imodholefinder(1) General Commands Manual imodholefinder(1)
NAME
imodholefinder - Find regularly spaces holes in carbon film
SYNOPSIS
imodholefinder options input_image output_model
DESCRIPTION
Imodholefinder finds regular spaced holes in a carbon film in order to
locate target positions for cryoEM data acquisition. It is principally
designed as a test harness for the Holefinder module shared with Seri-
alEM, but may also be useful for offline analysis of target positions.
It supports square and hexagonal lattices.
The main output is an IMOD model file in which the first object con-
tains points for the set of holes found on each section, the second
object contains positions located within the regular pattern for which
a point was not found with sufficiently low error, and the third object
contains points that were found close to some of those missing posi-
tions. Each point is in a separate contour, and one general value is
stored for each contour. This value will be the relative correlation
peak strength by default, but with the intensity option, the value can
instead be the mean or standard deviation of pixels within the hole, or
the fraction of dark pixels that appear to be outliers. Points below a
threshold value can be turned off and deleted using the Bead Fixer mod-
ule in 3dmod, and points above a value can also be turned off with
the Values panel of the Edit-Objects dialog of the Model View window.
However, there is currently no way to delete the points above that
threshold in 3dmod.
A text file with information about each located point, including all
four of those value metrics, can be output with the -summary option.
See its description below.
When the input images are based on a montage, the raw montage file can
also be provided in order to improve the result and overcome bad align-
ments between overlapping pieces. In this case, the summary output
file will also contain the coordinates within a piece for each point,
probably the best form for importing these positions into SerialEM.
The program locates holes in images that are scaled down if necessary
so that the holes are no bigger than 50 pixels. It starts with a stan-
dard method: filtering to reduce noise, application of a Sobel filter
and a Canny edge detector, and then a Hough transform of this edge
image to find circular features. Filter can be done with either Gauss-
ian smoothing with a specified sigma value, by median filtering with a
given number of iterations. The Hough transform is implemented by
cross-correlation of the edge image with a circular template via
Fourier transforms, which is essentially equivalent to but much faster
than direct summation and allows further efficiencies when transforms
are cached for reuse. To overcome uncertainty in the actual hole diam-
eter and variability in the appearance of hole edges, the program does
a series of scans with circles of given thickness. By default, it
starts with a fairly thick circle and a potentially wide-ranging scan
to find the best diameter; it then refines the diameter with three
steps using a circle of medium thickness, and finally repeats the
process with a thinner circle at the refined size. To improve the
result, an average is generated from the edge image at the half of the
locations with the strongest correlations, and this edge average is
correlated with the edge image to get a new set of positions.
The positions obtained from searching for peaks in these cross-correla-
tions are constrained to be separated by at least 3/4 of the nominal
spacing between holes, which greatly reduces the number of false corre-
lation peaks being considered. The next stage is to analyze these
position to determine the orientation and spacing of the regular grid
and eliminate points that do not fall near positions of that grid. In
addition, a robust fit to the positions of up to a 5 by 5 array of
points is used to generate a predicted position for each location on
the grid if possible, and a point is eliminated if it is not close
enough to its respective predicted position. Now that most points can
confidently be considered actual holes, an alternative analysis is done
if possible by averaging from the 90% of the positions with top corre-
lation scores after removing bright outliers, which should correspond
to empty holes. In this case the actual image is averaged, and the
average template is correlated with that image. The detected points
are subjected to the same analysis to determine which ones fall on a
regular grid. Results from these two different average template corre-
lations are then merged and analyzed again to find the best of both
sets of positions.
All of the above depends on two critical parameters at the start of the
process: the initial filtering and the threshold for the Canny edge
detector. The best results are obtained by a brute force method of
doing the entire analysis with a range of values for each parameter and
picking the values that give the most points. Once the final set of
points is obtained, bright outliers are removed, and dark outliers are
also removed with a very conservative outlier criterion.
When the input file contains montages images and a raw montage file is
also supplied, the program follows the above with a simplified analysis
of each montage piece. It applies the filter and edge detector with
the final parameters used for the full image, then correlates the
existing edge average template with the edge image and the existing
image average template with the actual piece image. These correlations
are separate analyzed for points being on a regular grid, results are
merged thet same way as for the full image, and the grid analysis is
repeated on the merged points. Outlying bright and dark holes are
removed with the same intensity criteria detreimined in the outlier
removal for the full image. After all pieces are analyzed, the posi-
tions in the pieces are considered in conjunction with those from the
full image. Positions within the overlap zones between pieces are sub-
stituted from the appropriate piece if possible to correct for any dis-
tortions of the hole image in those locations of the full image.
