alignframes(1) General Commands Manual alignframes(1)
NAME
alignframes - Aligns and sums camera movie frames and stacks sums
SYNOPSIS
alignframes options
DESCRIPTION
Alignframes aligns frames from direct electron detectors and other cam-
eras that can output a set of frames from an acquisition. It can take
input from multiple frame files and produce a single stack that is
ready to use for tilt series processing. It implements two basic
alternatives for an initial alignment that can optionally be refined:
aligning each frame to a reference accumulated from previously aligned
frames; or solving for the shifts of individual images by fitting to
shifts measured between many pairs of frames. The latter approach has
several advantages and can be applied in various ways. The advantages
are:
1) It involves multiple measurements for every image and thus can
average over more information for aligning the first few frames than
the cumulative method can.
2) Robust regression is used for the fitting, so a small proportion
of bad alignments can be tolerated and should not degrade the result.
Thus, this method should be more resistant to occasional failed corre-
lations due to fixed pattern noise.
3) The fitting yields an estimate of the residual error for each mea-
surement, from which it is possible to derive an error measure that
reflects the overall quality of the alignment and that can be used to
compare results with different parameters.
The fitting to shifts between pairs of frames is done for successive
sets of frames, 7 by default. With that setting, each frame is aligned
to all preceding frames for the first 7 frames, then the first fit is
done and a best shift determined for the first frame. The 8th frame is
then aligned to frames 2 through 7, and the next fit yields a shift for
the second frame. It is possible to align every frame to every other
one and do one fit to find all of the shifts at once, but this approach
does not seem to give any advantage in the typical case, and the number
of correlations can become quite large because it is proportional to
the square of the number of frames. When pairwise fits with sets of
more than 7 frames are indicated, another alternative strategy is to do
pairwise fits with sets of half the frames (see below).
The program allows one to test the quality of the fits with different
high frequency filter cutoffs, and even with different binnings. Mul-
tiple filters can be tested quickly in one run through the data, but
testing multiple binnings require multiple runs through the data.
After the initial alignment, it is possible to realign each frame to a
reference consisting of all other aligned frames. (For high-noise
data, it is essential to leave the frame being aligned out of the ref-
erence, or it would dominate the alignment.) This refinement can help
with after alignment to a cumulative reference, but has seemed super-
fluous in most tests with initial alignments from pairwise shifts.
All frame data are maintained as Fourier transforms, so each additional
alignment only involves one inverse FFT. Frames are shifted into
alignment in Fourier space to avoid losses from interpolation, and they
are reduced in size for the final sum (if at all) by cropping in
Fourier space to avoid aliasing.
If you want to compute a Fourier ring correlation, enter either the
-lines option with a value of 3 or the -frc option with a filename.
Fourier transforms of even and odd frames are summed separately and a
Fourier ring correlation is computed. The program reports the frequen-
cies at which the FRC crosses 0.5 and 0.25 in cycles/pixel of the
summed images, and also reports the mean value around a frequency of
0.25/pixel (half-Nyquist). The FRC is the only tool for comparing
results with the cumulative alignment to those with pairwise alignment
or for assessing the change from refining a pairwise alignment at the
end. The FRC can also be used for validating the choice of filter or
binning suggested by trying multiple values. However, changes in the
FRC may generally be quite small, so it will usually be helpful to
assess a change a parameters with a number of frame stacks. The pro-
gram Subtractcurves can help in this assessment as described below.
Quite strong high-frequency filtering is needed for typical frame
alignments. The filter cutoff is entered in frequency units of the
unbinned data so that a particular value has about the same effect at
different binnings. The default filter (0.06/pixel) is close to what
is typically needed, but smaller values (down to 0.05) or possibly
larger values (up to ~0.1) may give better results. Binning (actually
antialiased reduction in size) accomplishes most of the removal of
high-frequency noise prior to the application of the frequency filter,
so these two operations are partly redundant. Most of the motivation
for binning is to speed up the alignment; however, after a certain
point, additional binning will somewhat reduce the accuracy with which
the correlation peak position can be measured. Thus, the program
facilitates testing with different binnings, although such testing is
probably only needed when getting started with a particular class of
data, whereas testing with multiple filters is likely to be used more
routinely.
Alignment Strategies
The big challenges in aligning frames are the low signal-to-noise ratio
and interference from fixed pattern noise. There are some significant
distinctions between tilt series and single particle data when consid-
ering what methods to apply. First, tilt series may have a lower dose
per frame, but they are likely to have more features in the image than
some single-particle images, and thus more signal to align with. Sec-
ond, there can be beam damage such as doming of the ice within a series
of single-particle frames, which would never happen for a set a tilt
series frames.
One can think of a series of possible strategies for dealing with
increasingly difficult data:
1) When there is strong signal and no appreciable fixed pattern
noise, the simple method of aligning to a cumulative reference of
already aligned frames may be adequate. Here, refinement at the end
may help if it improves the shifts for the first few frames, which were
subject to the noisiest correlations. 2) Images with reasonable SNR
and little fixed pattern noise should work with the default method of
aligning all pairs among successive sets of 7 frames. 3) If the noise
is higher or signal lower, or if there is serious fixed pattern noise,
then pairwise alignment among much larger sets of frames will be
needed.
3A) For tilt series data, using all pairs of frames would generally
be appropriate.
3B) For single particle data, it is better to do pairwise alignment
among half the total number of frames, to avoid correlating across
large changes in the specimen. However, if fixed pattern noise is a
problem, it may be necessary to use all pairs instead, so that some
correlations with large shifts will be available for all frames.
(Fixed pattern noise makes the correlations unreliable or inaccurate
for small shifts; even when the filtered peak at the origin is smaller
than the true correlation peak, it can displace the peak position if
the two peaks overlap.)
3C) Refinement at the end is risky if there is serious fixed pattern
noise.
4) If the noise is just too high for alignments between single
frames to work, then grouping can be used. A group size of 3 can help
considerably. Refinements can be done at the end by correlating either
single frames or, if necessary, just the grouped frames with sums of
other frames.
Text Output with Single Parameter Settings
In all cases, output for a file starts with a line like
File 1 (Feb21_10.12.15.mrc): 11 frames
When there is only one filter and binning being used, the following two
lines present summary statistics for the results of the doing robust
fitting to the shifts for each set of pairwise alignments. All of the
values and distances are in unbinned pixels. The first line has these
items:
Weighted residual mean: The mean residual error value, averaged over
all of the fits. This is a weighted error, so aberrant shifts that are
completely down-weighted are not reflected here.
SD: The standard deviation of those weighted mean errors.
mean max: The mean value of the maximum weighted residual, averaged
over all the fits
max max: The maximum weighted residual seen in any of the fits
The second line has these items:
Max unweighted resid mean: The average of the maximum unweighted
residual values seen in the fits
max: The maximum unweighted residual seen in any fit
Dist: Raw sum of distances from one frame position to the next
smoothed: Sum of smoothed distances. If spline smoothing is used,
this is the distance for the smoothed shifts that are used to sum the
images. Otherwise, this is based on a local polynomial smoothing that
is not very good.
Finally, there is a summary of the output from Fourier ring correla-
tion. This line reports the frequencies (in cycles/pixel) at which the
curve crosses below 0.5, 0.25, and 0.125. The last number on the line
is the mean value of the curve around 0.25/pixel (half-Nyquist). If
the sum is reduced in size, these frequencies are in terms of the
binned pixels.
The most important value on the first line is the weighted residual
mean. After each fit, the shift between each pair of images is com-
puted from the solved shifts of the individual images, and the residual
for that shift is the difference between the computed and measured val-
ues, multiplied by the weighting factor applied to the measurement.
