Define a Template Stack

Template (a.k.a. reference) images should be in the form of an MRC stack, which can be created in several ways:

1. systematic 2D projections from a 3D model reconstruction;
2. class averages from a set of pre-selected particles;
3. representative particles manually selected from a few micrographs (see the next section on Manual Particle Selection).

Input A Template Stack

An MRC template stack (3D) can be opened from Data: Template: Load MRC. Upon completion, the system will automatically switch to Template OpPanel. In order to obtain meaningful correlation statistics, each template image should have at least 32x32 pixels. It is critical that the contrast (namely black or white particles) of the template images should be the same as that of the micrograph.

A set of image processing functions - for example, band-pass filtering, image resampling, rotational averaging - can be accessed from Image Stack Editor for template pre-processing. When necessary, load the template stack into the editor first, and output (the Save button) the processed image stack to a new MRC file.

Circular Masking

Both LCF and SCF Circular Profile radii should be comparable to the particle size. SCF Center Blocking masks out the central region of the auto-correlation function, which is always close to unity. This procedure can improve the sensitivity of the template-matching algorithm. Click Apply to update the specified parameters.

Manual Masking

The mask of each template image can be manually customized. Set Frame (press ENTER to activate) and click the Edit button, the specified template frame will be displayed with its mask and alignment center overlaid. Use the drawing tools (accessed from Toolbar) to make necessary modification. The alignment center (see below) can be changed to a new location by pressing CTRL and left-click. The mask display can be toggled by the F1-key, and the alignment center display can be toggled by the F2-key. Upon completion, deactivate the Edit button to update the setup.
The Sync button will apply the masking of the currently selected frame to all the succeeding frames. The Clear button will clear the masks of all template frames. When the L2 masking is necessary (see below), change the masking level to L2 from Target and repeat the manual masking procedure.

Level-II Screening

Signature supports a coarse-to-fine template-matching procedure to improve the accuracy of automated screening. The level-I (L1) mask normally contains a larger region for feature recognition, and the level-II (L2) mask zooms in to finer detail of the particle of interest. The scores from both levels will be combined at the end for the automated screening. To include level-II screening, check Activate L2 Screening box at the bottom of the panel.

Rotational Search

Rotational interval varies, depending on the expected accuracy. The finest step size can be estimated from the pixel size and the particle size. For example, given a particle of ~100 Å in diameter inside a 5 Å/pixel micrograph, a 5-degree rotational interval should suffice. No rotational search would be necessary if the template images have already been rotationally averaged. In that case, uncheck the Rotational Search box.