EMTAR

Overview

EMTAR is a front-end for alignment and reconstruction of EM tomography data. The basic recipe for generating a reconstruction is:

  1. Enter the file name for the tilt series in the field adjacent to the "Tilt series" button, or press the "Tilt series" button to select the tilt series file name with a file dialog.
  2. If you've added multiple resolution to the input tilt series using AppendRes with a z scaling of one, then you can enter the resolution level (zero is the highest) to process in the "Resolution" field. This can be especially useful if you are doing preliminary reconstructions of large datasets.
  3. As a guide for selecting a region of interest for reconstruction, reconstruct a single slice. To do so, turn on the "setup to find reconstruction bounds" toggle button and then press the "Do It" button.
  4. Select the region of interest for reconstruction; the "Select region" topic describes how to do so in more detail.
  5. Reconstruct the region of interest. To to do so, turn off the "setup to find reconstruction bounds" toggle button and press the "Do It" button.

If you wish to use an alignment based on fiducial markers (gold beads, for example), you can follow the same recipe but turn off the rough alignment and alignment calculations and use the result from the markers instead as massnorm's input alignment parameter file.

The actual processing is implemented as a series of steps, and the EMTAR dialog reflects this by having a toggle button to turn on or off each step and a button adjacent to that toggle button for opening a dialog with the parameters specific to that step. The steps are:

Median filter
This is a 2D median filter (identical to the median filter in Priism's Filter2D). It is included here because it can be useful for low signal-to-noise cases to use a filtered version of the raw tilt series in the mass normalization and alignment calculations.
Rough alignment
Computes an initial guess at the alignment so that it is possible to restrict the region of analysis in the mass normalization calculation. For data from the UCSF tomography data collection on TECNAI systems, it is possible to extract this information from the tilt series' extended header. As a fallback, the full cross-correlation alignment procedure can be used but with the parameters suitable for a quick and rough alignment calculation.
Mass normalization
Computes the relationship between intensities in the projections and sample mass densities.
Determine tilt axis
Determines the orientation of the tilt axis in the projections.
CTF correction
Can correct the tilt series for the effects of the contrast transfer function.
Alignment
Uses cross-correlation calculations to determine the alignment parameters for the tilt series.
Apply parameters
Takes the alignment and mass normalization results and applies them to the raw tilt series to give an aligned tilt series where the individual pixel values have been converted to accumulated mass density.
EWBP
Performs a weighted backprojection of the aligned and normalized tilt series to arrive at a 3D reconstruction.
TAPIR
Uses an iterative algorithm to arrive at a 3D reconstruction. Use this instead of the weighted backprojection when you want a higher fidelity reconstruction at the expense of significantly more computation.
GPURECON
This runs a client program that contacts one or more servers to perform the reconstruction. The servers use graphics cards to accelerate the calculation. You can use this instead of EWBBP or TAPIR.

EMTAR executes the steps by writing out a short text file with the commands to be run. By default, when you press the "Do It" button that text file is executed immediately on the current machine. To change the name of the command file, the name of the log file generated during processing, or how the command file is executed, Use the "Options" button in EMTAR's main dialog. It opens a dialog in which you can control set those parameters.

Topics

Overview | Select region | Parameters | Other controls | Parallel execution

Related Priism Topics

Priism | Alignment with markers | Filter2D | Alignment | Mass normalization | Determine tilt axis | CTF correction | Apply parameters | EWBP | TAPIR | GPURECON


SelectRegion

To select the region of interest in XY, do the following:

  1. If you already have a reconstruction and want to use it for selection of the region, first flip the reconstruction if you have not already done so and then turn on the "use flipped reconstruction" toggle button in EMTAR's main dialog. By default, EMTAR assumes that the flipped reconstruction has the same name as the tilt series but with a .xyzw extension. If that is not the case, press the "Change file names" button in EMTAR's main dialog and use the dialog that appears to change the "Flipped reconstruction for XY" file name. When the "use flipped reconstruction" toggle button is off, EMTAR uses the raw tilt series for selection.
  2. Load the target data (either the raw tilt series or the flipped reconstruction) into an image window. You can use ViewFile or CopyRegion in Priism to do that. For the raw tilt series, you could also use the "Tilt series" entry in the EMTAR's "Display" menu (which is simply a shortcut for calling CopyRegion with the correct file name).
  3. Press the "XY" button next to "Select region" in EMTAR's main dialog. That will open up a dialog and display the selected region in the image window. You can adjust the selected region with the mouse. To shift the entire region as a unit, position the mouse pointer in the center of the region, press the right mouse button, drag the mouse, and then release the right mouse button when done. To shift one edge of the region, position the mouse pointer at the edge, press the right mouse button, drag the mouse, and then release the mouse pointer when done. You can also change the bounds for the selected region by directly editing the values in the "EMTAR: Select XY" dialog. Pressing the "Apply" button in that dialog transfers the current parameters to the main dialog and leaves the dialog open. The "Close" button is similar but closes the dialog as well. The "Cancel" button closes the dialog and does not transfer the region parameters to the main dialog.