The mean of these errors gives an indication of how accurate the shifts
should be, on average. Good values are in the range of 0.05 to ~0.3,
but some sets may give values in the range of 1-3. The latter cases
are a sign that one should try analyzing a higher number of pairwise
shifts or even try grouping. Using more pairwise shifts will not
improve the mean residual but will allow greater averaging over these
random errors; grouping should improve the residual.
The maximum residual values on the first line should not be many times
larger than the mean; these values indicate that there may be a shift
in error by that amount.
The Max unweighted resid mean on the second line reflects how often
there are bad shifts measured between pairs of images. With no bad
shifts, it should be not very much bigger than the maximum weighted
residual; values above a few pixels may indicate that there are bad
correlations in most fits. Reducing the filter cutoff, reducing the
maximum allowed shift, and grouping may improve this value; increasing
the number pairs being fit will increase the ability of the program to
reject the bad shifts as outliers. (As long as the weighted maximum
residuals are not high, the program is already able to reject the bad
shifts.)
The distance values on the second line reflect the total specimen move-
ment, and comparison between raw and smoothed distances gives some
indication of how jittery the solved shifts are. They become more
important when trying different filter settings, as explained below.
Text Output with Tests of Binning and/or Filters
If you specify either multiple filters with the -vary option or multi-
ple binnings with the -test option, there will be output similar to
what was just described for each condition being tested. An initial
line for each condition shows the binning, the filter cutoff value, and
the sigma for the filter falloff. The latter is varied in proportion
to the cutoff value.
When there are multiple filters, the program will compose a "hybrid"
solution that is based on the filter that gives the lowest residual
error after each fit to a set of pairwise shifts. The set of results
from this hybrid solution appear after the ones for the various fil-
ters, with an initial line showing Hybrid results, bin =. This solu-
tion is not used by default, only if the -hybrid option is given.
After all of the results, there will be a line indicating which condi-
tion is considered best, such as:
File 1: Best at bin = 8 rad2 = 0.060 sig2 = 0.0086 mean res = 0.121
However, the selection of a best solution must be treated with caution.
If fixed pattern noise is significant, the fit may improve dramatically
with high filter cutoffs. One sign that fixed pattern noise could be
taking over is a substantial decline in the distance travelled with
higher filter settings.
If the program is run on more than one file, then at the end there will
be a report of the number of times each combination of binning and fil-
ter cutoff gave the best solution. For example:
Number of times each condition is best (rad2 in parentheses):
bin = 6 3 (0.050) 1 (0.060) 0 (0.080)
bin = 8 3 (0.050) 1 (0.060) 1 (0.080)
This indicates that a cutoff of 0.05 is generally better than 0.06, and
that there is not much difference between binnings 6 and 8.
Not all combinations of binning and filter cutoff are meaningful. Fil-
ter cutoffs that are at or above the Nyquist frequency for a particular
binning will have little or no effect. Here are the Nyquist frequen-
cies for common binnings:
Binning Nyquist frequency
2 0.25
3 0.167
4 0.125
6 0.083
8 0.062
Support for Frame Files with Extended Header Data
If the frame files have an extended header (as files saved by UCSFtomo
do), the program will look for several features:
1) If the header is large enough to contain a gain reference, then
these data will be extracted and used to gain-normalize the frames,
unless a different gain reference is supplied with the -gain option.
The reference will be assumed to be in the correct orientation to apply
to the frames; but if it is not, the -rotation option can be used to
reorient it.
2) If the header appears to contain valid tilt angles, the program
will use these values to break the frames into separate sets for align-
ing and summing, unless the -break option is given. It will recognize
one place where there are two sets at the same angle and make two sums
there, provided that there are at least 5 sets of frames of the same
size prior to that place. This, if you use -frame to try aligning a
subset of tilt angles right around the starting point of a tilt series,
you may have to use -break to prevent these two images from being com-
bined. The tilt angles will be placed into the extended header of the
output file unless provided by another source (the -tilt or -stack
options).
3) If the extended header contains valid entries for pixel size and
tilt axis rotation angle, the pixel size will be placed into the stan-
dard header location and a title will be added with the rotation angle.
Note that although multiple files can still be entered when there is an
extended header, the program will insist that their properties match.
Dose Weight Filtering
The program can do dose weight-filtering of frames before summing for
sets of frames from either tilt series or single-particle acquisition.
The filter is as described in Grant and Grigorieff, 2015
(DOI:10.7554/eLife.06980) and the implementation follows that in their
"unblur" program. At any frequency, the filter follows an exponential
decay with dose, where the exponential is of the dose divided by 2
times a "critical dose" for that frequency. This critical dose was
empirically found to be approximated by a * k^b + c, where k is fre-
quency; the values of a, b, c in that paper are used by default but can
be modified with the -critical option. This filter function is applied
for all frequencies (complete attenuation above the "optimal dose" is
no longer considered appropriate). For each set of frames, the program
will apply a weighting that changes through the set of frames as dose
accumlates. If just an overall dose for the image is specified, this
dose is divided equally among the frames. However, if frames are not
of equal duration, it is possible to specify the different doses of the
frames. For a tilt series, the weighting also changes through the
frame sets according to the amount of dose prior to each set of frames.
A variety of options are described below for providing dose information
to the program. The ".mdoc" file from SerialEM can be a convenient
source of such information, particularly if frames are not of equal
duration. For tilt series, it provides cumulative dose information.
If an ".mdoc" is supplied for single particle data with the -dfile
option, it can contain entries for many images; the program will pick
out the dose information that applies to the frame sets being aligned.
Computer and GPU Memory Usage
The amount of computer memory required for this processing depends
mostly on the size of the images and whether all of the frames will be
held in memory until all alignments are completed. Each frame held in
memory requires 4 times as many bytes as pixels. Frames will be held
in memory if only one binning is used and any of the following options
are used: multiple filters (unless the -hybrid option is used to allow
the final setting of one shift after each pairwise fit), pairwise
alignment among all frames, refinement after initial alignment, or
smoothing of shifts (which occurs by default with 15 or more frames).
Otherwise, the number of frames held until the end will equal the num-
ber of frames used for pairwise fits (plus the group size minus one, if
grouping). However, when more than one binning is tested, the program
will not hold any frames but instead read them in again on a second
pass.
The amount of memory needed for the binned images being aligned can
also become large if the binning is small. These images are all held
until the end if there is refinement of the initial alignment; other-
wise the number retained will be the number used for pairwise fits.
When using a GPU, the size of unbinned images dominates the usage, but
space is needed for only 3 or 4 image-sized arrays. This can approach
1 GB for ~8K images. The frames are actually held in computer memory
until their shifts are finalized. Binned images for alignment will
also consume significant amounts of the GPU memory; these images stay
on the GPU instead of in computer memory, and the requirements there
are the same as they would be in computer memory. If binning is only
to ~2Kx2K and there are 60 frames, with refinement at the end, then 1
GB would be needed; fewer frames or more binning (both typical) would
result in half this requirement for the aligned frames. Thus, 2GB of
memory should suffice for typical usage, but 4 GB would handle almost
any anticipated need.
OPTIONS
Alignframes uses the PIP package for input (see the manual page for
pip). Options can be specified either as command line arguments
(with the -) or one per line in a command file (without the -).
Options can be abbreviated to unique letters; the currently valid
abbreviations for short names are shown in parentheses.
