pickbestseed(1) General Commands Manual pickbestseed(1)
NAME
pickbestseed - Selects best seed points for autofidseed
SYNOPSIS
pickbestseed options
DESCRIPTION
Pickbestseed is used by Autofidseed to select an optimal set of seed
points fof the desired number, using bead models tracked starting from
several different views and using information on the quality of the
tracking, the shape of the beads, and on which of two surfaces they are
located.
The program operates with the following steps:
1) It reads in the different tracked models and identifies cases where
the same bead was tracked in different models, as signified by tracks
that are sufficiently close to each other over the whole range of views
tracked (controlled by parameters 1 and 6 below).
2) Each unique bead becomes a candidate, and a single score is obtained
by taking a weighted mean of four measures of the consistency and qual-
ity of tracking: the mean residual from alignment during tracking; the
fraction of models in which the bead is missing; the fraction of points
missing from each track of the bead; and the mean distances between
corresponding points in the tracks, computed from each pair of tracks.
By default, these measures are give equal weighting. A lower score is
better. When beads are located on two surfaces, each candidate is
assigned to a surface based on which assignment predominates in the
multiple surface analyses available.
3) The total area to be analyzed is measured, so that it is possible to
convert between number of beads and average density per unit area.
4) Information on the elongation of beads is analyzed, namely statis-
tics from the standard deviation of pixels around the bead and for the
elongation. An adjusted value for the mean of the elongation over the
11 views of a track is computed by, in effect, rotating a plot of mean
elongation versus the standard deviation of the elongation by an angle
(parameter 18 below) that removes much of the variability due to the SD
of the elongation, and using the new Y coordinate as the adjusted
value. The same operation is performed with the SD of pixels around
the beads to obtain an adjusted edge SD (rotation angle controlled by
parameter 19). The latter values are scaled to have the same standard
deviation as the adjusted elongation values, and then a plot of
adjusted elongation versus scaled, adjusted SD is rotated so as to com-
bine them into a single measure (parameter 20 below), with a final
elongation measure taken as the Y coordinate after rotation. This mea-
sure is analyzed by taking the median, finding the median absolute
deviation, and considering values as outliers if their deviation from
the median is more than the normalized median absolute deviation (MADN)
times a criterion (parameter 12 below). A candidate is marked as elon-
gated if it is an outlier or if the median elongation exceeds an abso-
lute threshold (parameter 16 below).
5) Beads are identified as clustered if they are within a criterion
distance (parameter 5 below) of any other bead on the same surface.
This distance is evaluated at the highest tilt angle of the series,
assuming that the distance between beads perpendicualr to the tilt axis
is foreshortened by tilting.
6) Elongation is analyzed again, considering only points that are not
identified as clustered. When there are many clustered points, which
also tend to have high elongation values, this can skew the criterion
for identifying outliers, so analyzing unclustered points separately
can identify additional outliers. Each unclustered point is given the
maximum of the elongation score from the two analyses.
7) Beads are sorted in order by their overall score and two different
procedures are used to accept points in the final model, in a set of
phases. Only unclustered, unelongated beads are considered for the
first 4 phases. The median and MADN of all the scores are used to
define a maximum acceptable score in this phase (8 MADNs from the
median, by default). Once some points have been accepted, the program
computes a continuous 2D density function from the points using kernel
density estimation with a triweight kernel. Specifically, for each
bead, a component proportional to (1 - (dist / H)^2)^3 is added for a
point at distance "dist" from the bead. H is chosen based on the tar-
get spacing to be achieved between points in the current phase, times
parameter 10 below.
7.1) Phase 1: The target density is converted to an equivalent spacing
and beads are accepted in order by their score, provided that they are
not closer to an already-added bead than a certain fraction of this
spacing (parameter 14 below). This procedure is then repeated with the
best half of the candidates, adding them if they are not closer to an
added bead than a lower fraction of the target spacing (parameter 15
below). When there are beads on two surfaces, this procedure is done
separately for each surface.
7.2) Phase 2: A gap-filling routine is used to add further points up to
a desired density, if necessary. This routine repeatedly finds the
point with lowest density then searches out from that point in succes-
sively wider rings for a bead to add. The ring spacing is the target
spacing times parameter 3 below. If multiple beads are found in a
ring, they are prioritized by an adjusted score, which is their overall
score divided by the distance to the nearest accepted bead. In addi-
tion, if clustered and overlapped points are being accepted (in a later
phase), the score is increased for a clustered or overlapped point, and
a point within the clustering distance of another accepted point is
simply excluded. After a search is done in one location, points within
a certain distance of the density minimum are excluded from further
consideration on that call of the gap-filling routine. (This criterion
distance is the target spacing times parameter 2 below.) The search is
terminated when all density minima below a fraction of the target den-
sity are examined. This fraction is parameter 8 below, but if there
are two surfaces and the ratio of minority to majority surface is less
than parameter 13 below, it uses the higher fraction in parameter 9
instead for the minority surface. The gap-filling routine is called
twice, once allowing two rings, then allowing the number of rings in
parameter 4 below.
