Bead Fixer Special Module
This module provides several functions to help generate tilt series fiducial
models. It has an autocentering capability, so that an added point
will be centered
on the nearest fiducial. It has an option when generating the seed model which
will automatically start a new contour for each point when appropriate.
It has a function for finding the next
gap or untracked place in the model and making that be the
current point. It also will read the log file from running
Tiltalign and move to points with high residuals, or to contours with high
mean residuals. In addition, this module
can be used to examine contours with values, adjust a threshold, and delete
contours below the threshold.
Hot Key Summary
- spacebar goes to next gap.
- ' (apostrophe) goes to next big residual (point or contour).
- " (double quote) Backs up to last point or contour.
- ; (semicolon) Moves point by residual.
- U Undoes move of current point.
- : (colon) Moves all points in current local area by residual.
- Ctrl+: (Ctrl+colon) Moves all points in current and
following local areas by residual.
- / (slash) Toggles color overlay of two views in seed mode.
Toggle the pin button on to keep the Bead Fixer window on top of
The Operation radio buttons select the mode of operation; only
the controls relevant to an operation will be shown. The three modes
of operation are making a seed model, filling gaps in a tracked model,
and fixing big residuals.
Autocentering is available when making a seed model, where it can be
quite useful. It is also available when filling gaps but is much less
likely to be useful there, except when tracking a long sequence of
Turn on Autocenter and set the Diameter spin box to the average
bead diameter (in pixels) to use autocentering. In this mode:
- When you press the second mouse button or the Insert key on the numeric
keypad, the module will search up to 3
bead diameters away (but no more than 50 pixels) for a bead and position the
new point at the center of gravity of the bead.
- The center of gravity is computed the same as in Beadtrack and will
be thrown off by adjacent density in just the same way.
- When you press the third mouse button and the Shift key, the module will
use autocentering to modify the position of the current point.
- Turn on Light to use autocentering with beads that are
lighter rather than darker than the background.
Seed Model Mode
This mode has the following features:
- Autocentering is available and should be tried.
- Points can be inserted with the Insert key on the numeric keypad.
- If you turn on Automatic new contour, the module will
start a new contour when you insert a point. It will always do this if the
current contour already has a point on the current view. Otherwise, it will
decide whether to start a new contour based on the distance between the new
point and the point in the current contour on the nearest view. If this
distance is less than two bead diameters, it will add the point to the same
contour; otherwise it will start a new contour.
- If you turn on Overlay, the current section will be
displayed along with a different view in a color overlay mode, with
one view shown in magenta and the other in green. The colors are
managed so that the beads on the current view are in magenta and those
on the other view are green,
regardless of whether the beads are light rather than dark.
You will then be
able to tell if a bead is on the top or the bottom surface by whether
it appears to the right or the left in the other view. Use the
view spin box to adjust the interval between the views;
negative numbers can be entered. You can also use the slash hot key
to toggle the overlay on and off.
- Overlay mode has in independent contrast setting, so you can
adjust the Black and White sliders to give the best definition of the
beads in overlay and this will not affect the contrast setting in
normal display. Select Reverse overlay contrast to show
the overlay in reverse contrast, which can make dark beads show up better
in a color overlay. The colors are exchanged in reverse contrast so
that the beads on the current section are still magenta.
- When you add a point to a contour, the program will automatically
keep the points in order by Z.
Gap Filling Mode
This mode provides controls for finding the gaps in the model, so that
you can fill them if appropriate.
- Autocentering is available.
- Points can be inserted with the Insert key on the numeric keypad.
- Press the Go to Next Gap button or hit the space bar key to
move to the next gap or incomplete place in the model. If there is a gap, the
point BEFORE the gap will be displayed and a upward pointing arrow will be
shown. If a contour does not start on the first section, the first
point in the contour (which is AFTER the gap) will be displayed, a message
will be printed, and a downward pointing arrow will be shown. To get to the
view with the missing point, press PageUp when the arrow points up and
PageDown when it points down.
- Use the Go to Previous Gap button to go backwards to a
- If you accidentally detach from the model point before the gap and lose
track of it, you can reattach to it with the
Reattach to Gap Point button.