To select the region of interest in XZ, do the following:

  1. First, you need to generate a reconstruction. A single slice is sufficient, and you can turn on the "setup to find reconstruction bounds" in the EMTAR main dialog and then press the "Do It" button in the EMTAR main dialog to generate that slice.
  2. By default, EMTAR assumes that the reconstruction will have the same name as the tilt series but with a .xzyw extension. If that is not the case, press the "Change file names" button in EMTAR's main dialog and use the dialog that appears to change the "Reconstruction for XZ" file name.
  3. Load the reconstruction into an image window. You can use ViewFile or CopyRegion in Priism to do that. You could also use the "Display" menu in EMTAR's main dialog: if the reconstruction was from EWBP, then select "EWBP reconstruction" from the "Display" menu; otherwise, select "TAPIR reconstruction".
  4. Press the "XZ" button next to "Select region" in EMTAR's main dialog. That will open up a dialog and display the selected region in the image window. You can adjust the selected region with the mouse. To shift the entire region as a unit, position the mouse pointer in the center of the region, press the right mouse button, drag the mouse, and then release the right mouse button when done. To shift one edge of the region, position the mouse pointer at the edge, press the right mout button, drag the mouse, and then release the mouse pointer when done. You can also change the bounds for the selected region by directly editing the values in the "EMTAR: Select XZ" dialog. Pressing the "Apply" button in that dialog transfers the current parameters to the main dialog and leaves the dialog open. The "Close" button is similar but closes the dialog as well. The "Cancel" button closes the dialog and does not transfer the region parameters to the main dialog.

Topics

Overview | Select region | Parameters | Other controls


Parameters

The following parameters are set in EMTAR's main menu.

TiltSeries
This is the name of the file containing the raw projection data stack from the CCD (i.e. measured in terms of electron counts; data stacks with the contrast inverted can not be handled).
Resolution
Selects which resolution to process from the input tilt series. 0 is the highest (full) resolution. You can use Priism's AppendRes to add resolutions to a tilt series; when you do so, use a z scaling of one since you do not want the lower resolutions to combine information from different projections. When you process a resolution other than the full resolution, any size-related parameters are still specified in the full resolution units: the backend processing will scale the values appropriately. Similarly, the generated alignment parameters will be in terms of the full resolution coordinate system and are transformed, as necessary, when working with a lower resolution.
Shift (XYZ)
Specifies the center of the reconstructed projection. A good way to select the values for the shift parameters is to select the region of interest with the mouse (see the "Select region" topic for details).
Size (XYZ)
The first value is the lateral dimension of the reconstruction in pixels. The second is the number of 2D reconstruction slices. The third is the thickness of the reconstruction in pixels and should be greater than the thickness of the object of interest. As with the shifts, you can interactively set these with the mouse (see the "Select region" topic for details).

Topics

Overview | Select region | Parameters | Other controls


Other Controls

Other controls in EMTAR's main dialog are:

NX:NY:NV
The first two values display, respectively, the x and y dimension of the projections. The third value displays the number of projections in the tilt series.
Display menu
The display menu is a shortcut for displaying the input tilt series or output image or plot files within Priism. The entries in the menu are:
Tilt series
Displays the image file specified by the "Tilt series" field in EMTAR's main dialog.
Filtered series
Displays the output of the median filter, i.e. the image file specified by the "FilteredSeries" field in the median filter dialog.
Aligned & normalized series
Displays the output of apply parameters, i.e. the image file specified by the "AlignedSeries" field in the apply parameters dialog.
CTF corrected series
Displays the output of CTF correction.
EWBP reconstruction
Displays the reconstruction generated by the weighted backprojection calculation, i.e. the image file specified by the "Reconstruction" field in the EWBP dialog.
TAPIR reconstruction
Displays the reconstruction generated by the iterative algorithm, i.e. the image file specified by the "Reconstruction" field in the TAPIR dialog.
Stretched and apodized series
Displays the optional output, specified by the "Stretched series" field in the alignment special parameters dialog, from the alignment calculation.
Cross correlations (alignment)
Displays the optional cross-correlation peak images output from the alignment calculation. The name of the image file is listed in the "Cross correlations" field of the alignment's special parameters dialog.
Cross correlations (tilt axis)
Displays the optional cross-correlation images generated by the calculations to determine the tilt axis orientation. The name of the image file is listed in the "Cross correlations" field of the tilt axis determination's special parameters dialog.
MASSNORM fit
Displays the optional graphs generated by the mass normalization calculation. The name of the file is set with the "Fit graph" field in the mass normalization special parameters dialog.
Tilt axis (composite)
Displays the optional composite cross-correlation graphs generated by the calculations to determine the tilt axis orientation. The name of the file is set with the "Composite plots" field in the find axis special parameters dialog.
Tilt axis (pairwise)
Displays the optional cross-correlation graphs for each tilt angle pair generated by the calculations to determine the tilt axis orientation. The name of the file is set with the "Pairwise plots" field in the find axis special parameters dialog.

Topics

Overview | Select region | Parameters | Other controls