7.3) Phase 3: If there are two surfaces, it now tries to beef up the
number on the majority surface to make up for the deficiency. The tar-
get number for this surface is the full target number minus the number
on the minority surface, unless the "-nobeef" option is entered, in
which case the target is still half the full target. First it calls
the routine that considers points in order by score and adds them if
their distance from other points is high enough. Then calls the gap-
filling routine, but now density is computed from points on both sur-
faces so that it can fill gaps left by the beads on the minority sur-
face preferentially. Again, the gap-filling routine is called twice
with two different numbers of rings.
7.4) Phase 4: If points are still deficient, it calls the gap-filling
routine, examining points with density up to the higher fraction
(parameter 9) of the target density. A revised target is used for the
majority surface unless "-nobeef" is entered, and the original target
is used for the minority surface. Densities are computed per surface,
and the routine is called only once with the full number of rings.
7.5) Phases 5-8: If clustered and/or elongated points are allowed to be
included, then it runs the same procedure as in phase 4, first allowing
clustered points if they are allowed, and then elongated points with
progressively higher elongation numbers, which are based on the frac-
tion of tracked models in which the bead was identified as elongated.
In these phases, a larger maximum score is allowed (12 MADNS above the
median by default), since most beads with high scores fall in these
categories.
8) Accepted points are put into the output model, along with a general
value equal to the inverse of the score. With two surfaces, points on
the top surface are given surface number 1, which is assigned magenta
color.
OPTIONS
Pickbestseed 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.
-tracked (-tr) OR -TrackedModel File name
Name of tracked model file from one Beadtrack run. This entry
is needed for each run to be included in the analysis. (Succes-
sive entries accumulate)
-surface (-su) OR -SurfaceFile File name
Name of file with surface information from one Sortbeadsurfs
run. There must be the same number of surface file entries as
tracked models. (Successive entries accumulate)
-resid (-re) OR -ElongationFile File name
Name of file with residual and elongation data from one Bead-
track run. There must be the same number of elongation file
entries as tracked models. (Successive entries accumulate)
-output (-o) OR -OutputSeedModel File name
Name of final output model file
-append (-a) OR -AppendToSeedModel
Read in existing output seed model and add points to it. All
points will be retained from this model. Candidate points that
match these points will be accepted before phase 1, then the
regular sequence of phases will be followed to reach the target
number.
-size (-si) OR -BeadSize Floating point
Diameter of beads in pixels in the images where beads were found
-image (-i) OR -ImageSizeXandY Two integers
X and Y dimensions of image file used for finding and tracking
beads
-border (-bor) OR -BordersInXandY Two integers
Number of pixels to exclude on each side in X and in Y
-middle (-m) OR -MiddleZvalue Integer
Z value of middle section for tracking, numbered from 0
-zseed (-z) OR -SeedZvalue Integer
Z value of seed for one Beadtrack run, numbered from 0. If this
option is entered at all, it must be entered for each tracked
models. (Successive entries accumulate)
-two (-tw) OR -TwoSurfaces
Try to sort beads onto two surfaces then select a seed model
that has equal numbers of beads on the two surfaces if possible.
-boundary (-bou) OR -BoundaryModel File name
Name of model file whose first object contains contours enclos-
ing areas in which to use or to exclude beads, depending on
whether -exclude is entered. If more than one contour is drawn
on a view, points inside any one of the contours will be consid-
ered inside the area. This program will use only the contours
on the view closest to the middle section for tracking.
-exclude (-ex) OR -ExcludeInsideAreas
Use the contours in the boundary model to define regions to
exclude from analysis rather than regions to include.
-counting (-cou) OR -BoundaryForCounting
Use the contours in the boundary model just for counting candi-
dates inside and outside the boundary when outputting a candi-
date model.
-number (-nu) OR -TargetNumberOfBeads Integer
Desired total number of beads to choose for output seed model.
If beads are on two surfaces, the program will seek to find half
the target number on each surface, then pick more beads on
either surface to reach the target. Either this option or -den-
sity must be entered.
-density (-d) OR -TargetDensityOfBeads Floating point
Desired density of beads in final seed model per 1000 square
pixels of area, excluding the area outside boundary contours if
any. This option provides an alternative way of specifying the
target that is independent of data set size.
-nobeef (-no) OR -LimitMajorityToTarget
Do not increase the number of beads on the surface with more
beads to make up for a deficiency on the other surface. Aut-
ofidseed(1) uses this option to limit the number of beads on the
majority surface in response to its -ratio option.
-elongated (-el) OR -ElongatedPointsAllowed Integer
Enter 1, 2, or 3 to include beads identified as elongated in up
to 1/3, up to 2/3, or all of the Beadtrack runs, respectively.
-cluster (-cl) OR -ClusteredPointsAllowed Integer
Enter 1 to include clustered beads. i.e, ones that appear to be
located within 2 diameters of other beads, where foreshortening
perpendicular to the tilt axis is taken into account in comput-
ing this separation. Only one of a pair of clustered points
will be accepted. If -elongated is not entered, 2, 3, or 4 can
be entered to also include beads identified as elongated in up
to 1/3, up to 2/3, or all of the Beadtrack runs, respectively.