Points found in the piece analysis but not in the full image analysis
are added to the collection.
OPTIONS
Imodholefinder 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 (-inp) OR -InputImageFile File name
Name of input image file. If it is not entered with this option
it must be entered with the first non-option argument.
-output (-o) OR -OutputModelFile File name
Name of output model file. If it is not entered with this
option it must be entered with the second non-option argument.
-boundary (-bo) OR -BoundaryModel File name
Model with contours enclosing areas to analyze, one per section
to be constrained. A contour will be applied on the sections
where it is drawn.
-summary (-su) OR -SummaryOutputFile File name
Name of output file for points and values found on each section.
The columns of this file are:
X position in input image in pixels
Y position in input image in pixels
Z value (section number, from 0)
Correlation peak value, relative to highest one found on sec-
tion
The mean value within the hole
The standard deviation of the pixels in the hole smoothed by
3x3 averaging
The fraction of smoothed pixels in the hole considered to be
negative outliers
The X position index within a regular grid, numbered from 0
The Y position index within a regular grid, numbered from 0
If raw montage entered, the montage piece on which the point
is found
If raw montage entered, the X coordinate on that piece, in
piece pixels
If raw montage entered, the Y coordinate on that piece, in
piece pixels
-diameter (-di) OR -DiameterOfHoles Floating point
Nominal diameter of holes. Enter a positive value to specify
the diameter in microns, or the negative number of pixels to
specify it as pixels
-spacing (-sp) OR -SpacingOfHoles Floating point
Nominal spacing between holes. Enter a positive value for a
spacing in microns, or a negative number of pixels.
-hex OR -HexagonalGrid
Holes are arranged in a hexagonal rather than a square lattice
-error (-e) OR -MaximumError Floating point
Maximum error allowed between a point and a prediction of where
it should be located, based on fitting to up to a 5x5 array of
surrounding points. Enter a positive value for the number of
microns, or a negative number of pixels. If this option is not
entered, the default is 0.05 times the nominal hole diameter, or
a different fraction if entered with -control.
-thresh (-thr) OR -ThresholdPercentiles Multiple floats
Set of thresholds to try for the Canny edge detector. The
thresholds are specified as the percentile of the highest edge
gradients to consider as strong edges; thus small values like
2-6 are appropriate. The lower threshold, for considering
points as weak edges, is taken as twice this value. The default
is 2.,3.2,4.4. When both multiple threshold and multiple fil-
ters are to be tried, the program exhaustively tries all combi-
nations.
-filter (-f) OR -FilterSigmaOrIterations Multiple floats
Set of filter values to try: a positive value specifies the
sigma in pixels to use for Gaussian smoothing; a negative values
specifies the negative of the number of iterations for a 3x3
median filter. The default is 1.5,2,3,-3, which means try
Gaussin smoothing with sigmas of 1.5, 2, and 3, then a median
filter with 3 iterations. If multiple median filter iterations
are to be tried, enter them in order of increasing iteration
number for maximum efficiency.
-thickness (-thi) OR -CircleThicknesses Multiple floats
Set of thicknesses of circles to correlate with the edge image,
in pixels. The default is 4,2,1.5. Multiple values should be
entered in order of decreasing thickness. If the number of val-
ues entered is not 3, then corresponding entries must be made
for -number and -step.
-number (-n) OR -NumberOfCircles Multiple integers
Set of numbers of circle sizes to try for each thickness. The
default is 7,3,1. When more than 5 is specified, as few as 5
will be tried if the summed correlation score drops monotoni-
cally to both sides of the first one tried. If the number of
values entered is not 3, then corresponding entries must be made
for -thickness and -step.
-step (-st) OR -CircleStepSize Multiple floats
Set of increments between circle sizes to try for each thick-
ness, in pixels. The default is 3,1.5,1. The increment is
irrelevant when only one size is being used. Increments should
be less than their respective thicknesses. If the number of
values entered is not 3, then corresponding entries must be made
for -number and -thickness.
