By first establishing the average gray-scale range of the particles of interest, contrast can be maintained while the fine texture of the background is smoothed out. The smoothed-out background is then subtracted.
The next steps involve a procedure called segmentation, developed by Adiga and his colleagues. After the background is subtracted, the micrograph is rendered in high contrast. Only shapes of a certain size and brightness are retained; all the rest are thrown away in a step called binarization, or thresholding. "You need not know how the particle looks before you set out to pick good images of it, only how big it is," says Adiga.
The thresholding procedure is iterative, but eventually the processed high-contrast particle images can be matched unambiguously with their originals in the more highly detailed, low-contrast micrograph. Some images may still remain problematic -- for example, some particles may be so close together they appear to be touching; in these cases, an additional procedure called "pinch-off" separates candidates that aren't actually connected and discards those that are. Boxes are drawn around the final picks and their image quality is enhanced by an operation called "shrink-wrapping."
If a portion of an adjacent particle protrudes into the box, it is automatically discarded and replaced with a pattern textured like the rest of the background. At this end stage of the procedure -- although not at the beginning -- it may be advantageous to use templates (which include shape information about the particle) to refine identifications.
Scores of micrographs are needed to supply the hundreds of thousands of particles in a typical large-molecule reconstruction, but a program user needs to set parameters like particle size and gray-scale range only once, on a single micrograph. Thereafter the program runs on its own, sorting through each micrograph i
Source:DOE/Lawrence Berkeley National Laboratory