EMRECON

Overview

EMRECON is the second step in EM tomographic image processing, which yields the 3-D reconstructions. It requires that the alignment parameters be supplied by BALIGN.

EMRECON is composed of four modules (programs):

• MASSNORMdetermines the relationship between electron counts in the raw projections and mass density of the specimen.
• APPL_PRM creates the mass-normalized and aligned data stack for reconstruction from the raw data stack in one step.
• EWBP makes the weighted back-projection reconstruction.
• TAPIR provides the iterative reconstruction result.

Topics

Overview | User interface | Recipe for use | Parameters

Related Priism Topics

Priism | MASSNORM | APPL_PRM | EWBP | TAPIR | Alignment

User Interface Features

• Each module is an independent program. So any combination of the four modules can be run in a given execution (by clicking the corresponding toggle buttons in the Master setup menu).
• When you change the content of a widget in a menu, the contents of some other widgets in the menus downstream can change correspondingly.
• All the files will have the same name as the input data file, IdatFile, except with their own standard extensions. For an optional file, there is a toggle button to the right of the file name widgets: toggling the button on will cause the standard file name to be entered in the file name field and toggling it off will enter none which flags that the file will not be used.
• The setup menu of each module has a submenu for special parameters. The default values in this submenu are normally fine.
• When you click the DoIt button, a *.COM file is created in your home directory and is executed with an accompanying *.log file created in your home directory. If you make the *.log file name = none in the Options submenu, the content of the *.log file will be diverted to the screen. You will find this convenient for monitoring execution when you do troubleshooting.

Topics

Overview | User interface | Recipe for use | Parameters

Suggested Recipe For Use

1. Do a partial run to determine the z shift for the shiftXYZ input parameter. This is usually done by reconstructing a single slice and selecting a region of interest from it. A procedure to do this is:
   1. For IdatFile, enter the name of the file containing the unaligned projection stack.
   2. Turn on the toggle labeled setup: to find shiftZ.
   3. If the results of BALIGN are in a different place than the unaligned projection series or have a nonstandard name, change the IprmFile parameter for APPL_PRM. You could also adjust the names of the output files for APPL_PRM and EWBP if you wanted to put the results in a different place.
   4. Press the DoIt button.
   5. When the reconstruction is finished, turn the toggle labeled setup: to find shiftZ off.
   6. Select Display: OrecFile 1 from the main dialog. This will open a dialog to load the reconstructed slice into a window.
   7. Next to Pick XYZ press the XZ button to open a dialog to select the region of interest. The region of interest is shown in red on top of the reconstructed slice; by depressing the right mouse button while the pointer is over an edge or corner of the region and then dragging the mouse, you can extend the region. You can shift the region by moving the pointer to the center of the region, depressing the left mouse button, and dragging the mouse. When you are satisfied with the selected region, use the Apply button or the Close button to record your settings (the Close button also dismisses the region selection dialog).
2. Choose the y bounds of the region to reconstruct. This can be done with the unaligned data or a previous reconstruction which has been flipped. Since step 1 hasn't generated a full reconstruction, you'll want to pick from the unaligned data so make sure the toggle labeled use .xyzw file is off and then select Display: IdatFile from the main dialog to view the unaligned data (or use ViewFile or CopyRegion). Then press the XY button next to Pick XYZ. This will open a dialog to select the x and y region for reconstruction; the selected region will be highlighted in red. You'll typically want to do this selection from a projection whose tilt angle is close to zero (the selection dialog displays the tilt angle for the unaligned projection currently shown; simply step through the projections to get to the desired tilt angle). To alter the selection, move the mouse pointer over one of the corners (to change the extent of the region in both directions) or over an edge (to change the extend of the region in a direction perpendicular to the edge), depress the left mouse button, and then drag the mouse until the region is the desired size. It is also possible to shift the region without changing its dimensions: position the pointer over the center of the region, depress the left mouse button, and drag the mouse until the region reaches the desired position. When you are satisfied with the selected region, use the Apply button or the Close button to record your settings (the Close button also dismisses the region selection dialog).
3. Now that you have selected the region to reconstruct, you'll turn on the applications to do the full reconstruction. Turn on APPL_PRM and EWBP if they are not already on. Also turn on MASSNORM. You may want to change the Fit: average option in MASSNORM to Fit: background if there is clear background in all projections. When fitting the background you may want to use different shifts for MASSNORM (set by the shXYZ field in the MASSNORM dialog) and different dimensions (set by the dimxy field in the MASSNORM dialog) to choose a region dominated by background. When fitting the average, it is preferable to use the same shifts in MASSNORM as in the other applications by turning on the toggle next to the shXYZ field in the MASSNORM dialog.
4. You may also want to turn on TAPIR as well to iteratively refine the reconstruction. Many users do not use it since they haven't found a set of parameters for their data so that it improves the reconstruction. If you do use it, you should adjust the setting for the severity of the limited detector problem (how much structure is not in the region of interest for tilt angles close to zero but is present for larger tilt angles).
5. Start the reconstruction by pressing the DoIt button.

Topics

Overview | User interface | Recipe for use | Parameters

Parameters

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

IdatFile

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).

NX:NY:NV

The first two values are, respectively, the x and y dimension of the projections. The third value is the number of projections in the data stack.

Pick XYZ

Pressing the XY button will open a dialog to interactively select the region of interest (in x and y) for reconstruction. When the toggle labeled use flipped reconstruction is on, the selection is done from a previous reconstruction which has been flipped. The flipped reconstruction is assumed to have the same file name as IdatFile but with a .xyzw extension; you may change the file used by pressing the Change file names... button. When the toggle is off, the selection is done from the unaligned data in IdatFile. Pressing XZ button has a similar effect, but you use it to select the region of interest in x and z. The selection is done from a reconstruction (this is assumed to have the same file name as OrecFile for EWBP; the name of the file used may be changed by pressing the Change file names... button). It is sufficient to generate a single z slice of the reconstruction and use that for selection. To do that, turn on the setup: to find shiftZ toggle and press "Do It". Remember to turn off setup: to find shiftZ before doing the full reconstruction.

shiftXYZ

This switch is used to choose and to center the object structure of interest in the reconstruction volume. A good way to select the values for the shift parameters is to select the region of interest with the mouse (see the Pick topic). Actually, the x, y, and z shifts move the imaginary 3-D tilt axis of the projection data from its default location. This is achieved by transforming the alignment parameters before applying them to the unaligned data stack. The default 3D tilt axis goes through a point whose x and y coordinates are the center of the unaligned reference projection and whose z coordinate is the z coordinate of the center of mass of the reference markers used in alignment. The x and y shifts center the region of interest for reconstruction. These shifts are judged by the location of the region of interest relative to the center of an unaligned projection (the reference projection - normally a projection with a tilt angle close to zero degrees is used). The z shift centers the region of interest in the center of the reconstructed volume. The z shift is usually set by looking at the reconstruction of a single y slice; if the region of interest is low in the y slice, a negative z shift should be used.

dimenXYZ

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 and should be greater than the thickness of the object of interest.

Topics

Overview | User interface | Recipe for use | Parameters