Table 1 Glossary definitions of analysis metrics for single particle tracking
From: SlimVar for rapid in vivo single-molecule tracking of chromatin regulators in plants
Metric/Object | Definition |
|---|---|
Foci | Spot-like local intensity maxima in a single frame, each corresponding to a localised group of labelled molecules. Associated properties include spatial/temporal location, total intensity, and signal-to-noise ratio. |
Track | A set of foci in adjacent frames which are spatially close enough to form a contiguous trajectory. Associated properties include those of the set of foci, plus stoichiometry, diffusivity, and signal-to-noise ratio. |
Characteristic molecular brightness | The average number of photon counts per frame associated with a single fluorescent reporter molecule (e.g. GFP), under a fixed imaging condition. Equivalent to the number of photons in the most common intensity step observed for tracks in the final stage of photobleaching. |
Integrated nuclear intensity | The total fluorescence intensity, including autofluorescence, of an entire nuclear segment in photon counts, normalised by the characteristic molecular brightness. Dimensionless; described in fluorescent protein equivalents. |
Total protein number | The average number of labelled molecules in a nucleus, not including autofluorescence, estimated from the difference in integrated nuclear intensity from that of an unlabelled negative control. |
Stoichiometry | The number of labelled molecules in a track, as estimated by dividing the track’s initial intensity by the characteristic single-molecule brightness. |
Periodicity | The number of labelled molecules in a repeat unit within tracked objects, as estimated by the consistent stoichiometry intervals between nearest-neighbour peaks in the stoichiometry distribution. |
Diffusivity | An average measure of the rate of random microscopic motion of a track based on the increase in its mean-squared displacement over time. |
Signal-to-noise ratio (SNR) | A measure of the signal strength of foci or tracks compared to background noise. Higher SNR implies higher confidence of a true positive detection. |
Sifting | Sifting is the postprocessing step which imposes a minimum SNR threshold on foci and tracks, and a minimum track length, to improve robustness of the track-wise metrics. |
