. In the special parameters for the "Apply parameters"
stage change the pcBase value to 0.001.
- Turn on the "EWBP" step in the EMTAR dialog. EWBP is an acronym
for elliptically weighted backprojection. It is the less computationally
intensive reconstruction available in the EMTAR interface; the other
reconstruction method is TAPIR. In the EWBP parameters, change the name of
the generated reconstruction, in the "Reconstruction" field, so the
last part fo the file name is "Cent_ufix_001_b4.xzyw" rather than
"Cent_ufix_001.xzyw". Adjust the "Y range reconstructed" to be "996 999".
Because the second lower resolution (i.e. binned by a factor of 4) of a
2000 x 2000 tilt series is used, that choice of values will generate a
single central slice through the reconstruction volume. Set z size of the
reconstruction, which is the second value in the "Output XZ size" field",
to be 600 rather than the default value of 150.
- Press the "Do It" button at the bottom of the EMTAR dialog to generate
the alignment parameters, aligned tilt series, and reconstruction slice.
When the processing completes, the "Display" menu in EMTAR's main dialog
(see Image 5 for a screenshot) is a convenient shortcut for viewing the
results in a Priism window. With the exception of the "MASSNORM fit" option,
the choices in that menu are shortcuts for bringing up CopyRegion for the
file in question.
Image 6. Shows a screenshot of Priism and EMTAR highlighting EMTAR's
After this stage of processing, you would usually look at the reconstructed
slice and the aligned and normalized tilt series. I'll discuss the
reconstructed slice in the next section. By playing animations of the
aligned tilt series (the Movie option in the Image menu of the image window is
convenient for that), you can qualitatively assess the alignment. Below is a
QuickTime move of the aligned and normalized tilt series from the sample data.
Movie 2. Displays the aligned and normalized tilt series for the
sample centrosome data. The QuickTime movie can be downloaded from
It is 6.7 megabytes long.
7. Correct Problems with Centering and Angles
The image below is the reconstructed slice computed for the example dataset
in the previous step. When looking at slices like this, focus on the following:
- Are the features of interest well contained in the reconstruction? If
not, note the displacement from the center of the reconstruction in
the vertical dimension (z, since the reconstruction has a flipped
orientation). A shift upwards from the center is a positive shift. The
"Point Values" option in the File menu of the image window is an easy way
to see the coordinates corresponding to the current mouse cursor position
in an image window.
- Does the sample appear to lie flat in the reconstruction or is it at an
angle? If it is at an angle, note the angle it makes with the horizontal
(treat a clockwise rotation as a positive value and a counterclockwise
rotation as a negative value). You can adjust the tilt offset later to
put the sample in a horizontal orientation.
- Estimate the approximate thickness of the sample as it appears in the
reconstruction slice. That is useful for assessing whether the z size you
chose for the reconstruction is appropriate.
- Are the missing wedge artifacts (x-shaped features about bright points)
symmetric? Are the bars of the x-shaped features straight? Systematic
asymmetries or bent bars are indicative of problems in the alignment:
an error in the assumed value for the tilt axis orientation or systematic
errors in the alignment shifts are possible causes.
Image 7. Shows the central reconstructed slice for the example dataset.
For the example image, the contrast is poor, but the features (for instance,
the broad smudge about 30 to 70 pixels from the left edge and another,
similarly shaped smudge another 50 pixels to the right of that) are roughly
centered in the vertical bounds of the slice. It is difficult to see the
bounds of the sample in the image, so I'd not make an adjustment to the tilt
offset on the basis of this slice along. The missing wedge artifacts appear
To apply corrections for the tilt offset or centering (for problems with
the alignment, you'll want to check the tilt axis orientation value and, if
you didn't run the tilt axis determination yet, run it to check against the
value you had used) do the following:
- In the EMTAR dialog, turn off the alignment stage.
- Make sure the apply parameters stage is on in the EMTAR dialog.
In the parameter dialog for that stage, add four times the shift in z
you noted from the reconstruction slice to the third value in the
"Output shift" field. The factor of four is there because the reconstruction
was done at two resolution levels lower than the full resolution; in
general, the multiplicative factor would be two raised to the selected
resolution level. In the special parameters for that stage, enter the
tilt offset you determined from the reconstruction slice.
- Make sure the EWBP stage is on in the EMTAR dialog.
- Press the "Do It" button at the bottom of the EMTAR dialog to regenerate
the aligned and normalized tilt series and the reconstruction slice.
With the regenerated slice, look again to see if the sample features are
centered vertically in the slice and that the sample appears to be
approximately horizontal. If not, repeat the process as necessary until
the sample does appear to be centered and horizontal in the reconstruction
If you found that you had to adjust the tilt offset to get a horizontal
sample in the reconstruction slice, take the tilt offset you determined
and rerun the alignment and apply parameters steps:
- In the EMTAR dialog, turn on the alignment stage. In the special
parameters for that stage set the tilt offset to the value you determined
- In the EMTAR dialog, make sure the apply parameters stage is on.
In the special parameters for that stage, set the tilt offset to zero
(when the alignment is run it will incorporate the offset into the tilt
angle values in the alignment parameters; therefore, you don't want to
add a further adjustment in the apply parameters stage).