- The program starts at the
beginning of the model and, after each gap, it
searches forward from where it left off, even if you change contours
or detach from the current point.
Press Start from Beginning to start the search at the
beginning of the model again. To start the search from a chosen
place, make that point be the current point and press
Start from Current Point.
- When you add a point at the beginning of a contour, the point will
automatically become the first point of the contour, thus keeping the
contour points in order by Z.
- If you skipped views when tracking and you do not want to go to those
views when filling gaps, make sure Ignore gaps at views is turned on and
insert the list of skipped views in the text box. Turn this option off if you
want to fill gaps in all the skipped views, or remove specific views from the
list if you want to fill gaps just on those views.
Residual Fixing Mode
This mode lets you correct bead positions that give large alignment
It has the following features and controls:
- Press the Open Tiltalign Log File button to open a log
file. The Info window will show the mean residuals from global and local
tracking, if any, and the number of points to be examined with big residuals.
- Press the Go to Next Big Residual button or hit the
apostrophe key to move to the next point in the log file
with a big residual. The Info window shows the residual length and X and Y
components of the residual length; these are always in pixels even when
mean residual values are shown in nanometers.
- The X and Y displacements implied by the residual are printed
in the Info window. Also, the Zap window will draw an arrow
corresponding to these displacements. If the arrow ends at the place
where the point should be located (i.e., the center of the bead),
then press the Move Point by Residual button or push the semicolon key
to move the point by those amounts in X and Y.
- After moving a point by its residual, press the Undo Move
button or push the U key to undo the move.
- Press the Reread Log File button to reread the file, or a
new version of it after rerunning Tiltalign.
- Press the Save & Run Tiltalign button to save the model, rerun
Tiltalign, and read in the new log file. Most buttons will be disabled
while Tiltalign is running and reenabled when it is done, so you will
be able to tell that something is happening. If you change the
alignment parameters in Etomo, be sure to compute the next alignment
using the button in Etomo rather than this button.
- Press the Go to Next Local Set button if you have a log file
with a series of local alignments and want to skip to the
residuals from the next local alignment.
- When local alignments were used, the next button is labeled
Move All in Local Area and can
be used to move all remaining points with big residuals in the current local
area. Without local alignments, the button is labeled Move All Points by
Residual and will move all remaining points in the big residual list
by their residuals. In this case, holding down the Shift key while pressing
this button will cause the program to iterate on this operation by saving the
model, running Tiltalign, reloading the log, and moving points by their
residual; this process repeats until there are no points left to move or you
stop holding down the Shift key.
In either case, points that would ordinarily be skipped over because they have
been examined already will not be moved. This approach should be used with
caution or abandoned in favor of robust fitting; see the section below
on Moving All Points by Residual
- When local alignments were used, Move in All Local Areas can be
used to move the remaining points in the current local area, and all points in
the following local areas, by their residuals. Again, points that have been
examined before will be skipped if appropriate. Also, holding down the Shift
key while pressing this button will cause the program to iterate on this
operation, rerunning the alignment and moving points in local areas until
there are no more points to move, or until you stop holding down the Shift key.
- Select the Back Up to Last Point button if you want to go back to
the last residual that you examined. This will not necessarily be the
previous residual in the file, if you skipped over points by going to
the next local set, or if the program skipped over points that you saw
before. However, you can then work forward through the
points that you skipped over.
- If robust fitting was used in Tiltalign, Skip low weights is
enabled, and when it is selected, the points with fitting weights less than or equal
to the value in the Threshold spin box will be skipped over when
going through the points. Turn this button on if you want to avoid the
effort of fixing points that are essentially excluded from the alignment
solution already. Turn the button off if you do want to fix such points.
The robust fitting is still valuable in this case because a fit that
excludes an aberrant point should give a more accurate estimate of the
point's correct location than a fit that includes and accommodates to the
- The program keeps a list of each point whose residual has
been examined. The list is retained when the log file is read again.
If the Examine Points Once checkbox is
selected, then any point already on the list is skipped over
when you Go to Next Big Residual. A point is added to the
list regardless of whether Examine Points Once is selected.
If you turn this checkbox off, you will see every point when going
forward or backward.
