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.

Controls

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

• 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

• 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

• 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 previous gap.
• 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

• 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 bad position.
• 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 read in.

Contour Mode

• 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 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:

1. 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.
2. 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.
3. 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 local residuals.

Seed Model Mode with Contour Values

• The Threshold peak value slider can be used to adjust the threshold value.
• 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 Imodfindbeads 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 Imodfindbeads 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 each object.

Help Index