-lower (-l) OR -LowerTargetForClustered Floating point
Include clustered and elongated points as allowed by the -clus-
ter and -overlap options only when the total number of beads is
still below the reduced target given here. The value entered
should be in the same form as the regular target was specified,
i.e, a number of beads if -number was entered or a bead density
if -density was entered.
-rotation (-rot) OR -RotationAngle Floating point
Angle of rotation of the tilt axis in the images; specifically,
the angle from the vertical to the tilt axis (counterclockwise
positive).
-highest (-hi) OR -HighestTiltAngle Floating point
Absolute value of highest tilt angle
-weights (-w) OR -WeightsForScore Multiple floats
Alternative weights for composing a score for each candidate
bead. Enter 4 weights: for fraction of points missing in a
track; for fraction of Beadtrack runs from which the point is
missing; for mean residual during bead tracking; and for the
mean deviation between the different tracks of the same bead.
The default weights are all 1.
-control (-con) OR -ControlValue Two floats
Parameter number and value for setting algorithm control parame-
ters. Parameters and their numbers (and default values in
parentheses; float parameters have decimal points) are:
1: Deviation between points as fraction of bead diameter for
tracks to be close (0.5)
2: Multiple of target spacing at which to exclude points from
further searches (0.75)
3: Width of rings for finding points when filling gaps, as
fraction of target spacing (0.25)
4: Number of rings to search (4)
5: Maximum # of bead diameters separation for points to be con-
sidered clustered (2.0)
6: Fraction of points that must be close in two tracks for them
to be considered same (0.6)
7: Scaling factor for the two elongation criteria (parameters
12 and 16), applied to the default or entered values (1.0).
8: Maximum fraction of target density at which to add points in
initial phase (0.9)
9: Higher fraction of target at which to add points in more
desperate searches (1.1)
10: Scaling from desired spacing to H for kernel density compu-
tation (1.3)
11: Scaling from desired spacing to density grid spacing (0.2)
12: Criterion for edge SD values or elongations to be consid-
ered outliers (2.24)
13: Ratio of minority to majority for using higher density fac-
tor (0.65)
14: Fraction of nominal spacing allowed for initial addition of
points (0.85)
15: Fraction of spacing for adding best half of points on next
phase (0.7)
16: Absolute threshold for elongation to be considered overlap
(2.5)
17: Option flags: the sum of 1 for fitting elongation measures
versus bead integral and replacing measures with the residual of
the fit (which does not help), and 2 for analyzing the elonga-
tion measure in successive groups of at least 50 values, when
values are arranged in order by bead integral (which does not
help)
18: Angle to rotate plot of mean of edge SD versus SD of edge
SD to obtain an adjusted edge SD (-59.0 degrees, the mean from 7
data sets)
19: Angle to rotate plot of mean versus SD of elongation mea-
sure to obtain an adjusted elongation measure (-67.0 degrees,
the mean of 8 data sets)
20: Angle to rotate plot of adjusted elongation measure versus
adjusted edge SD to obtain the final elongation measure to ana-
lyze for outliers (45.0 degrees, corresponds to simply averaging
the two adjusted values)
21: Maximum number of MADNs above the median score allowed to
accept a candidate point in phases 1-4 (8.0).
22: Maximum number of MADNs above the median score allowed to
accept a clustered or elongated candidate point in phases 5-8
(12.0). (Successive entries accumulate)
-phase (-p) OR -PhaseOutput
Color output points by the phase in which they were added as
well as by their surface. Available colors in order are green,
magenta, yellow, cyan, red, solid blue, orange, purple, dark
blue, salmon, dark red. One or two colors will be used for each
phase, depending on whether beads are sorted into two surfaces.
If more than 10 colors are needed the 9 after green are reused.
-root (-roo) OR -DensityOutputRootname Text string
Root name for output of density maps in gnuplot format
-candidate (-ca) OR -CandidateModel File name
Filename for an output model with all candidates beads sorted by
clustering and elongation scores. Points will be assigned to up
to 8 model surfaces. Surface numbers 0 to 3 are for non-clus-
tered points with elongation values of 0 to 3, and are colored
dark green, magenta, bright green, and yellow, respectively.
Numbers 4 to 7 are for clustered points with elongation values
of 0 to 3, and are colored mustard green, red, light blue, and
orange. Open the Surface/Contour/Point dialog in 3dmod (Edit-
Surface-Go To) to navigate to contours within and between sur-
faces and to see labels for the surfaces.
-verbose (-v) OR -VerboseOutput Integer
1 for verbose output including lists of candidates and their
properties; 2 for more verbose output from addPointsInGaps rou-
tine.
-help (-he) OR -usage
Print help output
-StandardInput
Read parameter entries from standard input
AUTHOR
David Mastronarde
SEE ALSO
autofidseed
Email bug reports to mast at colorado dot edu.
IMOD 4.12.61 pickbestseed(1)