INPUT CONTROL OPTIONS
These options specify the input to the program or are related to
input options
-input (-in) OR -InputFile File name
Input file with images to correlate. Non-option arguments will
also be used for input files, with those entries used after any
names entered with this option. If -foutput is entered, all
non-option arguments will be used for input files; otherwise all
but the last will be. Input files need not be entered if an
".mdoc" file is entered with the names of the frame files.
(Successive entries accumulate)
-output (-ou) OR -OutputImageFile File name
If this option is not entered, the last non-option argument will
be used for this output file. An output file is required unless
-nosum is entered.
-list (-lis) OR -ListOfInputFiles File name
Name of file with list of input files, one per line. Filenames
entered this way are equivalent to ones entered with -input or
as non-option arguments; the latter two entries cannot be used
along with a file list.
-break (-br) OR -BreakFramesIntoSets Integer
If the input consists of a series of single-frame files, this
option must be used to combine them into one or more sets of
frames to be aligned and summed. Additionally, the option can
be used to break a file with many frames into multiple sets of
frames, each of which will be aligned and summed. The input
frames (either the whole collection of single-frame files, or
the frames in one multi-frame input file) will be divided into
groups of the given size, with any extra frames distributed
among the initial groups. For example, for 50 single-image
files or one file with 50 frames, and an entry of 8, there will
be 6 summed images, with 9 frames in the first two and 8 frames
in the rest. There must be at least as many single-image files,
or as many frames in each input file, as the number given. For
single-image files, this option cannot be entered with the
-frame or -assess options. For multi-frame files, the option
cannot be entered with -assess and will work with -frame. In
either case, the option should work with -stack or -mdoc unless
frame filenames are being taken from the mdoc file. Frame files
with tilt angles in the extended header will automatically be
broken into sets by tilt angle, so this option is not needed in
that case.
-saved (-sa) OR -SavedFrameListFile File name
Name of file with list of frames saved from a fast-incremental
single exposure tilt series where blanked frames were not saved.
There must be only a single frame input file and it must have
the same number of frames as lines in this list file. Every set
of contiguous values is assumed to be from one tilt angle.
There are several changes in IMOD 4.10.36: 1) A negative entry
means that the frame should be skipped; this should occur only
at the start or end of a frame set. 2) The frame numbers need
not increase within a frame set, so the list could use the same
number for all frames in a set. 3) The frames numbers need not
be sequential within a frame set; there can be gaps as large as
the value entered with the -gap option, and the program will not
start a new frame set at such a gap. 4) The program will auto-
matically eliminate the first or last frame if there is a large
enough gap between it and the adjacent frame and the average
size of frame sets is at least 4, and if there are no negative
values in the file. SerialEM has to write these frames, and as
of 3.8beta7 it will mark them with negative frame numbers, but
this automatic detection should work with older frame lists. If
data are lost from entire tilt angles because they fell below
the threshold for saving, Alignframes can also use relative
starting and ending frame numbers from a tilt angle file or an
".mdoc" file to sort out which angles were lost (this informa-
tion is output by SerialEM as of 12/16/19). This entry cannot
be used with -frame, -assess, or -break.
-gap OR -MaxGapWithinFrameSet Integer
Maximum size of a gap in sequential frame numbers allowed before
starting a new frame set, when using a saved frame list. The
value is thus the number of frames that might have been lost
within a frame set because the threshold was set too high during
acquisition. The default is 0.
-skip (-sk) OR -SkipFileChecks
Skip initial check that all input files have the same size and
data mode; this check can take significant time with many non-
MRC single-frame files. This option is allowed only with sin-
gle-frame input files.
-stack (-sta) OR -CorrespondingStack File name
Name of image stack of sums corresponding to the input files,
such as a tilt series where each image is a sum of unaligned
frames. This file will be used for the basic header information
of the output file, thus preserving titles and extended header
data.
-mdoc (-md) OR -MetadataFile File name
Name of a metadata autodoc (mdoc) file with a section for each
input file to be aligned and stacked. This file is an alterna-
tive way to get basic header information for the output file, as
well as tilt angles into the extended header. In addition, if
there are no input filenames entered as arguments, input file-
names will be obtained from all of the sections in the ".mdoc"
file with "SubFramePath" entries. However, the paths in those
entries are ignored; the frame files must all be in the current
directory unless the -path option is entered with an alternative
path. This capability is useful for bidirectional tilt series
or if a Record image was acquired more than once at a tilt
angle, since only the frame file for the last Record image will
be used. If input filenames are entered as arguments, there
must be at least as many sections in the ".mdoc" file as input
files if tilt angles are to be obtained from the ".mdoc" file
(i.e., if -tilt is not entered). The exception to this is when
a saved frame list file is entered with the single input frame
stack from a tilt series. In this case, the ".mdoc" file from
SerialEM is a frame stack mdoc file with a "FrameSet" section
followed by a brief section for each tilt, with the angle,
expected relative starting and ending frames, and dose informa-
tion.
-path (-pat) OR -PathToFramesInMdoc Text string
Current path to the frame files listed in an ".mdoc" file, when
these are being used as the input filenames. If this option is
not entered, the program must be run in the directory where the
frames are located to access files listed in an ".mdoc" file.
-ignore (-ig) OR -IgnoreZvaluesInMdoc
Take sections in order from the ".mdoc" file instead of by Z
value. With this option, ".mdoc" file sections can be removed
or rearranged to control which frame files are stacked. Other-
wise, sections must exist for all Z values being accessed,
starting at 0.
-adjust (-ad) OR -AdjustAndWriteMdoc
Correct entries in the input ".mdoc" file for changes in image
size, binning, pixel size, or data mode, and write a new file
with the name of the output file plus ".mdoc". This option has
no effect unless an ".mdoc" is entered. If tilt series data are
processed in order by tilt angle, the new file will be written
in the new order and it can be used later for dose-weighting in
Mtffilter.
-reorder (-reo) OR -ReorderByTiltAngle Integer
Process sets of frames in order by increasing or decreasing tilt
angle. Enter 0 for no reordering, 1 to reorder by from negative
to positive unless the angles already decrease montonically, 2
to reorder always from negative to positive, -1 to reorder from
positive to negative unless angles already increase monotoni-
cally, or -2 to reorder always from positive to negative. The
default is 1 unless -ignore is entered, in which case it is 0
and this option cannot be entered.
-pixel (-pi) OR -PixelSize Floating point
Pixel size in nanometers. A pixel size is needed for dose
weighting. This entry is needed only if the pixel size in the
image file header or files entered with -stack or -mdoc is
incorrect; it overrides any other source of pixel size.
-eer (-e) OR -EERSuperResZSumPadding Three integers
Amount of super-resolution to retain (0 for none, 1 for 2x, or 2
for 4x), summing in successive groups of frames, and amount of
"padding" for row/column defects when reading from an EER file.
The super-resolution reduction and the Z summing both occur in
the TIFF reading module and these two entries determine what
size the EER file appears to be to Alignframes, both the size in
X and Y and the number of frames. All pixel-related entries and
outputs from Alignframes are in terms of the pixels returned to
the program from that module. When the summing value does not
evenly divide the total number of frames, the specified frame
summing is the maximum summing that will occur, and the frames
will be distributed as evenly as possible, with the summing
lower by 1 at the beginning of the stack. When a gain reference
is provided with a defect list included, the number of addi-
tional physical pixels specified by the third entry will be cor-
rected for extreme super-resolution bias by averaging over the
super-resolution pixels in a physical pixel in the direction
perpendicular to the defect. If you need to adjust the defect
list more than that, you will have to run Clip with the -ed
option to output the defect list in SerialEM format, modify
that, and enter it here with the -defect option. The defaults
are 1 for super-resolution, 10 for summing, and padding by 1
pixel.
