Trajectory Analysis in Single-Particle Tracking: From Mean Squared Displacement to Machine Learning Approaches.

Single-particle tracking is a powerful technique to investigate the motion of molecules or particles. Here, we review the methods for analyzing the reconstructed trajectories, a fundamental step for deciphering the underlying mechanisms driving the motion. First, we review the traditional analysis based on the mean squared displacement (MSD), highlighting the sometimes-neglected factors potentially affecting the accuracy of the results.

Impact of temporal resolution in single particle tracking analysis.

Temporal resolution is a key parameter in the observation of dynamic processes, as in the case of single molecules motions visualized in real time in two-dimensions by wide field (fluorescence) microscopy, but a systematic investigation of its effects in all the single particle tracking analysis steps is still lacking. Here we present tools to quantify its impact on the estimation of diffusivity and of its distribution using one of the most popular tracking software for biological applications on simulated data and movies. We found important shifts and different widths for diffusivity distributions, depending on the interplay of temporal sampling conditions with various parameters, such as simulated diffusivity, density of spots, signal-to-noise ratio, lengths of trajectories, and kind of boundaries in the simulation.

Quantitative determination of fluorescence labeling implemented in cell cultures.

Background: Labeling efficiency is a crucial parameter in fluorescence applications, especially when studying biomolecular interactions. Current approaches for estimating the yield of fluorescent labeling have critical drawbacks that usually lead them to be inaccurate or not quantitative.

Results: We present a method to quantify fluorescent-labeling efficiency that addresses the critical issues marring existing approaches.

Setting up multicolour TIRF microscopy down to the single molecule level.

Investigating biological mechanisms in ever greater detail requires continuous advances in microscopy techniques and setups. Total internal reflection fluorescence (TIRF) microscopy is a well-established technique for visualizing processes on the cell membrane. TIRF allows studies down to the single molecule level, mainly in single-colour applications.

Choosing the Probe for Single-Molecule Fluorescence Microscopy.

Probe choice in single-molecule microscopy requires deeper evaluations than those adopted for less sensitive fluorescence microscopy studies. Indeed, fluorophore characteristics can alter or hide subtle phenomena observable at the single-molecule level, wasting the potential of the sophisticated instrumentation and algorithms developed for advanced single-molecule applications. There are different reasons for this, linked, e.

Fast-diffusing p75 monomers support apoptosis and growth cone collapse by neurotrophin ligands.

The p75 neurotrophin (NT) receptor (p75) plays a crucial role in balancing survival-versus-death decisions in the nervous system. Yet, despite 2 decades of structural and biochemical studies, a comprehensive, accepted model for p75 activation by NT ligands is still missing. Here, we present a single-molecule study of membrane p75 in living cells, demonstrating that the vast majority of receptors are monomers before and after NT activation.