Particle Selection from a Single Image

Access: CP::Particle::Single Image (Tab)

PARTICLE supports both computational and manual particle selection from micrographs.  While computational screening has the potential for significantly higher throughput, manual selection can often achieve improved accuracy because of human's still superior ability in pattern recognition (which, unfortunately, could introduce subjective bias too).  In PARTICLE, these two functions can operate collaboratively to identify particles with both quantity and quality.  As always, visual inspection of the result is recommended because the accuracy of a computational screening highly dependents on the image quality (contrast, SNR, etc.).

EM micrographs, and cryo-EM in particular, often have low SNR.  Consequently, the result of particle screening will likely contain false-positives.  By design, PARTICLE integrates the process of particle selection, alignment and classification to identify "good" particles.  Thus, the program is able to tolerate substantial amount of false-positives in the initial particle dataset, thereby relaxing the quality requirement at the stage of particle screening (and reducing the number of false-negatives as well).  Note that a large number of false-positives will also unnecessarily increase the overall data volume.  Therefore the investigator's discretion for an appropriate balance remains indispensable.

Computational Screening

PARTICLE applies a template-matching algorithm in computational particle screening, which requires that a set of parameters to be defined in the Particle Parameter group:

• Particle Size : approximate particle diameter (in Å).
• Seperation : the minimal boundary-to-boundary distance between two particles (in Å).
• Image Binning : a downward resize factor to speed-up the screening process.  In practice, the algorithm works effectively when the binned pixel-size being around 1nm.  For large molecules, the binning factor can be even higher.
• Frame Size : the square frame dimension for particle boxing (in pixels).
• Screening Threshold : the scoring threshold for particle identification.

In addition, a set of particle templates should be defined in the Reference Image group:

• Template Stack : load a reference (image stack) for the template-matching calculation.
• Circular Mask Radius : a circular mask that encircles the region of interest in the reference images.
• Rotational Search : when checked (the default), rotational search will be activated according to the search range and angular step defined in the group below.
• Testing : when checked, all the reference images will be rotationally averaged into one frame for a quick test of the parameter setup.
• Display : a toggle button on the top, when activated, the references will be displayed in a montage.

Once the particle parameters and the reference stack have been set, the Screen button will initiate the calculation of a scoring map.  Upon completion, candidate particles will be marked according to the screening threshold.  The threshold can be modified afterwards, and the screening result will be updated dynamically via the Screen button.  The number of identified particles is displayed at both the Particle Parameter::Count and the DP::DataInfo::Particles value-boxes.

Manual Annotation

The function of manual annotation is facilitated by the following keyboard/mouse combination in the DP::MainDisplay window:

• Select Particle : CTRL+LeftClick at the spot of interest in the image.
• Delete Particle : SHIFT+LeftClick at the spot of interest in the image.
• Auto Centering : when a scoring map has been calculated, activate the auto-centering button in DP::ToolBar, and put the mouse cursor close to the spot of interest with CTRL+LeftClick, the optimal location of the particle marker will be automatically determined.
• Pan Image : LeftClick+Drag in the display window.
• Zoom Image : RightClick+Drag in the display window.
• The intensity and contrast of the image display can be adjusted via the dials at the lower-right corner of the DP window.

When CP::Particle is activated, a toolbox becomes accessible in DP::ToolBar, which contains toggle buttons (on/off) that control data visualization in particle annotation and inspection:

• : display an in-set image at the upper-left region of the DP::MainDisplay window, showing the current particle selection.
• : label the selected particles with a cross marker.
• : flash the display of particle labels (shortcut: ALT+x).
• : particle auto-centering in manual annotation (shortcut: ALT+c).
• : display the image ROI beneath the mouse cursor at the original resolution in a pop-up window (shortcut: ALT+m). The window dimension can be customized at Particle Parameter::Frame Size.
• : measure the size of an object in the image by  LeftClick+Drag to mark the dimension.  The measurement will be displayed in DP::MessageBoard by both pixels and the physical size in Angstroms.

Particle Stack

The coordinates of selected particles in a micrograph can be saved into a data file, and the particle images can be grouped into a stack.  These functions can be accessed from the buttons located at the bottom of the page:

• Load : import particle coordinates from a data file.
• Save : export the coordinates of selected particles to a file (internally named by PARTICLE).
• Clear : clear the current particle annotation and reset the particle counters (in CP and DP) to "0".
• Stack : box selected particles according to the Frame Size and group the particle images into a stack.  Then, switch to the Particle Stack Editor CP::Editor::PTK and display the particle frames in a montage.  Particles deleted in the PTK editor will be automatically synchronized with the annotation in the corresponding micrograph (if present in CP::Editor::IMG).

CMX-Mode

To run computational particle screening in the CMX-mode, the CMX parameter setup can be exported and imported via the buttons on top of the Particle Parameter group:

• : load a CMX file, which will restore the screening result from the CMX-mode into the program GUI for further processing.
• : save a CMX file for the subsequent CMX-mode execution.