-super (-su) OR -SuperGainFactorFile File name
File with factors for adjusting the gain reference for an EER
file to correct for bias among the super-resolution pixels
within each physical pixel. These factors can be calculated
from all the pixels in one or more EER files using the "clip
supergain" command, which produces the file to be input here.
The adjustment is quite minor when reading with 2x2 super-reso-
lution, where the factors range up to ~1.7%, but may be more
significant with 4x4 super-resolution, where the factors range
up to ~7%. See the FILES section of the Clip man page for
the format of this file.
OUTPUT CONTROL OPTIONS
These options specify the output of the program or are related to
output options
-binning (-bin) OR -AlignAndSumBinning Two integers
Image reductions to apply when aligning and when summing. The
default for summing is 1, and the default for aligning is chosen
by seeing which binning out of 2, 3, 4, 6, or 8 brings the size
being correlated closest to 1250, or to 1560 for frames in a
size range that could come from a K3 camera. Enter a negative
number for the alignment binning to use the default instead of
having to specify it. If -test is entered with one or more bin-
nings, an entry for alignment binning is ignored.
-target (-tar) OR -TargetAlignSize Integer
Use a reduction factor that brings images for alignment close to
the given size. The default is 1250 pixels, or 1560 pixels for
frames recognized as from a K3 camera.
-frames (-fra) OR -StartingEndingFrames Two integers
First and last frame in each file to align and sum, numbered
from 1. The default is to do all the frames. The starting
frame number must be no bigger than the smallest number of
frames in any file.
-partial (-par) OR -PartialFrameThresholds Two floats
Relative thresholds for skipping one frame at the start and the
end of a frame set, when using a saved frame list. The values
must be less than 1 and greater than 0. The mean of the middle
frame of the set is taken as a reference; the first frame is
skipped if its mean is less than the first threshold times the
reference; the last frame is skipped if its mean is less than
the second threshold times the reference. A higher threshold
might be appropriate for the first frame if there tends to be
excessive drift then. The only reason to skip a partial frame
at the end of a set is to avoid including a frame with insuffi-
cient signal-to-noise ratio. There is no test for statistical
significance, so do not set the threshold so high (e.g., 0.99)
that you risk dropping frames just due to random variations in
the means.
-drift (-dr) OR -DriftLimitDistAndNumber Two floats
Maximum shift between frames above which to drop initial frames
of the set, and either the maximum fraction or the maximum num-
ber of frames to drop. The shift is in unbinned pixels. If a
maximum fraction is entered, it must be between 0 and 0.5. With
this option, the program will do the alignment, then if initial
shifts are above threshold, it will redo the alignment with
those frames dropped.
-sets (-se) OR -RangeOfSetsToDo Two integers
First and last frame set or file to process, numbered from 1.
The default is to process all input data. The file or set num-
bers correspond to the numbers shown in the text output and
apply after reordering by tilt angle, if any. This option is
here for convenience in testing and assessment, and it may not
work in all cases. The correct tilt angles should be placed in
the the file header, but an adjusted ".mdoc" file will not be
correct.
-mode (-mo) OR -ModeToOutput Integer
Mode for output image file: 0 for bytes, 1 or 6 for signed or
unsigned integers, or 2 for floating point. The default is to
use the mode of the input file unless is it 0, in which case the
default is to use mode 1.
-scale (-sc) OR -ScalingOfSum Floating point
Amount to scale summed values before output. The default is no
scaling; however, note that reduction of the output size will
scale the data up by the square of the reduction factor. Such
scaling mimics the summing of counts by binning during data
acquisition.
-total (-to) OR -TotalScalingOfData Floating point
Search the titles of the first input file for a scaling factor,
and apply an additional scaling to the summed values to bring
the total scaling to the amount entered. If no scaling is found
in a title, it is assumed to be 1 and the full scaling specified
here will be applied. A default total scaling of 30 or 32 will
be applied if the input data consists of bytes or 4-bit values,
gain normalization is being applied, and the output mode is not
set to 2. A default of 32 is used if the gain reference is
clearly from a K3 camera.
-meansd (-mea) OR -MeanAndSDtoScaleTo Two floats
Scale each summed image to the given mean and standard deviation
if the given SD is greater than 0, or just scale to the given
mean if SD is 0 or less.
-rfsum (-rf) OR -SumRotationAndFlip Integer
Rotation and flip operation to apply to sum before output.
Enter a number from 0 to 7, chosen by taking the rotation angle
counterclockwise divided by 90, plus 4 for a flip around the Y
axis before the rotation. (This corresponds to the RotationAnd-
Flip property used in SerialEM for several kinds of cameras.)
-tilt (-til) OR -TiltAngleFile File name
File with tilt angles to insert into the header of the output
file. The file should have one tilt angle per line, and must
have at least as many angles as frame files or sets being
stacked. Tilt angles will be placed into the extended header in
the UCSF/FEI format, one floating point value per section. With
this entry, tilt angles will not be used from a corresponding
stack or mdoc file. If this entry is used with a list of saved
frames and a single input frame stack, each line of the file can
also have the expected relative starting and ending frame num-
bers for that tilt following the tilt angle.
-axis (-ax) OR -AxisRotationAngle Floating point
Rotation angle from the tilt axis to the Y axis, counterclock-
wise positive. This angle will be placed into a title in the
output file readable by Etomo. It will override an angle from
an mdoc file or from a corresponding tilt series.
-xfext (-x) OR -TransformExtension Text string
Extension for output file(s) with image transformations having
shifts in columns 5 and 6. One file will be produced for each
input file, with the input file extension replaced by the given
extension. These files have the absolute shifts being applied
to each frame, in unbinned pixels, not relative shifts between
successive frames.
-frc OR -FRCOutputFile File name
Output file for Fourier ring correlations between sums of even
and odd frames, which are computed when a sum is produced. The
file will have a series of lines, each with the file number, the
frequency at the center of ring, and the correlation coeffi-
cient. When a GPU is used, the program may not compute the FRC
if there is only enough memory to sum into one buffer on the GPU
instead of two.
-ring (-ri) OR -RingSpacingForFRC Floating point
Spacing between the rings of the Fourier ring correlation, in
cycles/pixel of the summed images. The default is 0.005, which
is needed for resolving closely spaced CTF oscillations.
Smaller values like 0.02 - 0.05 will provide more averaging for
situations with more widely spaced oscillations. See the sec-
tion below, Evaluating and Visualizing Differences with FRC
Curves.
-lines (-lin) OR -LinesOfAlignSummary Integer
Number of lines summarizing the fitting and FRC results. Set to
3 for full output, 2 to eliminate the FRC line, or 1 for a con-
densed output with the weighted mean residual, maximum of maxi-
mum weigted residual, and the raw and smoothed distances. When
using the -saved option to enter a saved frame list file, the
output will also include the shifts of the first four frames;
enter a negative value to suppress this output.
-plottable (-pl) OR -PlottableShiftFile File name
Filename for output file with raw and smoothed shifts in
unbinned pixels. The smoothed shifts will be from spline
smoothing if it was done, otherwise from local polynomial
smoothing. The shifts will be put into the file one per line,
starting with a type number of 10 times the file number for the
raw shifts or that value plus 1 for the smoothed shifts (e.g.,
10 and 11 for the first file).
-nosum (-nos) OR -NoSumsOutput
Do alignments without making a summed image; no output filename
should be entered.
