Clicker

Clicker is a tool made for tilt series alignment in 3D space that allows visualization of EM images and tilt series.

Table of Contents Installation Algorithm Parameters Input image types (single frame) Input image types (stack) Usage View single frame image or tilt stack Visualization settings Align tilt series stack Tilt settings Select gold beads Parameters Alignment Troubleshooting Reconstruction assistant

Installation

After setting up the CMake project, run "make Clicker" in your build directory.

Algorithm

Tilt series alignment, once fiducials have been clicked, is done through MarkerAlignator.

Parameters

Input image types (single frame)

• *.dm3 (Currently not working)
• *.dm4
• *.tif
• *.ser
• *.mrc / *.st
• *.lsm

Input image types (stack)

• *.dm3 (Currently not working)
• *.dm4
• *.mrc / *.mrcs / *.st

Usage

View single frame image or tilt stack

EM-data can be opened using the file dialogs from the File -> Open ...-menu.

Visualization settings

Center and Width	sliders permit contrast and brightness adjustment for all loaded images.
Min:	shows the minimum pixel value of the entire tilt series.
Max:	shows the maximum pixel value of the entire tilt series.
Mean:	shows the mean pixel value of the entire tilt series.
Std:	shows the standard deviation of the pixel values from the entire tilt series.
Linear interpolation	applies a linear interpolation over the loaded images so in case of a tilt series, over the entire tilt stack.
Show scale bar	reads the pixel size form the loaded image and shows a scale bar with the corresponding length value.
Gauss filter 5x5	applies a Gauss filter over the loaded images so in case of a tilt series, over the entire tilt stack.

Align tilt series stack

Tilt settings

'Page up' and 'page down' keys allow scrolling through the tilt series.

Tiltnr:	shows the tilt number of the active image and the total number of tilts.
Tilt angle:	shows the tilt angle of the active image.
Min:	shows the minimum pixel value of the active image.
Max:	shows the maximum pixel value of the active image.
Mean:	shows the mean pixel value of the active image.
Std:	shows the standard deviation of the pixel values of the active image.
Sort by tilt angle	sorts the projection in the tilt series from highest negative angle to the highest positive angle value.
Auto loop	permits looping through the tilt series.
Skip unaligned p.	if the stack was aligned it allows hiding of the unaligned projection.
Loop speed:	slider allows adjusting the speed of looping through the tilt series.

Select gold beads

Left-click on the image and scoring with the mouse wheel induces a zooming in respectively out of the image. Holding left mouse button and moving of the mouse permits translation of the image.

New-button on the lower right allows gold bead selection. After pressing 'New' in the Allingment menu Markers: should have value '1'. Usually, the first marker to select is the central gold bead on the 0° Projection. Selection is done by right-clicking on the gold bead.

Marker size: slider permits to adjust the size of the circle surrounding the gold bead.

Gold bead selection from original image - left. The corresponding fit - center. Binned and cropped version of the original image - right.

The gold bead needs to be selected through the entire tilt series. Projections that should not contribute to the final alignment should not contain the first marker.

Add-button allows adding of gold bead markers.

Remove-button allows deleting of the active gold bead marker.

Parameters

Parameters that should be set are:

• Magnification anisotropy factor
• Magnification anisotropy angle

The order of alignments should be set:

• Tilts first
• Magnification first

The total number of iterations can be set in the Iteretions:-box and after which iteration the next alignment should be done by writing a value inside the Switch at iteration:-box.

Alignment

The following alignments can be done for a tilt series:

• Tilt angles
• Magnification
• Image rotation
• Beam declination

Usually, the beam declination alignment is done first without alining for 'Tilt angles', 'Magnification' and 'Image rotation'. That for, a constant image rotation is needed (Image rotation and Constant img. rot. both checked. Tilt angles and Magnification remain unchecked).

Press Start to align. A pop out window shows the beam declination value and it is automatically saved in the Set beam decl. to: box.

The second alignment round is for Tilt angles, Magnification and Image rotation (uncheck Constant img. rot., Beam declination but the beam declination box should contain the determined value).

Magnification normalization is done during the magnification alignment procedure and it has the following options:

• Set 0° projection to 1 (the other tilts are aligned depending on the 0° projection)
• Set minimum to 1 (the other tilts are aligned depending on the projection with the smallest magnification)
• Set maximum to 1 (the other tilts are aligned depending on the projection with the biggest magnification).

Troubleshooting

After alignment, a pop-up window shows the alignment report consisting of:

• Alignment score [pixel]
• Error per projection
• Error per marker
• Marker positions

This errors can reveal problems of the tilt series, for example, a particular gold bead marker that was badly clicked or a projection image that might be problematic.

Reconstruction assistant

The reconstruction assistant creates the configuration file (EmSART (cfg file)) needed for running EmSART. Never the less it allows also reconstructions by WBP and WBP with high contrast.

Category:Software