KEYWORD Resolution ApplyMassnorm ReconstructionShift ReconstructionZSize TapirCyclesPerRun TapirSmoothing RestartRecon NumberOfRuns TemporaryDirectory InitialGuess CrossCorrelationImages GuessResolution StartingRunNumber AutomaticTermination ResolutionScale FullSize AlignedSize SectionRange Positivity CoerceInput Multires AlignmentMethod SimplexFilter Offset PhaseWeighting WienerCoefficient CenterPeakDistance 2ndPeakThreshold SimplexDeviations CommandLine DESCRIPTION EMTIAR is a front-end for the iterative alignment and reconstruction of EM tomography data. It differs from the other alignment and reconstruction front end, EMTAR, in that it can do multiple runs of reconstruction, reprojection, and alignment of the data to the reprojection in order to refine the alignment parameters and reconstruction; EMTAR does not refine the alignment parameters and therefore requires less time. The basic recipe for generating a reconstruction with EMTIAR is: 1) Use the mass-normalization and alignment components of EMTAR to generate the final values for the mass- normalization parameters and initial guess for the alignment parameters. If your data has fiducial markers, you can use BALIGN instead to generate the initial guess for the alignment parameters and then use the mass-normalization component of EMTAR to add the mass-normalization values. 2) Start EMTIAR. Enter the name of the tilt series in the "TiltSeries" field at the top of the dialog. Enter the name of the file with the mass- normalization parameters and initial guess at the alignment parameters in the "InAlignParam" field at the top of the dialog. 3) Adjust other parameters. Parameters that are commonly changed are: The resolution level to process If you've added multiple resolutions 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. The generated alignment parameters will always be in terms of the coordinates of the highest resolution. The generated reconstruction will be at the selected resolution level. Size-related input parameters are automatically scaled by the resolution factor so you should choose values for those that are appropriate for the highest resolution. Performing initial refinement at lower resolutions can significantly speed up the process because the reconstructions are smaller. Bounds of the reconstructed volume The bounds are controlled by the "Reconstruction shift" and "Reconstruction Z size" fields. The x and y dimensions of the reconstruction are constrained to be the same size as the x and y dimensions of the input tilt series. EMTAR has methods to interactively select the bounds; you can use them and then copy the values from EMTAR's "Shift (XYZ)" field to the "Reconstruction shift" field in EMTIAR and the third value in EMTAR's "Size XYZ" field to the "Reconstruction Z size" field in EMTIAR. Number of runs This is the number of times the steps of reconstruction, reprojection, and alignment to the reprojection are repeated. Parallel execution If you have a cluster of computers or a single machine with multiple processors, you can speed up the reconstruction step by having pieces of the reconstruction done by different processors. 4) Press the "Do It" button to start the alignment and reconstruction. Before pressing "Do It" you way want to alter the job submission settings by pressing the "Options" button at the bottom of the EMTIAR dialog. The job submission settings are described in BatchRegion.hlp. Besides a log file, the iterative alignment and reconstruction generates two outputs. The first is an alignment parameter file for each iteration. The names for these files are the contents of the "OutParamBase" field with an underscore, the iteration number, and the extension of the input alignment parameter file appended. The second output is the data for the reconstructed volume. The file name for the reconstructed volume is in the "Reconstruction" field. Resolution The resolution parameter selects which resolution to process from the input tilt series. 0 is the highest (full) resolution. Normally all that you need to do to process a different resolution is set the resolution parameter. There are other parameters in the special parameters dialog that affect the handling of different resolutions (guess resolution, resolution scale, and full size), but in typical cases the user interface chooses correct defaults for those parameters. ApplyMassnorm The default behavior for the iterative alignment and reconstruction has an initialization step where the mass-normalization parameters are used to generate a mass-normalized (but not aligned) version of the input tilt series. The toggle button next to "Apply massnorm" controls whether or not to use the initialization step. If you have already applied the mass-normalization parameters to the input tilt series, turn the toggle button off; otherwise, make sure it is on. Two controls affect the application of the mass- normalization parameters during the initialization step. The menu at the end of the "Apply massnorm" line allows you to select the image formation model. You have three choices: logarithmic The mass density is linearly related to the logarithm of the electron counts. scaled linear The mass density is linearly related to the electron counts. linear The electron counts are used without modification as a measure of the mass density. The other control is the pcBase field in the special parameters dialog. For the scaled linear or logarithmic image formation models, the value in that field controls the contribution of an additive