PREPROCESSING OPTIONS
These options provide for initial processing of the data before
aligning
-titles (-tit) OR -RefAndDefectFromTitles
Normalize the data using a gain reference and possible defect
file listed in the header titles of the first frame file. The
title with the reference name must contain "ref" or "Ref" and
end in ".mrc", ".dm4", or ".tif". The title with the defect
name must contain "defect" and end in ".txt". Each file must
exist in the directory with the frame file. This entry also
causes the rotation and flip operation found in a title to be
applied, so -rotation -1 need not be entered. An entry with the
-gain, -defect, or -rotation option overrides the respective
information found in a title.
-gain (-gai) OR -GainReferenceFile File name
Gain reference for normalizing unprocessed or dark-subtracted
frames. The gain reference should be a floating point file with
a mean of 1. If this option is entered, it supercedes a gain
reference found in the extended header of the frame files. If
the gain reference is a TIFF file or the frame input is an EER
file, then the gain is allowed to be exactly 2 or 4 times
smaller than the frames. In that case, the gain reference is
expanded by replicating the value for a pixel to all the super-
resolution pixels within it.
-rotation (-ro) OR -RotationAndFlip Integer
Rotation and flip operation that needs to be applied to the gain
reference to match the orientation of the frames being cor-
rected. Enter a number from 0 to 7 by taking the rotation angle
counterclockwise divided by 90, plus 4 for a flip around the Y
axis before the rotation. (This corresponds to the RotationAnd-
Flip property used in SerialEM for a K2 camera, but it is also
possible to save such frames without the rotation and flip.)
Enter -1 to have this number taken from an "r/f" entry in the
title of the first input file.
-dark (-da) OR -DarkReferenceFile File name
Dark reference to be subtracted before multiplying by a gain
reference when the frames are saved as unprocessed data.
-defect (-def) OR -CameraDefectFile File name
File of camera defects to correct. The defect file is put out
by SerialEM for versions of DigitalMicrograph from GMS 2.3.1 and
higher when frames are not gain-normalized. The program will
determine the binning of the image relative to these defect
coordinates by assuming that the images are more than half the
camera size. It will decide to scale the coordinates in the
defect list up by 2 if necessary for super-resolution frames.
These decisions will be reported and can be overridden with the
next two options. If this option is entered, it supercedes the
defect list found in a TIFF gain reference file.
-double (-do) OR -DoubleDefectCoords
Scale camera defect coordinates by 2 if they are not already
scaled. This option should not be needed.
-imagebinned (-im) OR -ImagesAreBinned Floating point
Binning of images, which could be needed for defect correction
if frames are not bigger than half the camera size.
-truncate (-tru) OR -TruncateAbove Floating point
Replace values above a limit with the mean of surrounding val-
ues. The mean is taken from pixels in a 7x7 area, excluding the
center 9. Enter a positive number to specify an absolute limit
in counts that applies to all frames being processed. Enter a
negative number to specify the number of standard deviations
above the mean at which to truncate (e.g., -8 for 8 SD's above
the mean). This limit will be determined separately for each
frame file or set of frames, using the first frame of the set.
When the saved option is entered with a saved frame list file,
the limit is determined separately again for the second frame
and that limit is used for the rest of the frames in the set, in
case the first frame is a partial exposure.
ALIGNMENT OPTIONS
These options control the strategy and main parameters of the align-
ment
-pair (-pai) OR -PairwiseFrames Integer
Number of frames or groups to use in successive pairwise align-
ments, or 0 to use alignment to a cumulative reference of
already-aligned frames. The default is 7. With an entry of -1
or a value equal to or bigger than the number of frames, the
program will align all pairs of frames or groups and do a single
fit. With an entry of -2, -3, or -4, it will do pairwise align-
ments among sets of one-half, one-third, or one-fourth of the
frames or groups, but with a minimum of 7 included.
-reverse (-rev) OR -ReverseOrder
Reverse order of processing and start with the last image. This
should make very little different when using pairwise align-
ments, but is a potentially useful option when using alignment
to a cumulative reference, unless there is substantial fixed
pattern noise.
-shift (-shi) OR -ShiftLimit Integer
Limit on distance to search for correlation peak, in unbinned
pixels. If the previous frame was aligned to the same reference
being aligned to, the center of the region searched corresponds
to the peak position for the previous frame. The default is 20.
-group (-gr) OR -GroupSize Integer
Number of frames to sum for correlations between groups of
frames; such groups are needed when correlations between single
frames are too noisy to give reliable results. Since correla-
tions are done only between non-overlapping groups, grouping
reduces the number of measured shifts from which each frames's
shift can be determined. Frames will be grouped in one of two
ways: in non-overlapping blocks, or in successive overlapping
groups, referred to as "slide grouping". The latter is used
only when the total number of frames is large enough to allow a
linear equation to be fit to the shifts; for example, this
requires 8 frames for a group size of 3. With slide grouping,
each frame will have a different shift. If the program has to
drop back to block grouping, all frames in a block will have the
same shift.
-radius2 OR -FilterRadius2 Floating point
High spatial frequencies in the cross-correlationd will be
attenuated by a Gaussian curve that is 1 at this cutoff radius
and falls off above this radius with a standard deviation speci-
fied by FilterSigma2. Unlike in other applications, this value
is entered in frequency units (1/pixel) of the input frames, not
of the reduced images being correlated. It is scaled by the
reduction before being applied to the reduced image, which means
that a particular value will give about the same amount of fil-
tering regardless of the binning. The default is 0.06.
-vary (-va) OR -VaryFilter Multiple floats
Set of radius2 filter values to test. This option can be
entered separately for each binning, but that should not be nec-
essary, for two reasons. First, because these values are in
unbinned frequency units, each one would have about the same
effect for the different binnings. Second, there is little cost
to applying extra filters, because different filters are applied
to a small subarea of an unfiltered correlation. Sigma2 will
automatically be set for each filter so that it is in the same
ratio to the particular radius2 value as the basic sigma2 is to
the basic radius2 value. Thus, to provide a different set of
sigma2 values for these filters, you need to enter -radius2 or
-sigma2. (Successive entries accumulate)
-hybrid (-hy) OR -UseHybridShifts
Derive a set of shifts while alignments are being done by using
the results from the best filter after each individual fit. By
default, when given multiple filters to test, the program will
decide on the best overall filter after all fits are done and
use the shifts from that filter. This option will reduce memory
requirements, unless the alignment is being refined at the end.
-refine (-ref) OR -RefineAlignment Integer
Refine an initial alignment based on pairwise correlations by
correlating each frame with an aligned sum of all but that
frame. The entry gives the maximum number of iterations that
will be run, but iterations will stop if the biggest change in
shift falls below a threshold.
-rgroup (-rg) OR -RefineWithGroupSums
When using group sums for the initial alignment, refine the
alignment with group sums as well, instead of single frames.
This may be needed if the signal-to-noise ratio is too low even
for the correlation between a single frame and the sum of other
frames. The shifts converge more slowly in this case, so more
iterations may be needed.
-stop (-sto) OR -StopIterationsAtShift Floating point
Maximum change in shift at which to stop iterating the refine-
ment of initial shifts by correlating with the sum of frames.
The default is 0.1.
-rrad2 (-rr) OR -RefineRadius2 Floating point
High frequency filter cutoff (radius 2) for refining the align-
ment. The default is to use the same filter that was used to
obtain the alignment, or the filter that gave the best overall
error value when a hybrid alignment was used.
