FluoSim: simulator of single molecule dynamics for fluorescence live-cell and super-resolution imaging of membrane proteins

Abstract

Fluorescence live-cell and super-resolution microscopy methods have considerably advanced our understanding of the dynamics and mesoscale organization of macro-molecular complexes that drive cellular functions. However, different imaging techniques can provide quite disparate information about protein motion and organization, owing to their respective experimental ranges and limitations. To address these limitations, we present here a unified computer program that allows one to model and predict membrane protein dynamics at the ensemble and single molecule level, so as to reconcile imaging paradigms and quantitatively characterize protein behavior in complex cellular environments. FluoSim is an interactive realtime simulator of protein dynamics for live-cell imaging methods including SPT, FRAP, PAF, and FCS, and super-resolution imaging techniques such as PALM, dSTORM, and uPAINT. The software, thoroughly validated against experimental data on the canonical neurexinneuroligin adhesion complex, integrates diffusion coefficients, binding rates, and fluorophore photo-physics to calculate in real time the distribution of thousands of independent molecules in 2D cellular geometries, providing simulated data of protein dynamics and localization directly comparable to actual experiments.

MDGAs are fast-diffusing molecules that delay excitatory synapse development by altering neuroligin behavior

“…dSTORM stacks were analyzed using the PALM-Tracer program, allowing the reconstruction of a unique super-resolved image of 32 nm pixel size (zoom 5 compared to the original images) by summing the intensities of all localized single molecules (1 detection per frame is coded by an intensity value of 1). The localization precision of our imaging system in dSTORM conditions is around 60 nm (FWHM) 71 . For the analysis protein enrichment at post-synapses, the average number of detections within Homer1c puncta was divided by the the average number of extra-synaptic detections, both normalized per unit area.…”

Dense Mapping of Intracellular Diffusion and Drift from Single-Particle Tracking Data Analysis

“…Evaluation on simulated datasets First, we used the Flu-oSIM software [13] to generate simulated fluorescence microscopy tracking data, by drawing a representation of a 2D crossbow-shaped cell (Fig. 1).…”

A computational approach for detecting micro-domains and confinement domains in cells: a simulation study

“…2, we illustrate the estimation of drift from trajectories on a artificial sequence depicting two circular trapping areas. A general view of trapping areas with variable sizes generated with Fluosim [Lagardère et al, 2019], is shown in Fig. 1(b)).…”

“…In Section 3, we present related works and focus on the Eulerian method proposed by [Hoze et al, 2012]; we emphasize the underlying assumptions, the key parameters, and the properties of this approach used to detect potential wells within cells. In Section 4, we describe the simulation framework corresponding to the Fluosim simulator [Lagardère et al, 2019] used in our experiments. In Section 5, we describe the procedure that combines the Dbscan clustering algorithm [Ester et al, 1996] with the test procedure described in [Briane et al, 2018].…”