term corresponding to a sheet of uniform mass density. The documentation for appl_prm, APPL_PRM.hlp, has more information about the pcBase parameter. ReconstructionShift These three values (they are in units of pixels) shift the imaginary 3D tilt axis of the projection data from its default location; they are used to center the reconstruction volume on an object of interest. The default orientation of the 3D tilt axis has it pass through the point whose x and y coordinates are the center of the reference projection. ReconstructionZSize This value sets the size of the z dimension, in pixels, for the reconstructed volume (the volume is written as slices parallel to the xz plane so this value sets the y dimension of a single slice). TapirCyclesPerRun EMTIAR uses tapir to calculate the reconstruction from the aligned data. The value in the "Tapir cycles/run" field is the number of iterations to tapir to perform for each run of the EMTIAR steps. TapirSmoothing EMTIAR uses tapir to calculate the reconstruction from the aligned data. The value in the "Tapir smoothing" field constrains how smooth the reconstruction should be. Consult the tapir documentation, TAPIR.hlp, for more details. RestartRecon If the "Restart recon" toggle button is on, the reconstructions in all iterations after the first do not start with an initial guess. If the "Restart recon" button is off, the reconstructions use the reconstruction result from the previous iteration as the initial guess. Use the "Initial guess" field to control whether or not the reconstruction during the first iteration uses an initial guess. By default, the "Restart recon" toggle button is on. That is different than the behavior of the iterative alignment and reconstruction in Priism 4.2.0, which always carried over the reconstruction between iterations. NumberOfRuns The value in the "Number of Runs" field is the number of iterations that should be performed to refine the alignment parameters and reconstruction. TemporaryDirectory The iterative alignment and reconstruction procedure will use the directory specified in the "Temporary directory" field for storage of the temporary results: the mass- normalized tilt series from the initialization step (a floating-point data set which has the same dimensions as the input tilt series), the aligned tilt series (a floating-point data set whose size is given by the "Aligned size" and "Section range" fields), the intermediate reconstruction (a floating-point data set whose size is given by the x and y dimensions of the input tilt series and the reconstruction z size), and the reprojection result (a data set which is the same size as the input tilt series). At any given time, the temporary files present will be the result of the initialization step, if any, and, at most, either an intermediate reconstruction and aligned tilt series, an intermediate reconstruction and reprojection result, or, if running in parallel mode, two copies of the intermediate reconstruction. If you are running in parallel mode with a list of machines, there may be additional files present if input files or outputs are not visible to all machines in the list. By default, the temporary directory is a directory named tmp in your home directory. If you are using the parallel execution mode, the temporary directory should be accessible under the same name to all the computers used. You might change the temporary directory if you were short on space in the default directory or the default directory was on a slow disk and you have a fast disk with enough space for the temporary files. InitialGuess To specify an initial guess for the reconstruction, enter the name of the file containing the initial guess into the "Initial guess" field or press the adjacent button to launch a file browser to help you select the file. The initial guess is optional, if you do not enter anything in the "Initial guess" field or use "none" as the name of the file, the reconstruction will proceed without an initial guess. If you do use an initial guess, its dimensions must match the dimensions of the reconstruction (i.e. have an x size equal to the x size of the input tilt series, a y size equal to the value in the "Reconstruction Z size" field, and a z size equal to the y size of the input tilt series). CrossCorrelationImages If you select an alignment method that includes Talign, the alignment calculations can generate images of the cross-correlation results, with one cross-correlation image per projection for the most recent iteration. If the field labeled "Cross correlations" is not empty and not set to "none", those cross-correlation images will be written to an MRC file with the name given in the field. GuessResolution The guess resolution parameter allows you to select which resolution to use from the file containing the initial guess. The dimensions of the resolution you select must be compatible with the dimensions you've specified for the reconstruction. StartingRunNumber The iterations of alignment and reconstruction will be numbered from the value in the "Starting run number" field to the value in the "Starting run number field" plus the number of runs minus one. That numbering only affects the names of the output alignment parameter files. If you had previously done n iterations and wanted to see the effect of performing m more with a record of the alignment parameters for all of the m plus n iterations, you would set EMTIAR so the input alignment parameter file was the output alignment parameter file from the