-smooth (-sm) OR -MinForSplineSmoothing Integer
Smooth the shifts with a spline curve whose smoothing parameter
is found with generalized cross-validation, but only if the num-
ber of frames is at least as big as the entered value. This
method requires a fairly large number of frames to be reliable;
the documentation for the cross-validation code being used sug-
gests 20 frames may be needed. Smoothing should not be used
with less than 10 frames. For numbers between 10 and ~20, a
minority of images may come out slightly worse with smoothing,
so it would be advisable to evaluate results with and without
smoothing, such as with an FRC. The default is currently 20;
enter 0 to disable smoothing.
-gpu (-gp) OR -UseGPU Integer
Use the GPU (graphical processing unit) for computations if pos-
sible; enter 0 to use the best GPU on the system, or the number
of a specific GPU (numbered from 1). If GPU memory is a limita-
tion, the program will prioritize forming the sum on the GPU
over doing the alignment there, and will compute odd and even
sums as the lowest priority. If alignment becomes possible on
the GPU only by deferring the summing, and if CPU memory is suf-
ficient for that, then it will keep the entire stack of frames
in memory and sum them after aligning.
-memory (-mem) OR -MemoryLimitGB Multiple floats
Limit on CPU memory usage, and optionally a limit on GPU memory
usage, in gigabytes or as a negative fraction of total memory.
Enter a value > 0.05 to specify that number of GB, or a value
between -0.05 and -0.95 to specify between 0.05 and 0.95 for
total memory. The default for the CPU limit is 3/4 of system
memory for system memory less than 16 GB, half of system memory
for system memory more than 24 GB, and 12 GB between those lim-
its or when system memory cannot be determined. When the memory
usage would exceed the limit for a set of input frames, the pro-
gram will run through the data in two passes, one to get the
alignment and one to make the sum. This may not be possible if
the -assess option is used. The default GPU limit is 0.85 times
the GPU memory.
DOSE WEIGHT FILTERING OPTIONS
These options control "dose weighting", which filters out high fre-
quencies as a function of the dose already applied to a cryo-specimen.
-dtype (-dty) OR -TypeOfDoseFile Integer
This option both enables dose weighting and indicates what kind
of file is being provided with the dose information. Type 4
indicates key-value information in the autodoc format, such as
an ".mdoc" file produced by SerialEM. If an ".mdoc" file has
been entered with the -mdoc option, this is the file that will
be used, and no filename can be entered with the -dfile option.
The other four types are simple text files with a line for each
output image (i.e. each set of frames being aligned). These
types are:
1: A single value per line, just the dose for each image;
2: Two values, the prior accumulated dose followed by the
image dose;
3: The prior dose followed by the cumulative dose at the end
of that image;
5: One or more pairs of entries indicating the dose per frame
and the number of frames with that dose. For example, "0.5 15
0.75 10 1.0 15" specifies a dose of 0.5 for 15 frames, 0.75 for
15 frames, and 1.0 for 15 frames. The total number of frames on
such a line must match the number of frames in the set.
For these four types of text files, if the files or frame sets
are being processed in order by tilt angle, the lines should be
in the same order as the processing would occur without this
reordering, i.e., the original order of the data, or of the Z
values in an mdoc file.
-dfile (-dfi) OR -DoseWeightingFile File name
Name of file with dose information for dose weighting. This
entry is required if -dtype is entered, unless a 4 is entered
there and a file was entered with the -mdoc option.
-dtotal (-dto) OR -FixedTotalDose Floating point
Total dose for each set of frames, in electrons/square Angstrom.
This option independently enables dose weighting and cannot be
entered with -dtype or -dframe.
-dframe (-dfr) OR -FixedFrameDoses Floating point
List of frame doses and numbers that applies to all sets of
frames. The list is a set of paired numbers: a dose in elec-
trons/square Angstrom and the number of frames taken at that
dose (e.g., enter "2,10,4,5" for 10 frames of 2 e/A^2 and 5
frames of 4 e/A^2). This option independently enables dose
weighting and cannot be entered with -dtype or -dtotal.
-dprior (-dp) OR -InitialPriorDose Floating point
Dose applied before any of the images in each frame set were
taken; this value will be added to all the prior dose values (if
any), however they were obtained.
-bidir (-bid) OR -BidirectionalNumViews Integer
Number of views in the first part of a bidirectional tilt
series, where the order of images in the input file is inverted
from their order of acquisition. This entry is essential for a
bidirectional series if a fixed dose is entered with -dfixed or
if a dose file of type 1 or 5 is entered. It is ignored for
types 2 and 3. It is not needed for type 4 and an ".mdoc" file
entered with the -mdoc option, where the correct doses are
available either from PriorRecordDose entries or from analysis
of DateTime entries. However, it would be needed for an ".mdoc"
file entered with -dfile unless the -accum option is entered
with a 2 to use prior dose information from the ".mdoc" file.
-accum (-ac) OR -DoseAccumulates Integer
This option can be used to override the program's assumptions
about whether images are from a tilt series where dose should be
accumulated between successive images. By default, dose accumu-
lates whenever -mdoc or -stack is entered; otherwise it does
not. When there is an ".mdoc" file entered with -mdoc, the
cumulative dose information is taken from either the prior
Record dose (if present) or the sum of doses of earlier images,
even when only a subset of frame sets (images) are being used.
However, if an ".mdoc" file is entered as a dose file, the dose
will not accumulate. For this option, enter 0 to prevent accu-
mulating dose or 1 or 2 to accumulate dose. The latter will
have the same effect except when a subset of images is being
aligned and an ".mdoc" file is entered with -dose instead of
-mdoc. In that case 1 will make it accumulate dose by summing
the doses over the images being aligned, while 2 will make it
use cumulative dose information from the whole set of images in
the ".mdoc" file.
-normalize (-nor) OR -NormalizeDoseWeighting
Normalize the dose weight filters so that they add up to to 1.0
within each set of frames. This option is designed for tilt
series where the true dose weighting of the tilt images is to be
done later (e.g., with Mtffilter). It compensates for the
difference in dose effect among the frames of an individual
image, which can be significant for some early images if the
dose is high enough. However, it results in no net filtering of
the summed tilt image, leaving this dose weighting for a later
stage. If this option is applied to a single-particle frame
stack, the high frequencies will be boosted in early frames to
compensate for their being attenuated in later frames.
-volt (-vo) OR -Voltage Integer
Microscope voltage in kV; this value must be either 200 or 300.
The default is 300; if 200 is entered, the computed critical and
optimal doses are multiplied by 0.8.
-optimal (-op) OR -OptimalDoseScaling Floating point
Factor by which to scale the computed optimal and critical doses
that determine how much to attenuate a spatial frequency for a
particular dose. Enter a factor greater than or less than 1 to
indicate that the specimen is more or less resistant to damage
than the equations indicate. Another use for this entry would
be to adjust the critical dose for a voltage other than 200 and
300 kV.
-critical (-c) OR -CriticalDoseFactors Three floats
Replacement factors a, b, and c in the equation
-unweight (-un) OR -UnweightedOutputFile File name
File name for output of summed images that have not been dose
weighted in addition to the dose-weighted images placed in the
main output file.
OPTIONS FOR ASSESSING ALIGNMENT AND OTHER TEST PURPOSES
This section includes options that are useful for assessing alignment
parameters (mainly binning and filters) and advanced options that
should not need adjustment.
-test (-te) OR -TestBinnings Multiple integers
Set of binnings at which to test pairwise alignments. Each bin-
ning involves a separate pass through the frames of an input
file, plus another pass to make a sum at the end with the best
binning.
