Algorithmic Screening

The first step of the automated screening is to evaluate the Local-Correlation-Function (LCF) between the micrograph and the specified template images. The threshold of acceptable LCF score can be set by the slidebar according to the displayed maximum and average values. The number of particles with LCF score above the threshold will be counted.

For the micrograph spots passing the LCF test, the second step of the automated screening is to evaluate the Spectrum-Correlation-Function (SCF) between the micrograph and the specified template images. The threshold of acceptable SCF score can be set by the slidebar according to the displayed maximum and average values. The number of particles with SCF score above the threshold will be counted.

The third step of the automated screening is to restrain the inter-particle boundary distance to eliminate particle overlapping. The parameter is set at Dist in the Particle Parameters box.

Test Run & Full-Range Screening

Before starting, carefully choose the downsize factor so that the resulting image has a resolution comparable to that expected for final model reconstruction. A smaller image will result in faster computing. Computerized particle selection will be labeled by red crosses.

Test screening () provides an "applicability" check. It computes the sum of rotational average of all template images and uses that as a single template image to screen the given micrograph. The user can judge the quality of the template images and set appropriate expectations according to the score printout.

Screening conducts a full-range automated screening according to the search parameters defined in the Template OpPanel. A full screening will be performed afresh when certain program parameters are changed: the downsize factor, the template dimension, the rotational search range and increment, and the LCF/SCF profile masking. In order to avoid time-consuming repetition, it is critical to set up these parameters properly at the start. If the screening thresholds are too low, some selection markers might appear at the boundary of true particles instead of at the center. Whenever this occurs, the LCF and SCF thresholds should be raised. Depending upon the accuracy to be achieved, the full-range screening may take a long time to complete. In this case, batching processing and distributed computing are recommended.

Upon completion, the total number of selected particles is displayed at Particle Count. These particles can be saved into a coordinate file and/or a particle stack. When saving to a particle stack, the image frame size can be customized at Frame in the Particle Parameter box. A functional button () is also provided to save the selected particles to a stack and then switch directly to the Image Stack Editor.

Manual Particle Selection

Some particle candidates might be visually acceptable but excluded nonetheless by the algorithmic screening. In that case, the user will have the option to select them manually. Manual particle selection is marked by blue crosses. Two monitors are provided to facilitate this effort by displaying particle images at various scales and blurring levels. Press CTRL and left-click to select, and press SHIFT and left-click to delete. Both operations can occur in any order, at any micrograph location. To assist precise positioning, a floating magnifier (accessed from Toolbar) is provided to zoom the region under the mouse cursor to its original resolution. Press CTRL and left-click, a marker will be drawn beneath the current cursor position.

In addition to the simple manual operation, the program also provides an CO-OP selection function under the condition that the micrograph has been screened with templates. Press ALT and left-click, the micrograph region under the mouse cursor will be searched automatically to identify the highest score. The corresponding spot will then be labeled as a particle candidate. (On certain keyboards, the ALT-key might need to be replaced by either the META-key or the Windows-key.)

Inspection Monitor

An inspection of those selected particles is optional but recommended. The Accept (CTRL+a) and Reject (CTRL+r) buttons are provided to navigate sequentially through the existing selection. The Undo (CTRL+u) button is for one-step back track only. The confirmed particle selection will be labeled by green crosses.

Imagery content of a micrograph can be examined at various scales on the two monitors. Images in each monitor can be independently resized and blurred (press ENTER key to activate a new parameter). There are four display-mode buttons (labeled +, +, S and T) equipped with the monitors. The "+" buttons are to toggle the cross-hair, and the distance between the pair of short bars on the cross-hair corresponds to the user-defined particle diameter. The "S" button is to set spectrum display instead of the image itself. The "T" button is to display the best matching template image. When "S" is on, the intensity and contrast adjustment knobs are associated with spectrum display; when "T" is on, the adjust knobs are associated with template image display.