Overview
This is the third step in automatic/interactive bead alignment. This
program attempts to dig out missing bead positions in the bead list
file created by BEAD_MATCHER.
The algorithm is to first calculate the shifts between projections.
Starting with the projection whose tilt is closest to zero, for each
projection i the shift parameters are derived from common bead
positions from the input .sl bead list between projection i and some
other projection in the searching range of n0,
or, if a .sbl file or alignment parameter file has been provided,
those are used to obtain the shift parameters. If the shift
parameters for certain projections cannot be achieved with one of
these three methods, the program will stop and ask to pick (extra)
single bead positions on these sections for a .sbl file.
Once the rough alignment parameters are available for the whole
data stack, start from the section whose tilt is closest to zero
and for section i with missing bead, Bij:
- Find the corresponding position (Bkj) on the closest
neighboring section k among the adjacent n0
projections. If Bkj is not found, Bij will not be searched.
It is optional to add the newly found positions to the .sl bead
list pool in searching Bkj for predicting Bij (this is controlled
by the new_bp toggle.
- Find the missing bead position Bij by searching a range
(set by the search range input parameter)
around the predicted position. The search procedure either uses the
local cross-correlation peak combined with other criteria on the
original projection or an adaptive threshold on the amplitude as used
in BEAD_FINDER. The reference bead
for the search can be a model bead or a region around corresponding
bead position in a previous projection (the program takes the first
found bead-like feature around the predicted position in digging each
bead position).
- With the list of features found in the previous step,
- Rank each bead-like feature by feature area minus expected
area.
- Rank each bead-like feature by its shape factor (the
ratio of its area to its circumference squared).
- Score each bead-like feature on the basis of the two calculated
ranks. The score is the simple sum of the area rank and the
shape factor rank.
- Choose the feature with the highest score.
- Merge the newly found positions with the existing list. If
any pair of positions is the same (or very close when the
reference bead is from Bkj), reject both.
Here's an example command file for BEAD_CHASER:
(time bead_chaser \
/mama/weiping/test/cent5r.stk \
/mama/weiping/test/cent5r.sl \
/mama/weiping/test/cent5r.sl2 -beadrad=3.5 \
-rot0=78:-1 -area_lub=.4:6 -shape lb=.4 \
-barea_pct_mult=2 -rad_search=8 -isblfile=none \
-xc_aat=0 -nO=10 -iv=0:22 -iref=-1
-iprmfile=none ) > /mama/weiping/BALIGN.log
IdatFile |
IslFile |
OslFile |
NX:NY:NV |
bead_radius |
rot0 |
area: lb,ub |
shape: lb |
bArea: mult_pct |
search range |
IsblFile |
chasing method |
new_bp |
nRange |
iv |
iRef |
res |
ppmin |
IprmFile |
Related Priism Topics
Priism |
Alignment |
Bead finder |
Bead matcher |
Bead align |
Bead align (interactive) |
Bead list editor |
Reconstruction
This is the name of the file containing the raw projection data stack
from the CCD (i.e. measured in terms of electron counts; data stacks with
the contrast inverted can not be handled).
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IslFile is the name of the input point list file which has the
sorted and matched bead coordinates from
BEAD_MATCHER.
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OslFile is the name of the file which BEAD_CHASER should create
to store its output (the input sorted and matched bead coordinates with
the additional beads that were found).
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The first two values are, respectively, the x and y dimension of the
projections. The third value is the number of projections in the data
stack.
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This is the average bead radius in pixel units. It sets the expected bead
area, area0, which is used to identify bead-like features. To specify this
parameter from the command line, use -beadrad=r;
the default value is 3.5 pixels.
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Let the orientation angle be the smallest, in absolute value, rotation
angle needed to rotate the image so that the tilt axis is parallel to the
y axis (the y axis is vertical when images are displayed in Priism). The
orientation angle is positive if the corresponding rotation is clockwise
and is negative if the corresponding rotation is counterclockwise.
The first value in the rot0 field is the estimated orientation
angle, in degrees, at a tilt angle of zero. The second value is the
estimated difference, in degrees, between the orientation angle at a tilt
angle of sixty degrees and the orientation angle at a tilt angle of minus
sixty degrees. To specify these parameters from the command line, use
-rot0=o0:ochange. The
default value for o0 is 78 degrees; the default value for
ochange is -1 degrees. You should substitute values that
more accurately reflect what happens at the magnification used when
collecting the tilt series.
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Sets the range on the area for a bead-like feature. The first value,
arealb in the field is the lower bound on the area;
the second value, areaub is the upper bound. Both are expressed
as multiples of the expected area, area0, which is set by
bead_radius. Thus, a bead-like feature will
have an area in square pixels between arealb*PI*bead_radius^2 and
areaub*PI*bead_radius^2.
