With alignment parameters and the conversion factors for electron counts to mass density (both are loaded from the file pointed to by the input alignment parameter file), APPL_PRM creates a mass normalized and aligned data stack for reconstruction.
Here's an example command file for APPL_PRM:
(time appl_prm \
/mama/weiping/test/centSr.stk \
/mama/weiping/test/centSr.MnAln \
-iprmfile=/mama/weiping/test/centSr.bprmMn \
-dimxy=480:480 -iv=0:22:1 -shxyz=0:0:0 -iref=-1 \
-imform=0 -pcbase=.05 ) \
> /mama/weiping/emrecon.log
Tilt series | Aligned series | NX:NY:NV | Input alignment | Output XY | Section range | Output shift | Reference projection | Image formation | pcBase | Tilt offset | Statistics | Apply alignment | Resolution | Full size | Resolution scale | Add multires
Priism | Alignment and reconstruction | MASSNORM | EWBP | TAPIR | Alignment with markers
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). On the command line, the tilt series file name is the first argument.
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This parameter names the file that will contain the aligned and mass normalized data stack. On the command line, the file name for the aligned result is the second argument.
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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.
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This parameter supplies the name of the file with the alignment information and, optionally, conversion factors for mass normalization. Usually you would generate the file with MASSNORM. You could also directly use the files generated by cross-correlation alignment or alignment with markers.
On the command line, use -iprmfile=filename
to supply the name of the parameter file. If the option is not set or
the filename is not supplied or is none, the program will
terminate with an error.
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These two values are, respectively, the x and y dimensions of the aligned projections. Usually the full dimensions of the input data are used, but when doing a quick reconstruction to determine an appropriate value for the z shift, it is convenient to generate an aligned projection with a y dimension of one.
On the command line, set the dimensions of the output data stack with
-dimxy=nx:ny. If not set,
the output x and y dimensions are the same as the x and y dimensions of the
input data stack.
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These three values set which of the input projections are aligned and mass normalized. 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.
From the command line, use
-iv=first:last:step to set the range of projections to process.
When not set, the default is to process the entire input stack: first is
zero, last is the number of input projections minus one, and
step is one.
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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 and whose z coordinate is the z coordinate of the center of mass of the reference markers used for alignment.
A good way to determine appropriate values for the shifts is to use the XY and XZ buttons for region selection in the main alignment and reconstruction dialog.
Use -shxyz=x_shift:y_shift:z_shift
to set the shifts on the command line. If the shifts are not specified, a
value of zero pixels for each shift is assumed.
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For calculating the effect of the x and y shifts (see the output shift parameter), APPL_PRM uses the center of the reference projection. Specify the reference projection by its zero-based index in the file (i.e. a value between zero and the number of projections minus one) or use a value of negative one to select the projection whose tilt angle is closest to zero.
The reference projection can be set on the command line with
-iref=index. If it is not specified or
index is less than zero or greater than or equal to the
number of projections in the input file, the projection whose tilt
angle is closest to zero is used.
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This parameter sets which model should be assumed for the process of image formation - how electron counts are related to the mass density of the specimen. The three options are:
For the logarithmic and scaled linear models, the relationship between the ith input image, P(i,j,k) after interpolation for the alignment parameters and the ith output image (i.e. the mass-normalized values), P'''(i,j,k), is P'''(i,j,k) = minimum(h(i), P''(i,j,k) - a - (b-a)/cos(tilt(i))). For the logarithmic model, P''(i,j,k) is 10000 * log(s(i) / (P'(i,j,k) - s(i) * c0)). P'(i,j,k) is f(i) / r(i) * P(i,j,k) - b(i). For the scaled linear model, P''(i,j,k) is f(i) * s(i0) / (r(i) * s(i)) * P(i,j,k) - b(i). The other terms are:
On the command line, use -imform=0 to use the logarithmic
model, -imform=1 to use the scaled linear model,
or -imform=2 to use the linear model. If the
input alignment file does not contain
mass normalization data, APPL_PRM ignores the -imform options
and always assumes the linear model. If you do not supply a
-imform option and the
input alignment file has mass normalization
data, APPL_PRM uses the logarithmic model.
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With the scaled linear or logarithmic image formation models, the pcBase parameter affects the contribution of an additive term corresponding to a sheet of uniform mass density.
On the command line, set pcBase with
-pcbase=cutoff. When it is not set, appl_prm uses a
a value of 0.05.
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The value of this parameter, assumed to be in degrees, is added to
the tilt angles stored in the header of the output data stack and used
in the mass normalization calculation. On the command line you can supply
the tilt offset by specifying -tilt_offset=angle.
When no offset is set, a value of zero is assumed.
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If the image formation model is not linear, APPL_PRM can write out a text file with some intermediate results for each projection: the 1/cos(tilt_angle) factor and the intensity corresponding to the cutoff for the lower 5% of the histogram. To cause these results to be written, enter a name different than "none" in the "Statistics" field. As a shortcut, you can turn on the toggle button adjacent to the "Statistics" field to insert the base name for the input tilt series plus a .applprm_stat extension. From the command line, use
-statfile=name
to specify the file name for the intermediate results; if you do not use
a -statfile option, APPL_PRM will assume name was
"none" and will not write out the intermediate results.
