The fundamental definition of a particle in DigImage is based on a region of an image (corrected for background and filtered as appropriate) which satisfies a given intensity threshold. Once such regions or "blobs" have been detected, their geometry is investigated to determine whether they have the expected characteristics determined by other options in this menu. Only if they satisfy all these criteria will they be considered as one, or possibly two particles.
DigImage infact utilises two distinct thresholds. Blobs satisfying all the criteria at the more stringent (higher) threshold are treated as particles unreservedly. Blobs which do not satisfy this but satisfy the less stringent (lower) threshold are treated with more caution in any matches to them. There are a number of reasons for this approach. One is that the intensity of illumination across the light sheet is not even, typically dying away towards the edges, and we may wish to be a little more cautious about matching to the edge of the region. It also provides a method of discrimating between high and low quality particles in a way which may be readily used during the final analysis of the tracking data.
In addition to satisfying the threshold criteria, a particle will generally have an average intensity which exceeds this threshold by some amount. The lower limit for this intensity excess is specified by [A Minimum average intensity excess].
This option specifies the upper, more stringent threshold. This is applied for locating particles before any attempt is made using the less stringent threshold. If, during particle location, more than 510 particles are found, then the upper and lower thresholds will be adjusted (for that buffer only) to more stringent values and the particle location repeated until 510 or fewer particles are found in the selected window. A warning is generated when this occurs.
Images are ALWAYS treated as bright particles on a dark background. If the situation is the other way around, as defined by [P Particle polarity] in this menu, then the image will be inverted prior to processing.