- Turn off the EWBP stage unless you want to double check the appearance
of the reconstruction slice.
- Press the "Do It" button to regenerate the alignment and aligned tilt
8. Use EWBP to Reconstruct the Full Volume
Up to now we've only reconstructed a single slice to avoid the overhead
of computing the full reconstruction while adjusting some key parameters.
We'll now proceed to reconstruct the full volume with EWBP:
- In the EMTAR dialog, turn off all stages except EWBP (we already have
an up-to-date aligned and normalized tilt series from the previous step).
- In the EWBP parameters, set the "Y range reconstructed" to cover the
full range of the input tilt series. For the example data which is
2000 x 2000, enter "0 1999" in the "Y range reconstructed" field.
- Press "Do It" at the bottom of the EMTAR dialog to generate the
- Since the reconstruction is in a flipped orientation, you typically
also want the unflipped orientation. Use Priism's
Flip (in the
Priism menus, go to the "DataViews" menu, select "Rotation", and then
select "Flip" to launch it) to generate the unflipped view.
The first movie below steps through the xz slices of the reconstruction for
the example data. The second movie steps through the xy slices of the same
Movie 3. Displays the EWBP result (.xzyw) for the example dataset. The
QuickTime movie can be downloaded from http://msg.ucsf.edu/em/EMNEW2/movies/xzyw_v2.mov. It is 9.7 megabytes long.
Movie 4. Displays the flipped version of the EWBP result for the example
dataset. The QuickTime movie can be downloaded from
It is 12.9 megabytes long.
9. Iterative Refinement of the Alignment and Reconstruction
Use this step to improve the alignment parameters and reconstruction that
you obtained earlier. This step is significantly more computationally expensive
then the earlier steps. I recommend running the process in parallel on a
cluster or multiprocessor machine.
- In the Priism menus, select "Processing", and then "EM Processing",
and finally "Iter. Alignment + Reconstruction" to open the dialog
for the iterative refinement of the alignment and reconstruction.
- Select the same tilt series (using either the "TiltSeries" button
or the adjacent field) that you used in the earlier steps.
- Use the "InAlignParam" or the adjacent field to select the file with
the input alignment parameters. For the example dataset, they are in
Cent_ufix_001.bprmMn in the same directory as the input tilt series.
- The output alignment parameters (you will get one set per every iteration
of the algorithm) and the output reconstruction are, by default, written
to the same directory as the tilt series. If you want to change that, edit
the fields labeled "OutParamBase" and "Reconstruction". To flag that the
reconstruction have been binned by a factor of four from the full
resolution, change the final part of the name in the "Reconstruction"
field from "Cent_ufix_001.xzy" to "Cent_ufix_001_b4.xzy".
- Use the same resolution level, 2, that you used earlier by entering 2
in the "Resolution" field.
- The menu next to the "Apply massnorm" button selects the image formation
model to use Use the same model as you used with EMTAR (for the example
dataset, that was the "scaled linear" model).
- In the "Reconstruction shift" field, enter the same values as you used
for the full reconstruction in the previous step.
- In the "Reconstruction Z size" field, you can use the same size as you
used for the full reconstruction in the previous step. For cases where
you could see the sample thickness in the earlier reconstruction, you
could use that (multiplied by by the resolution factor, two raised to
the selected resolution level) with some added slop (perhaps 20% of the
total though it hasn't been rigorously tested to see what is optimal)
as the size.
- Enter ten for the number of runs.
- In the special parameters, change the setting for "pcBase" to be .001.
The remaining special parameters are likely fine.
- Press the "Do It" button at the bottom of the main dialog to start the
At the end of the processing, you'll have a series of ten alignment
parameter files (one for each iteration) and a reconstruction.
10. Stepping up to Higher Resolutions
In the steps so far, you used a version of the tilt series binned down
by a factor of four. For a quick reconstruction (no further refinement of
the alignment parameters) using a higher resolution version of the tilt
series to get a higher resolution reconstruction you would:
- Open the EMTAR dialog.
- Select the tilt series.
- Enter the resolution level you want in the "Resolution" field.
- Turn on the apply parameters processing stage. Use the best alignment
and mass-normalization parameters that you determined at the lower
resolution. Use the same values for the image formation model and,
in the special parameters, pcBase that you used for the lower resolution
- Turn on either the EWBP reconstruction method or the TAPIR method
(takes more time but applies a positivity constraint which should
improve the resolution). Enter the same reconstruction shift and
size that you used for the lower resolution reconstruction in the
"Size (XYZ)" and "Shift (XYZ)" fields in EMTAR's main dialog.
- Press "Do It" at the bottom of EMTAR's dialog to generate the
higher resolution aligned tilt series and reconstruction.
To get more out from stepping up to a higher resolution, you will
need to refine the alignment parameters at that resolution. The procedure
is essentially the same as the iterative refinement at the lower resolution
- Use your best estimate of the alignment parameters from the lower
resolution as your initial guess for the alignment parameters.
- You may want to tune the number of iterations. Likely, you can
use fewer iterations (say 5 in the "Number of runs" field) since
the alignment parameters are closer to the optimal value.
Part 1 of the tutorial