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When the "Apply alignment" toggle button is on, APPL_PRM generates an
aligned data stack; when it is off, APPL_PRM only applies the conversion from
electron counts to mass density when generating the output data stack (if you
specify a change to the image size or shift the region of interest, however,
those will be applied as if there was no other alignment information
available). When run from the command line, generating an aligned data stack
is the default and to disable the alignment you must specify the option
-noalign.
If you want to disable the conversion from counts to mass density, use the linear image formation model.
The relationship between a coordinate, (x,y), in the unaligned projection k, to a coordinate, (x',y'), in the corresponding aligned projection is given by the linear transformation:
[ x ] [ cos(r(k)) -sin(r(k)) ] [ x'-0.5*(nx'-1) ] [ tx(k)+0.5*(nx-1) ]
[ ] = m(k) * [ ] [ ] + [ ]
[ y ] [ sin(r(k)) cos(r(k)) ] [ y'-0.5*(nx'-1) ] [ ty(k)+0.5*(ny-1) ]
m(k) is the magnification for the kth projection; the value is read from the third column of the parameter file. r(k) is the rotation angle for the kth projection; the value, in degrees, is read from the second column of the parameter file. tx(k) and ty(k) are the two components of the translation for the kth projection; the values for those are read from the fourth and fifth columns, respectively, of the parameter file. nx' and ny' are the dimensions, in pixels, for the aligned frame. nx and ny are the dimensions, in pixels, for the unaligned frame. The coordinates for the samples in the aligned image are integers drawn from the range, [0, nx'-1], for the first coordinate and the range, [0, ny'-1], for the second coordinate. The coordinates for the samples in the unaligned image are integers drawn from the range, [0, nx-1], for the first coordinate and the range, [0, ny-1], for the second coordinate.
When the alignment parameters are used to align a lower resolution version of the input data, appl_prm uses the linear transformation given above with values for the image dimensions and translations that have been corrected for the resolution change. If nxh, nyh, nxh', and nyh' are the dimensions of the unaligned and aligned images in the higher resolution, the dimensions of the images in the lower resolution are:
nx = truncate(nxh/s)
ny = truncate(nyh/s)
nx' = truncate(nxh'/s)
ny' = truncate(nyh'/s)
where s is the scaling factor between the two resolutions. The translations in the lower resolution are calculated from those, txh(k) and tyh(k), in the higher resolution as follows:
tx(k) = (txh(k) + 0.5*(nxh-1) - s*0.5*(nx-1)) / s ty(k) = (tyh(k) + 0.5*(nyh-1) - s*0.5*(ny-1)) / s
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The resolution parameter selects which resolution to process from the input tilt series. 0 is the highest (full) resolution. From the user interface, you would normally select the resolution from the main EMTAR dialog and allow the software to propagate the setting to the individual processing stages. You can also set the resolution to process specifically for the application of the alignment and mass-normalization parameters from the special parameters dialog for "Apply Parameters".
On the command line, use
-res=i
to apply of the alignment and mass-normalization parameters to the ith resolution. When run from the command line and no resolution is selected, the calculations will use the the highest resolution present in the input tilt series.
Two other parameters, the full size and resolution scale, affect the handling of resolution.
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Use the full size parameter to specify the dimensions, in pixels, of the full resolution tilt series corresponding to the input tilt series. In the typical case where you select a resolution level to process from the main dialog of EMTAR, the graphical interface will automatically fill in these values correctly. In the case where you are applying the alignment and mass-normalization parameters to a downsampled file but the input parameters are for the full resolution data set, you should adjust the full size parameters to be the dimensions of the full resolution data set.
On the command line, use
-fullsize=nx:ny
to specify the dimensions, in pixels, of the full resolution data set. When run from the command line without the -fullsize option, apply parameters will use the dimensions of the highest resolution present in the input tilt series for the full size.
Two other parameters, the resolution and resolution scale, affect the handling of resolution.
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The resolution scale parameter controls the scale factor applied to the alignment parameters and to the output size and shift. In the typical case where you select a resolution level to process from the main dialog of EMTAR, the graphical interface will automatically fill in the resolution scale correctly. In the case where you are applying the alignment and mass-normalization parameters to a downsampled file but the input alignment parameters are for the full resolution 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 set the resolution scale parameter to four).
On the command line, use
-rscale=i
to set the resolution scale to be i. When run from the command line without the -rscale option, apply parameters will use a resolution scale factor equal to two raised to the power of the resolution level selected with -res.
Two other parameters, the resolution and full size, affect the handling of resolution.
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Turn on the "Add multires" toggle button to cause apply parameters to add lower resolutions to the aligned and mass-normalized tilt series if the x and y dimensions of the tilt series are large enough. Turn off the "Add multires" toggle button to cause apply parameters to always generate a tilt series with a single resolution.
On the command line, include
-multires
in the command-line options for apply parameters to cause apply parameters to add lower resolutions to the tilt series if the x and y dimensions of the tilt series are large enough.
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