nth iteration, the number of runs was m, the starting run number was n plus one, and the initial guess was the reconstruction generated by the previous set of iterations. AutomaticTermination When the "Automatic termination" toggle button is off, the iterative alignment and reconstruction will terminate when the number of runs you specified has been completed. When the "Automatic termination" toggle button is on, processing will terminate early if the R-factor between reprojections of the reconstruction and the measured tilt series increases. ResolutionScale The resolution scale parameter controls the scale factor applied to the alignment parameters and to the size-related input parameters (reconstruction shift, reconstruction z size, tapir smoothing, aligned size, and characteristic lengths for the x and y shifts). In the typical case where you select a resolution level to process from the main dialog, the resolution scale factor will be set appropriately. In the case where you are running the iterative alignment and reconstruction on a downsampled tilt series but the input alignment parameters are from a higher resolution version of the data set, you should set the resolution scale to be the same as the downsampling factor (if the data is scaled down by a factor of four relative to the coordinates used for the alignment parameters, set the resolution scale parameter to four) and also adjust the values for the dimensions of the higher resolution data set (the "Full size" field). FullSize These two values set the x and y dimensions, in pixels, for the tilt series corresponding to the alignment parameters in the input alignment parameter file. For most cases the user interface can correctly determine these values from the dimensions of the input tilt series. One case where you will have to manually change these values is if you use a downsampled tilt series as the input to the iterative alignment and reconstruction but the input alignment parameters correspond to a higher resolution version of the same tilt series. In that case, you should enter the dimensions, in pixels, of that higher resolution tilt series in the "Full size" field and also adjust the resolution scale. AlignedSize These two values are, respectively, the x and y dimensions, in pixels, of the aligned projections. SectionRange These three values set which of the input projections are included in the reconstruction. The first value is the index of the first projection to use, the second value is the index of the last projection to use, and the third is the index step. Allowable values for the indices are between zero and the number of input projections minus one, inclusive. Positivity When this toggle is on, the pixel values in the reconstruction are forced to be non-negative. This a priori constraint is a powerful way to improve the reconstruction so it isn't generally useful to turn off the positivity constraint. CoerceInput When this toggle is on, the values from the aligned projection data are forced to be non-negative (negative values are set to zero). This is almost always the right thing to do. Multires Turn on the "Add multires" toggle button to cause the iterative alignment and reconstruction to add lower resolutions to the reconstruction if the x and y dimensions of the reconstruction are large enough. Turn off the "Add multires" toggle button to generate a reconstruction with a single resolution. AlignmentMethod Sets the method to use for alignment. You have three choices: Talign + Simplex Use a FFT-based cross-correlation to determine the translations followed by a simplex optimization of all parameters. Talign Use a FFT-based cross-correlation to determine the translations. The other parameters are not modified. This is the default. Simplex Use a simplex optimization of all parameters. SimplexFilter If the "Simplex filter" toggle button is on and you have selected an alignment method that includes the simplex optimization, a high pass filter will be applied to the data sets prior to the simplex optimization. When the "Simplex filter" toggle button is off, a high pass filter is not applied prior to the simplex optimization. Offset If the "Offset" toggle button is on and you have selected an alignment method that includes Talign, a shift along the tilt axis will be induced with the intent of keeping the peak corresponding to the best shift parameters (that peak would likely be near the origin if the input alignment parameters are sufficiently good) away from the origin which receives special treatment. Before the alignment parameters are written out, the program compensates them for the induced shift. When the "Offset" toggle button is off, no shift along the tilt axis is induced when using one of the methods which include Talign. PhaseWeighting If the "Phase weighting" toggle button is on and you have selected an alignment method that includes Talign, phase weighting will be performed to enhance the cross-correlation peak at the expense of less robustness in the face of noise. When the "Phase weighting" toggle button is off, phase weighting is not used to enhance the cross-correlation peak. WienerCoefficient When you use phase weighting of the cross- correlations, there is a term like that in a Wiener filter to keep things well behaved when the amplitude of the cross-correlation value in the frequency domain approaches zero. The term is an estimate of the high-frequency power spectral density times the value in the user interface's "Wiener coefficient" field. A higher value for the "Wiener coefficient" increases the suppression of the low amplitude components. The default value is 100. CenterPeakDistance With the alignment methods that include Talign, the alignment algorithm will try to cancel out the effect of a peak at the origin by subtracting out a radial average over the central 7 x 7 region from that region. The alignment application only performs that subtraction if the largest value in the central 7 x 7 region occurs at the center and the quadratic fit to that point and its four nearest neighbors gives a peak position, (xp, yp), where both the absolute value of xp and yp are less than a threshold value. The threshold value is, by default, 0.05 pixels, You can change the threshold by modifying the value in the "Center peak distance" field. 