These parameters are set on the command line with
-area_lub=arealb:areaub.
The default values are 0.4 for the lower bound and 3 for the upper bound.
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The second criteria that bead-like features must meet is that the
quantity (the shape factor), 4*PI*(feature area)/(feature circumference)^2,
must exceed a threshold. This parameter sets that threshold. By its
definition, the shape factor falls in the range [0, 1], and a circle has
a shape factor of 1.
On the command line, The threshold for the shape factor is set by
-shape_lb=lb. The default value for lb
is 0.4.
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This parameter set the upper limit on the area which is non-zero in
the binary image. If the limit, u is greater than one,
then the limit imposed on the area is u*(# of beads)*area0. If the u is less than one,
then it is used directly as the fraction of each projection which can
be non-zero.
The option -barea_pct_mult=u sets the limit from
the command line. The default value of u is two.
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This parameter sets the radius, in pixels, of the region about a
predicted bead position that is searched for a missing bead.
Use -rad_sr=search_radius
to specify this parameters on the command line. If not set, the default
is to use three times the expected bead radius
for the search range. Internally, the search radius is coerced to be
larger than two times the bead radius.
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This parameter gives the name of a file that contains the coordinates
of a single reference bead on all projections. These coordinates are used
to enable better prediction of bead positions in the matching step and can be
used to work around the problems created by large image shifts between
adjacent projections. Specifying rough alignment parameters with
IprmFile is another way to do this, but keep
in mind that the translational parameters from IsblFile have
priority over those from the alignment parameters file.
The typical way to generate IsblFile is to use
the bead list editor to interactively select
the positions.
This parameter is specified on the command line with
-isblfile=file_name. The default value is "none"
which indicates that no IsblFile is available.
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There are two options for the algorithm used to search for beads.
The first, chasing method: x-c, looks for peaks in the
cross-correlation of the projection data with the expected bead shape.
The second, chasing method: at, uses an adaptive thresholding
approach. By default, the cross-correlation algorithm is used.
On the command line, use -xc_aat=0 to select the
cross-correlation algorithm and -xc_aat=1 to select
adaptive thresholding.
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When this toggle is not set, only those locations present in the input
bead list are used when searching adjacent projections for known bead
positions in order to predict the position of a bead in a projection
where it was not found. This implies that, if a bead was only found
in projections i0 through i1 in the input list, that largest range
of projections where it will be found by BEAD_CHASER is
i0 - n0/2 to i1 + n0/2.
When the toggle on, the locations that have been found so far by
BEAD_CHASER are also considered which avoids the limitation on extending
the range of projections where a bead is found but increases the chance
the program will follow an erroneous prediction and misidentify bead locations.
On the command line, use -new_bp to use locations found
by BEAD_CHASER in addition to those present in the input list when predicting
missing bead positions. The default is not to include the newly found
locations.
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This is the number of adjacent projections to search when finding
common beads between projection to determine the shift parameters and
when finding a bead position in one projection to predict the missing
bead position in another.
The command line switch to set the number of projections searched,
n, is -n0=n. The default value for
n is ten.
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These two values, which are the index of the first projection to use
and the maximum index to use, can be used to limit which projections
BEAD_CHASER processes. This is frequently useful when experimenting with
different thresholds or bead feature criteria. The default is to process
all the projections so the index of the first projection is zero and the
maximum index is the number of projections minus one.
The range of projections to use is set from the command line with
-iv=first:last.
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This parameter sets the starting section for the algorithms to calculate
the shift parameters and locate missing beads. The default is to use the
section whose tilt angle is closest to zero.
The starting section, v, is specified on the command line with
-iref=v.
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This parameter sets which resolution to use from the input tilt series
if the input tilt series has multiple resolutions available. The resolutions
are numbered from zero with zero as the highest resolution and increasing
values correspond to decreasing resolution. The resolution to use is set
on the command line with -res=ires.
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This parameter sets a lower bound on intensity values included in the
calculation of the background statistics for the adaptive threshold search
method; it has no effect at all if you use the local cross-correlation
search method. The default value is 100 to match what previous versions
implicitly used. The command line parameter to set the lower bound is
-ppmin=bound.
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IprmFile is the name of an optional file which has the rough
alignment parameters is standard format. It can be from EM
calibration and stretched cross-correlation alignment. The alignment
parameters are used to predict the missing bead positions when
no common bead positions in the input .sl file are found in adjacent
projections and when no .sbl file is available
or it does not have beads to relate the position of the section of interest
to the previous one. If all three of these methods fail,
the program will stop and ask to pick (extra) single bead
positions on these sections for a .sbl file.
The rough alignment parameter file is set on the command line
with -iprmfile=file. The default is "none"
which specified that a parameter file is not available.
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