BackgroundNanoparticles can be used as markers to track the position of biomolecules, such as single proteins, inside living cells. The activity of a protein can sometimes be inferred from changes in the mobility of the attached particle. Mean Square Displacement analysis is the most common method to obtain mobility information from trajectories of tracked particles, such as the diffusion coefficient D. However, the precision of D sets a limit to discriminate changes in mobility caused by biological events from changes that reflect the stochasticity inherent to diffusion. This issue is of particular importance in an experiment aiming to quantify dynamic processes.ResultsHere, we present simulations and 3D tracking experiments with Gold Nanorods freely diffusing in glycerol solution to establish the best analysis parameters to extract the diffusion coefficient. We applied this knowledge to the detection of a temporary change in diffusion, as it can occur due to the transient binding of a particle to an immobile structure within the cell, and tested its dependence on the magnitude of the change in diffusion and duration of this event.ConclusionsThe simulations show that the spatial accuracy of particle tracking generally does not limit the detection of short binding events. Careful analysis of the magnitude of the change in diffusion and the number of frames per binding event is required for accurate quantification of such events.Electronic supplementary materialThe online version of this article (doi:10.1186/s13628-017-0035-8) contains supplementary material, which is available to authorized users.

Fluorescence correlation spectroscopy measures the time constants for rate processes giving rise to fluorescence intensity fluctuations observed from one or more microscopic sub-volumes of a nanomolar aqueous solution containing the biomolecules of interest. In our approach, the analyte molecules flow through an electrophoresis capillary under the influence of pressure driven flow or electrophoretic flow and are probed by two spatially offset probe volumes in such a way that the molecules flow sequentially from one probe volume to the next. Fluorescence fluctuations are analyzed from each probe volume independently using autocorrelation analysis, and from the two spatially offset probe volumes using cross-correlation analysis. In this way, we are able to resolve fluorescence fluctuation time constants arising from diffusion, flow, triplet blinking, and conformational fluctuations. Conformational fluctuations are monitored by quenching and unquenching of dyequencher molecules attached to DNA or RNA hairpin structures and probe the folding and unfolding kinetics of the hairpins. In this presentation, we will discuss recent results that show how base stacking within the loop region of the DNA and RNA hairpins alters the kinetics and thermodynamic stability of the hairpins. We will also discuss binding and unbinding of counterions to individual nucleotides as they flow through the capillary under the influence of an applied electric field. Emphasis will be placed on how the desired information can be extracted using our unique approach to fluorescence correlation spectroscopy.

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