-sections (-se) OR -SectionsToDo List of integer ranges
List of sections to analyze, numbered from 0 (ranges allowed)
-intensity (-int) OR -DiamForIntensityStats Two floats
The diameter of hole region to get statistics from, in microns,
and the type of value to store in the model in place of the cor-
relation peak strength: 0 for the mean, 1 for the SD of values
smoothed by averaging a 3x3 set of pixels, or -1 for the frac-
tion of those smoothed values that appear to be dark outliers.
If a negative value (e.g., -1) is entered for the diameter, it
will use twice the difference between the determined hole size
and maximum error.
-montage (-m) OR -RawMontageFile File name
Original montage file that is the basis for the input images.
With this entry, the program will find holes on each montage
piece and use this information to improve the number and loca-
tion of the holes found on the full images. The -piece option
must also be entered.
-piece (-p) OR -PieceListFile File name
File with list of nominal piece coordinates for the raw montage
-aligned (-a) OR -AlignedPieceList File name
File with list of aligned piece coordinates for the montage, as
put out with the -AlignedPieceCoordFile option to Blendmont,
so that the program can relate locations in the full images to
those in the pieces. If pieces were not aligned to produce the
full images (e.g., by Reducemont), then this entry is not
needed.
-binned (-bi) OR -FullImageIsBinned Integer
Binning of the input images relative to the raw montage. This
entry is not needed if both image file headers have valid pixel
spacings that differ by an integer factor.
-retain (-r) OR -RetainDuplicates
Keep all points that correspond in overlapping pieces in the
output model. Without this option, the point from one piece of
such a pair will be removed, leaving only the one that is poten-
tially substituted or added to the set of points from the full
image.
-show (-sh) OR -ShowPieceObjects
Turn on the objects containing points from the montage pieces
and turn off the objects containing the actual set of found
points, missing, and nearby ones. By default the latter are on
and the montage objects are off.
-control (-c) OR -ControlValue Two floats
Parameter number and value for setting algorithm control parame-
ters. Numbers in parentheses are default values.
1: Target hole diameter to reduce images to if holes are big-
ger than it (50.)
2: Fraction of nominal diameter to use as maximum error when
analyzing grid, if the maximum error is not entered (0.05)
3: 0 use filter/threshold values from first section for the
rest; 1 to use filter value from first section and keep varying
threshold; 2 to vary on all sections (2)
4: Retain FFTs of circle and average templates when appropri-
ate (1)
5: Minumum number of total points for making an average (5)
6: Fraction of points to average if there enough; more will be
done if < minimum (0.5)
7: Maximum change between nominal and found diameter allowed;
above this it do next set of circles at the nominal diameter
instead of the found one (0.2)
8: Criterion for omitting positive outliers in mean value from
the raw average (4.5)
9: Criterion for removal of negative mean outliers after the
final grid analysis (9.0)
10: Criterion for removal of positive mean outliers after the
final grid analysis (4.5)
11: Fraction of radius to take pixels from for this outlier
removal (0.9)
12: Fraction of average spacing below which points found on
separate montage pieces are considered to be the same (0.5)
13: Fraction of average spacing below which a point found on a
montage piece is considered to be the same as a point found in
the full image (0.5)
14: Fraction of radius used as criterion distance from edge of
overlap zone for substituting a point found on a piece for the
same point on the full image; 0 means a point right on edge
qualifies, a positive value admits points not in overlap (0.)
15: Fraction of radius used as criterion distance from edge of
image for using a point found only on a piece (adding or substi-
tuting it) (0.25)
16: Maximum fractional extent into overlap for adding a point
from a piece not found on the full image (and not eliminated
because the it was identified as the same as one on the overlap-
ping piece) (0.75) (Successive entries accumulate)
-verbose (-v) OR -VerboseOutput Integer
1 for verbose output; 2 for more verbose output
-debug (-de) OR -DebugImages Integer
1-3 for output of intermediate images. Most images come out
with any value; the splitfill and crosscorr files are specific
to the value: 1 for crosscorr from ideal hole templates, 2 or 3
for splitfill and crosscorr from the average edge or raw average
templates.
-help (-hel) OR -usage
Print help output
-StandardInput
Read parameter entries from standard input
AUTHOR
David Mastronarde
SEE ALSO
blendmont, reducemont
BUGS
Email bug reports to mast at colorado dot edu.
IMOD 4.12.62 imodholefinder(1)