2ndPeakThreshold With the alignment methods that include Talign, the algorithm prefers peaks in the cross-correlation that are away from the origin and only takes the peak at the origin if the next highest peak (after masking out the origin) has a height less than f times the height of the peak at the origin. The default value of f is .001. In the graphical user interface, alter the value in the "2nd peak threshold" field to alter the value of f. SimplexDeviations These six parameters set the characteristic length for each of the six alignment parameters that can be refined when aligning a section from the tilt series to a section from the reprojection. A characteristic length scale of zero for a parameter causes the simplex method to not refine that parameter; in the graphical user interface, you turn on or off the refinement of a parameter with the toggle button adjacent to the field which displays the characteristic length. The six alignment parameters are the x shift (in pixels), y shift (in pixels), rotation (in radians), isotropic magnification factor, the orientation angle for the anisotropic stretching axis (in radians), and the anisotropic stretching factor. The first four parameters have a direct effect on the output alignment parameters. The last two (the orientation of the anisotropic stretching direction and the anisotropic stretching factor) do not. CommandLine EMTIAR simply generates a script that runs the command-line application, emtiar. The command- line syntax for emtiar is (optional parts are enclosed in brackets): emtiar input_tilt_series input_align_param \ output_align_param_base output_recon \ [options] The options that emtiar understands are: -bkgopt=option Causes emtiar to use option with the command specified with -rshcmd= when that command is to be run in the background. By default, emtiar uses "-n" as the option. -cor_out=fname If fname is not empty and not equal to "none" and an alignment method including Talign is selected, images of the cross-correlation results for the most recent iteration will be written to an MRC file named fname. -cpd=d To reduce the effect of a central peak, the Talign algorithm will subtract off a radial average from the central 7 x 7 if the brightest point in the central 7 x 7 occurs at the center and a quadratic fit to the center and its four nearest neighbors gives a peak position, (xp, yp), where the absolute values of xp and yp are less than d. The default value of d is 0.05 pixels. -cycles=n Sets the number of cycles of tapir to use for every iteration to be n. By default, emtiar uses 20 cycles of tapir per iteration. -dev=xs:ys:rot:mag:axis:stretch Sets the characteristic length for each of the six alignment parameters that can be refined when aligning a section from the tilt series to a section from the reprojection. By default, emtiar uses a characteristic length of one pixel for the x and y shift, a characteristic length of zero radians for the rotation and orientation of the asymmetric stretching axis, and a characteristic length of zero for the magnification and asymmetric stretching factor. -dimxy=nx:ny Sets the x and y dimensions of the aligned projections, in pixels. By default, emtiar uses the same dimensions as the input tilt series. -fullsize=nx:ny Specifies the dimensions, in pixels, of the tilt series corresponding to the alignment parameters in the input alignment parameter file. If you do not set this option, emtiar will use the dimensions of the highest resolution in the input tilt series. A case where you would want to use values other than the defaults is when the input tilt series has been downsampled and the input alignment parameters are for a higher resolution of the tilt series. In that case you would also want to specify the -rscale option as well. -gres=i Selects the resolution i from the initial guess specified by -istrfile. i must be a non-negative integer less than the number of resolutions in that file. -imform=code Sets the image formation model used during the initialization step. Valid values for code are: 0 logarithmic (the default if the input parameter file contains mass-normalization parameters) 1 scaled linear 2 linear (the default if the input parameter file does not contain mass- normalization parameters) -imod=m Sets the method to use for alignment. m may be one of the values listed below. 0 Use Talign (a FFT-based cross-correlation) to determine the translations followed by a simplex optimization of all parameters. 1 Use Talign (a FFT-based cross-correlation) to determine the translations. The other parameters are not modified. This is the default. 