We applied single-molecule detection and tracking methods to study the factors that control the mobility and clustering of phagocytic Fcγ receptors (FcγR).
These problems can be overcome by single-molecule imaging and tracking, as discussed below.
As with our previous studies, diffusion trajectories of single fluorescent lipid and protein molecules were tracked and measured using the Particle Tracker plugin for ImageJ, yielding a per-frame quantitation of particle position and brightness.
The controlled photoconversion of these proteins provides unique opportunities to mark and track selected molecules in cells and organelles [14], [15].
SPECT has commonly been used to track molecules and cells including the radiolabelled annexin-V as an early marker of apoptosis [ 52].
Tracking molecules and their abundance is sometimes not an issue, but defining and acquiring phenotypic traits precisely can be very demanding.
The team identified 42 such molecules and settled on four to track with the nanoparticles: decane, trimethylbenzene, ethylbenzene, and heptanol.
In contrast to the "bulk" transport assay, the speed of barrier crossing (characteristic crossing time) can be measured directly using single molecule (sm) tracking assays, in which individual Importinβ molecules are fluorescently labeled and tracked while they cross from one side of the NPC to the other.
Single molecules were tracked and lifetimes measured using a custom Matlab script (MathWorks, Natick, MA).
Network-free simulation is a particle-based, or agent-based, approach that involves tracking individual molecules and molecular components; the cost of simulation depends on the number of molecules, molecular components, and rules considered but not the number of chemical species or reactions implied by rules [ 40, 41].