-assess (-as) OR -AssessWithFrames Two integers
Starting and ending frame to use during testing of parameters
with multiple binnings or filters, numbered from 1. The default
is to use all frames.
-good (-go) OR -GoodEnoughError Floating point
Combined error measure that is sufficient to stop testing bin-
nings. This error is a weighted sum of two measures: the mean
of the weighted mean residual errors from the set of fits, and
the maximum weighted residual seen in any fit. The latter is
weighted by the value entered by -weight option.
-weight (-w) OR -MaxResidualWeight Floating point
Weighting applied to maximum weighted residual from all fits
when combining with the mean weighted residual to obtain a sin-
gle error measure. The default is 0.1.
-trim (-tri) OR -TrimFraction Floating point
Fraction of image size to trim off each edge for correlations.
The default is 0.02, which is the same amount as the padding.
-taper (-tap) OR -TaperFraction Floating point
Fraction of image size to taper on each edge for correlations.
The image is tapered down to the mean at its edge. This taper-
ing is usually an important component for reliable correlations.
The default is 0.1. If this fraction is set to 0 and there is no
trimming, the program obtains the FFT for cross-correlation by
extracting it from the FFT of the full image instead of by
reducing, padding and tapering the image and taking an FFT of
that. The padding/tapering extent of the full image is
increased from 2% on each edge to 5% in this case.
-antialias (-an) OR -AntialiasFilter Integer
Type of filter for image reduction when trimming or tapering.
The standard values of 1 to 6 are available as in Newstack,
with 1 corresponding to binning. The default is 4 for a
Mitchell filter, which seems to be optimal on average for this
application.
-radius1 OR -FilterRadius1 Floating point
Low spatial frequencies in the cross-correlations will be atten-
uated by a Gaussian curve that is 1 at this cutoff radius and
falls off below this radius with a standard deviation specified
by FilterSigma2. Spatial frequency units range from 0 to 0.5.
This option is here for the sake of completeness; use Filter-
Sigma1 instead of this entry for more predictable attenuation of
low frequencies.
-sigma1 OR -FilterSigma1 Floating point
Sigma value to filter low frequencies in the correlations with a
curve that is an inverted Gaussian. This filter is 0 at 0 fre-
quency and decays up to 1 with the given sigma value. However,
if a negative value of radius1 is entered, this filter will be
zero from 0 to |radius1| then decay up to 1. The default is
0.03, expressed in frequency units (1/pixel) of the reduced
images being correlated.
-sigma2 OR -FilterSigma2 Floating point
Sigma value for the Gaussian rolloff below and above the cutoff
frequencies specified by FilterRadius1 and FilterRadius2. Like
radius 2, this value is entered in unbinned frequency units and
will be scaled by the reduction being applied for alignment.
The default is 0.0086.
-kfactor (-k) OR -KFactorForFits Floating point
K factor for robust fitting to pairwise alignments. The default
is 4.5; a smaller value will down-weight more outliers in the
fits.
-debug (-deb) OR -DebugOutput Integer
This entry is a sum of flags for particular kinds of output: the
last digit controls the printed output from the program (1 for
basic output, 2 for more verbose output from each fit and dose
weighting filters); 10 will give timing output, 100 will give
output of cross-correlations during initial alignment; 1000 will
give output of refining correlations; 10000 will give output of
the sums of odd and even frames. Images are output with names
"faimg-n.mrc" where n is increased sequentially.
-flags (-fl) OR -FlagsForGPU Integer
Flags to control which kinds of initial processing occur on the
GPU. These flags are for testing and supersede the decisions
that the program would make based on the memory available.
Enter a sum of
1 to do noise-taper-padding of full images on the GPU
10 to do reduction and taper-padding of alignment images on
the GPU
100 to stack full-sized, unpadded frames on the GPU, provided
that both the noise padding and reduction padding are to be done
there
1000 to do gain normalization, defect correction, and trunca-
tion on the GPU, provided that noise padding or reduction pad-
ding are to be done there
10000 times the maximum number of frames to stack on the GPU
-shrmem (-shr) OR -ShrMemTest
Use shrmemframe to do align frames (special Windows build only)
-help (-he) OR -usage
Print help output
-StandardInput
Read parameter entries from standard input
EXAMPLES
The example commands below would all be entered in one line, or as mul-
tiple lines with a backslash at the end of every line except the last,
as they are shown here due to line length limits. Most options could be
abbreviated more than they are.
Frames from Tilt Series
An image from a tilt series with ~4Kx~4K images might align well with a
command as simple as
alignframes Feb21_10.43.50.mrc Feb21_10.43.50_ali.mrc
where you can add "-gpu 0" to use a GPU on this or any of the following
commands. This will use a default binning of 3 and filter cutoff of
0.06/pixel. If you already know that you want a different binning
(say, 4) or filter (say, 0.05), then the easiest way to enter this is
with
alignframes -bin 4,1 -vary 0.05 Feb21_10.43.50.mrc \
Feb21_10.43.50_ali.mrc
where using -vary will scale the sigma for the filter rolloff automati-
cally.
If the frame files for your tilt series list in order from one high
tilt to the other and the tilt series file is available (say,
cell4.mrc),
alignframes -bin 4,1 -vary 0.05 -stack cell4.mrc Feb21_*.mrc \
cell4_ali.mrc
will process the entire tilt series and move information from the
header of the tilt series file into the new file. However, if the tilt
series was taken bidirectionally and the frames files do not list in
order, or if the there were any duplicate images taken, then you want
to supply the .mdoc file instead of the stack and input files:
alignframes -bin 4,1 -vary 0.05 -mdoc cell4.mrc.mdoc cell4_ali.mrc
which will process the frames in the right order and place essential
information in the output file header.
To explore filter settings, it is best to run on a collection of files,
perhaps about 20. Suppose "Feb21_10.4*.mrc" lists the desired number
of files, you could explore a range of filters at binning 3 with
alignframes -vary 0.05,.06,.08,.1 -nosum Feb21_10.4*.mrc
The summary at the end will tell which filter gave the lowest mean
residual. Be sure to scan through the results and look at whether
either the mean residual or the distance moved declines a lot with
increased filtering, which may be a sign that fixed pattern noise is a
problem. Also note whether the maximum unweighted residual is high.
If it goes over 5-10 pixels, you can problem reduce the occurrence of
bad fits by restricting the allowed shift with "-shift 10" or even
"-shift 5". Obviously, you do not want to set this limit lower than
the maximum possible shift from one frame to the next.
Evaluating and Visualizing Differences with FRC Curves
Suppose you want to use FRCs to evaluate the difference between two
conditions, such as filter 0.05 versus 0.06, or with and without
refinement. You need to make output sums to get an FRC.
alignframes -vary 0.05 -frc sample.frc Feb21_10.4*.mrc sample.mrc
alignframes -vary 0.05 -ref 5 -frc sample-refine.frc \
Feb21_10.4*.mrc sample.mrc
The ".frc" files have all of the FRC curves for a run, each with a
"type" number equal to its file number in the alignframes text output.
You can plot one curve (say, the fourth one) with
onegenplot -ty 4 -sym 0 sample.frc
To compare FRC's, run
subtractcurves -ave 10 -rad 2 sample-refine.frc sample.frc \
refine-diff.dat
which will subtract each pair of corresponding curves and average over
10 points to reduce the noise in the differences. You could have
avoided the averaging here with the option "-ring .05" to alignframes,
which will make the FRC curve much less noisy in Onegenplot. (The
default FRC output is good for seeing the CTF oscillations in single-
particle data, but a larger ring size will often be more appropriate
for tilt series.) You could examine each difference in a separate
graph with
onegenplot -ty 1 -sym 0 refine-diff.dat &
onegenplot -ty 2 -sym 0 refine-diff.dat &
etc.
