MASSNORM is the first step in EM reconstruction after the alignment stage. It calculates the relationship between observed electron counts and the specimen's mass density and corrects for electron dose fluctuations during data collection.
The algorithm is:
Here's an example command file for MASSNORM:
(time massnorm \
/mama/weiping/test/cent5r.stk \
-iprmfile=/mama/weiping/test/cent5r.bprm \
-oprmfile=/mama/weiping/test/cent5r.bprmMn \
-bckgfit=1 -dimxy=480:480 -trange=-60:60 \
-shxyz=0:0:0 -iref=-1 -sigma=5. -toffset=0 \
-nhist=400 -oplotfile=none -plot=1 ) \
> /mama/weiping/emrecon.log
IdatFile | NX:NY:NV | IprmFile | OprmFile | background fit | dimxy | tRange | shXYZ | iRef | sigma | tOffset | nHist | OplotFile | FindJumps
Priism | Reconstruction | APPL_PRM | EWBP | TAPIR | Alignment
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).
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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 is the name of the input alignment parameter file; the file can be generated with BALIGN.
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This is the name of the output file in which to store the alignment parameters. This file can be used as input to APPL_PRM and includes the information for converting electron counts to a mass density.
To specify the output parameter file from the command line use
-oprmfile=filename. none can be
used for the file name to signal that no output parameter file should
be generated; this is the default.
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Controls how the reference value for each projection is calculated. For the Fit: average option, the reference value is e^(average(log(projection i))). Use this option when there is no clear background in the projections. For the Fit: background option, the reference value is the data value where the peak in the histogram of the background is located. Use this option whenever there is a large area of background on all projections.
On the command line, use -bckgfit=1 to use the estimate from
the histogram of the background and use -bckgfit=0 for the
other method. If neither is supplied, the default is to use the histogram
of the background.
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Sets the x and y dimensions for the area used to estimate the reference average values. These dimensions are for a projection with a tilt of zero degrees; the area chosen on a projection with tilt t is ny * cos(t) by nx where nx and ny are the dimensions at a tilt of zero degrees.
On the command line, specify these dimensions with
-dimxy=nx:ny. If no dimensions
are specified, the dimensions used are the x and y dimensions of the
input projections.
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Sets the range of tilt angles included when fitting the reference average values. The first value is the minimum angle, in degrees, to use; the second is the maximum value.
On the command line specify the range of tilts with
-trange=min_angle:max_angle.
If the range is not specified, -60 degrees is used for the minimum tilt and
60 degrees is used for the maximum tilt.
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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; this can be used to choose a different area for computing reference values. 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 (see the iRef parameter) and whose z coordinate is the z coordinate of the center of mass of the reference markers used for alignment.
When using the Fit: background option for the bckgFit parameter, it is recommended to turn the toggle off next to the shift field (this forces shift values of zero to be used) since you want a large area of background to estimate the reference values. When the Fit: average option is used, you should turn the toggle on next to the shift field so the reference values are estimated from the same region that will be reconstructed.
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 shXYZ input parameter, the center of the projection whose index is given by iRef is used.
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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When fitting the background, if two adjacent projections have background intensities that differ by more than sigma times a local measure of the intensity variations, then the projections on either side of the unusual shift are fit separately. This is done to work around problems in data collection that frequently prevent maintaining the same beam intensity for all of the projections in a tomographic series.
To specify the threshold on the command line use,
-sigma=threshold. The value used for the threshold
if none is supplied is 5.
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If tOffset is not 90, it is the offset in degrees massnorm adds to the input tilt angles to get the tilt angles written to the OprmFile. If tOffset is 90, massnorm computes an offset from a fit to the intensities and adds that offset to the input tilt angles to get the tilt angles written to the OprmFile.
Use tOffset to correct for cases where the tilt angles in the extended header are offset from the true values. Normally, you would set tOffset to zero (i.e. the extended header values are known to be correct) or to 90.
To specify the tOffset on the command line, use
-toffset=offset. If you do not set tOffset via the
command line, massnorm determines the offset from the fit to the intensities.
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This is the number of bins used to calculate the histogram of the background when the Fit: background option is used for bckgFit.
The syntax for specifying this parameter on the command line is
-nhist=n. When it is not specified on the
command line, 400 bins are used.
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This is the name of the output file to generate with the projection reference values and the corresponding fitted values from the absorption model. You can view line plots of this data by loading the file into 2d_plot. This file is not used by any other part of the EM reconstruction package.
To specify the name of the plot file on the command line, use
-oplotfile=filename. If -oplotfile
is not supplied or filename is none, then no
output plot file is generated.
By default, the normalization process saves summary results for the
absorption model fit to filename.mnplt where filename is the base
file name of the input file. If you change the pulldown menu in the
Special Parameters dialog, from Plot: save only to
Plot: show and save, these results will be automatically displayed
with 2d_plot On the command line, use
-plot=0 to automatically display the fit results (this is
the default setting); otherwise, use -plot=1.
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Normally, the plot of the reference average value versus tilt angle will be
a smooth curve. For those cases where it is not smooth (typically because it
was necessary to collect the data in two or more stages and it was not
possible to restore the beam settings used in the first stage), turn on the
"Find jumps" toggle so that massnorm attempts to identify sharp jumps and
adjusts the curve fitting to account for them. On the cammand line, use
-find_jumps to cause massnorm to look for jumps.
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