- Press the Clear Examined List button to empty the list of
points that have been examined. The list is not cleared when
Examine Points Once is switched on or off. To see some
points for the second time, turn off Examine Points Once; to
see all points again, push Clear Examined List. When you Back Up
to Last Point, the current point is removed from the list so that
you will come back to it when you go forward again.
- The Progress lines shows a percentage that is based on the
number of the current residual point divided by the total number of residuals
This mode lets you move to contours with the highest mean residuals. With a
fiducial model, you can step through all the points in such contours and
adjust ones as needed, or choose to delete the whole contour. With a model
based on local patch tracking by
Tiltxcorr, you can delete the contours
where the tracked positions are least consistent with the overall fit. In
addition to the buttons to read a log file and run Tiltalign, this mode has
the following controls:
- Press the Go to Next Contour button to move initially to the
contour with the highest mean residual, and thereafter to the contour with the
next lower mean residual. If the Model View window is open, the selected
contour will appear thicker there.
- Press the Back Up Contour button to go back to the contour with the
next higher mean residual.
- Press the Delete Contour button to delete the current contour,
provided it is still the one selected by either of the two buttons above. Use
Ctrl+Z to undo a deletion made with this button.
Moving All Points by Residual
The guiding principle when deciding whether to move one or many points by
residual is that model points should not be moved away from the gold
particles. If many points are displaced from their proper location, this will
lead to a poorer alignment of the views where such points are located
(typically the high tilt views).
The robust fitting now available in Tiltalign is a more statistically valid
method of eliminating or down-weighting points with high residuals than
blindly moving points by the residual values from a fit that includes all
the points with equal weight. The robust fitting should produce a better
estimate of the true alignment solution. Nevertheless, more experience with
this method is needed to confirm this, so
here are some guidelines for moving large
numbers of points by their residuals:
- To avoid misaligning high tilt views, where residuals tend to be higher,
in favor of the lower tilt views, you should have the large residuals be
selected relative to other residuals on nearby views. Do this by choosing
Neighboring views rather than All views in the Residual
Reporting box in Etomo.
- You should always go through enough points manually so that you can
become confident that the large majority of points are being moved correctly.
If you are just doing global alignments, perhaps do one round manually after
switching to reporting residuals relative to neighboring views. If you have a
small number of local areas, perhaps do one complete round through all of them
manually. If you have many local areas,
perhaps examine the first few points
in each local area by hand to see that the suggested movements are appropriate,
then move the rest in that area automatically. On the next round, you could
then move points in all areas automatically.
- If you know that you are going to be using local alignments, do not
move points by the global residuals automatically, as it is quite likely to
move model points off of the gold. Do the automatic refinements only with
Seed Model Mode with Contour Values
If any objects in the model contain stored values (e.g., correlation peak
strengths for beads selected by
Imodfindbeads), then in seed mode
the dialog will have additional controls for setting a threshold and deleting
points below threshold.
- The Threshold peak value slider can be used to adjust the threshold
- When Turn off below threshold is selected, only the contours above
the threshold will be drawn. This option should be on when adjusting the
threshold, but can then be toggled to see points below threshold.
- Press Delete Below to delete contours below the threshold value.
You will see a message telling how many contours will be deleted and asking
you to confirm the deletion. If you answer Yes Always then you will
not see such a message again. These deletions can be undone with the standard
Undo button in the Info Window or the Ctrl+Z hot key. If there is a Zap
window with a rubber band or lasso, then the deletion will include only the contours
inside the rubber band or lasso. Specifically, for planar contours, they must be
completely inside the rubber band or lasso; for open contours that pass between
sections, they must have a point within the rubber band or lasso.
- When Delete on all sections is turned off, contours below
threshold will be deleted
only if they have a point on the current section. With the rubber band on,
that point must be inside the rubber band. When this option is on,
contours will be deleted regardless of the Z values of their points. With the
rubber band on, there must be a point at some Z value inside the rubber band
With this option, you can delete points found by
from all planes at once.
- When Delete in all objects is turned off, only contours below
threshold in the current object are deleted. When this option is on, the
program looks at each object with stored values, and compares the values of
its contours with the threshold specific to that object. Thus, if
produced points in multiple objects, you can use this option
together with Delete on all sections to delete all contours below
threshold at once, after examining the appropriateness of the threshold for