But to assess the overall benefit of the difference in conditions, it
is more efficient to look at a lot of points at once:
onegenplot -ty 1,2,3,4,5,6,7,8,9,10 refine-diff.dat &
onegenplot -ty 11,12,13,14,15,16,17,18,19,20 refine-diff.dat &
etc.
When looking at these curves, treat any improvements below about 1.3
times the cutoff frequency with caution because they could arise from
overfitting. Improvements past this point would not reflect overfit-
ting, although if the fit is locking in on fixed pattern noise, it
would boost high frequency correlations.
Frames for Single-Particle Reconstruction
For single-particle data, the best initial approach is to fit to pair-
wise shifts among about half of the frames. So for a set of 34 frames,
if you wanted to do an initial assessment with two filters and look at
the FRC,
alignframes -vary 0.05,.06 -pair 17 -frc test.frc \
Feb15_10.13.15.mrc test.mrc
which will use a default binning of 6 for ~8K frames. A more flexible
command for fitting to shifts among half the frames, regardless of the
exact frame count, is
alignframes -vary 0.05,.06 -pair -2 -frc test.frc \
Feb15_10.13.15.mrc test.mrc
If you had some indication that fixed pattern noise was a problem, or
if the data seemed particularly noisy, giving mean residuals above 1,
you could use pairwise shifts among all frames with
alignframes -vary 0.05 -pair -1 -frc test.frc Feb15_10.13.15.mrc \
test.mrc
For even noisier situations, you can use grouping to reduce the mean
residual and improve the fits:
alignframes -vary 0.05 -pair -1 -group 3 -refine 5 -frc test.frc \
Feb15_10.13.15.mrc test.mrc
where the refinement at the end may or may not be beneficial, depending
on just how noisy the single frames are. The option specifies up to 5
iterations of refinement, which is almost always sufficient unless
refining the data as groups with the -rgroup option.
Processing of Raw Frames
If you have collected dark-subtracted data from a K2 camera into MRC or
compressed TIFF files, you can process them with
alignframes -gain SuperRef_Feb20_20.26.21.dm4 -scale 39.3 -rot -1 \
-defect defects_Feb20_20.26.21.txt -pair 17 Feb21_10.32.56.tif \
Feb21_10.32.56_ali.mrc
using the gain reference copied into the data directory and the defect
file written there by SerialEM. The option "-rot -1" specifies that
the gain reference needs to be rotated by the value for rotation and
flip indicated by "r/f" in a label in the file header. MRC files
started having this label in SerialEM 3.4, TIFF files in SerialEM 3.5,
so check either kind of file by running "header" on the file. If the
label does not show "r/f", the number to use in the "-rot" entry would
be the RotationAndFlip value in the SerialEM properties file. If
frames were saved without rotation, do not include this option.
Either the "-scale" option as shown here, or the "-total" option with a
total scaling, or the option "-mode 2", should be entered to preserve
the precision of the data when they are written. The program will use a
default total scaling of 30 when normalizing if the input data are byte
values and data are not being written as floating point with "-mode 2".
However, it is probably better not to rely on the default and instead
scale the electron counts by the same factor that is applied in Seri-
alEM (39.3 in this example, a typical value when not dividing by 2).
You could add an option such as "-trunc 7" to replace values above 7
with the local mean. Use the command
clip hist Feb21_10.32.56.tif
to see the distribution of pixel values. There will be a point at
which the values stop falling rapidly and then have a long tail;
removal of values above there is indicated.
Assessing Fixed Pattern Noise
This procedure is not convenient, but does work. The first step when
fixed pattern noise is suspected to affect the correlations is to out-
put the individual shifts with the option "-deb 2". Use only a single
filter for simpler output. You will see a series of lines like
1 to 0 -1.95 -0.17 near 0.00 0.00
2 to 0 -1.57 0.02 near -1.95 -0.17
2 to 1 -0.60 -0.77 near 0.00 0.00
3 to 0 -1.79 0.44 near -1.90 -0.30
3 to 1 0.44 -1.32 near -0.28 -0.45
3 to 2 0.36 0.02 near 0.00 0.00
Each line shows a shift between a pair of frames (numbered from 0),
then the shift that it was assumed to be "near" when comparing with the
maximum allowed shift. If you see many shifts close to 0 (but not
exactly 0), then they could be due to fixed pattern noise. (shifts of
exactly 0 might indicated thatthe maximum shift was too low.
Try with some higher filter values and see if the incidence of near-
zero values increases. Set a binning lower than the default (e.g., 4
instead of 6, 2 instead of 3) if necessary to make a filter value be
below Nyquist. In the above example, a higher filter gave:
1 to 0 -0.80 -0.22 near 0.00 0.00
2 to 0 -0.71 0.07 near -0.80 -0.22
2 to 1 -0.08 -0.38 near 0.00 0.00
3 to 0 -0.92 0.42 near -0.77 -0.15
3 to 1 0.17 -0.81 near -0.02 -0.15
3 to 2 0.30 -0.29 near 0.00 0.00
To visualize the correlation peak from fixed pattern noise, you need to
run the program with options that will make it save unbinned correla-
tions with no high-frequency filtering. This is available only when
not using a GPU and when specifying more than one filter.
alignframes -bin 1,1 -vary 0.05,0.06 -deb 102 -frame 1,5 -nosum \
Feb15_10.13.15.mrc
There will be series of lines indicating the names of saved images and
the measured shift between a pair of frames. Pick one with more than a
few pixels of shift, which may end up being the one between frames 0
and 4. For example:
Saved lf correlation image frame 4 in ./faimg-18.mrc mean -0.00
Saved correlation image frame 4 in ./faimg-19.mrc mean -0.03
4 to 0 -0.59 -2.70 near -1.90 0.14
The last line shows the shift between frames 4 and 0, then the shift
that it was assumed to be "near" when comparing with the maximum
allowed shift. You want to examine the "lf correlation image" in
"faimg-18.mrc" ("faimg-19.mrc" is a small subarea with high-frequency
filtering, centered on the expected shift, so is not suitable). You
can open this file directly in 3dmod, but it will easier to load a cen-
tered subarea. Run "header faimg-18.mrc" to see its size, NX and NY.
Determine these values:
xst = NX / 2 - 200
xnd = NX / 2 + 199
yst = NY / 2 - 200
ynd = NY / 2 + 199
and open the file with
3dmod -x xst,xnd -y yst,ynd faimg-18.mrc
Open a slicer window and close it; this will place the current point
marker on the middle pixel. Zoom the Zap window up to about 6 and turn
off the high-quality display (the checkerboard) to see individual pix-
els. Adjust black and white levels to 0 and 255. The degree to which
this central pixel stands out indicates the amount of fixed pattern
noise. Increase the black level to see just how much it stands out.
If you can see 4 brighter pixels around the central one, the situation
is particularly bad.
You may wonder where the real correlation peak went! It is very dif-
fuse and the high-frequency filtering is essential. Use Edit-Image-
Process to open the processing dialog and select the Fourier filtering
panel. Set the high frequency cutoff to your filter value and the
falloff to one-seventh of that and press "Apply" to filter. The real
peak will now be prominent. If it is close to the origin, you might
see its position change with different filters as it merges with the
fixed pattern peak.
AUTHOR
David Mastronarde
SEE ALSO
Email bug reports to mast at colorado dot edu.
IMOD 4.11.0 alignframes(1)