2 Use a simplex optimization of all parameters. -istrfile=file Specifies that the initial guess for the reconstructed volume should be read from file. -iv=ivstart:ivend:ivstep Sets the range of input projections included in the reconstruction. By default, emtiar includes all the projections present in the input tilt series. -machinefile=file If you wish to use parallel execution on a cluster of machines, use -machinefile= to set the name of the file containing the list of processors to use. The format of the file is described in the documentation describing parallel execution. If you wish to use parallel execution on a machine with multiple processors, do not use -machinefile= or use it with an empty file name. -mult=s Sets the scale factor, s, for the noise component when performing phase weighting in an alignment method that uses Talign. The default value for the scale factor is one hundred. -multires Causes the generated reconstruction to have multiple resolutions if the x and y dimensions of the reconstruction are large enough. -no_nodecheck If a list of machines has been supplied with -machinefile=, disables the checks on whether all of the compute nodes can see the temporary directory and whether copies of input or output files have to be kept in the temporary directory for the compute nodes that would otherwise not be able to see those files. Only use this option if you know the temporary directory and other files are visible to the compute nodes and you want to avoid the extra overhead of the additional checks. -nofilter If specified, a highpass filter is not applied when you use simplex optimization in the alignment; otherwise, a highpass filter is applied when you use simplex optimization. -nooffset If specified, no offset along the tilt axis is applied when you use one of the alignment methods which include Talign; otherwise, an offset is applied along the tilt axis and then compensated for prior to writing out the alignment parameters. -noterm Disables early termination if the R-factor between reprojections of the reconstruction and the measured tilt series increases. -np=n Sets the maximum number of processors to use when parallel execution is enabled. If -machinefile= is used with a non-empty file name, emtiar will by default use all the processors listed in that file; otherwise, emtiar defaults to using two processors. -parallel Enables parallel execution. By default, emtiar does not attempt to use parallel execution. -pcbase=b Sets a factor that comes into play if the scaled linear or logarithmic image formation model is used during the initialization step. By default, emtiar assumes a value of 0.05. -phaseweight If specified, phase weighting is done when you use an alignment method that includes Talign; otherwise, phase weighting is not done. -positivity=i Turns on the positivity constraint for the reconstruction if i is any integer. By default, the positivity constraint is not enforced. -proj_pos=i For the reconstruction, causes any negative values in the input projections to be set to zero if i is any integer. By default, the reconstruction does not force the input projection data to be non-negative. -reconz=nz Sets the z dimension of the reconstruction, in pixels. By default, the z dimension is one fourth the size of the x dimension of the input tilt series. -res=i Selects i as the resolution to process from the input tilt series. i must be a non- negative integer less than the number of resolution in the tilt series. Other command- line options affecting the processing of different resolutions are -rscale, -fullsize, and -gres, but, in normal circumstances, it is sufficient to use -res by itself. If you do not specify this option, the highest resolution (zero) is processed. -restart_recon If specified, reconstructions for iterations other than the first do not start with an initial guess. -rscale=i Controls the scale factor (1.0 / i) applied to the alignment parameters and size-related input parameters to compensate for processing a lower resolution. i must be a positive integer. If you do not set this option, i defaults to be two raised to the power of the resolution level selected with -res. A case where you would want to use a value different than the default is when the input tilt series has been downsampled and the input alignment parameters are for a higher resolution of the tilt series. In that case you would also want to specify the -fullsize option as well. -rshcmd=cmd Sets the command used to start launch processes on remote machines in the parallel execution mode. By default, emtiar will use "ssh -x". -runs=n Sets the number of iterations of refinement to perform. By default, emtiar will perform a single iteration. -shxzy=xshift:yshift:zshift Specifies the shift, in pixels, of the reconstructed volume from the default position. By default, emtiar does not shift the reconstructed volume. -skip_init Disables the initialization step for computing a mass-normalized tilt series. -smooth=s Sets the factor constraining the smoothness of the reconstruction. By default, emtiar will use a value of zero. -spth=t In the Talign algorithm, cross-correlation peaks away from the origin are preferred. A peak at the origin is only accepted if the next highest peak (after masking out the origin) has a height less than t times the height of the peak at the origin. The default value for t is .001. -start=i Sets the number to associate with the first iteration. By default, emtiar will use a value of one. -tmpdir=directory Sets the directory used for temporary files. By default, emtiar places the temporary files in a directory named tmp within your home directory.