Determination of diffusion coefficient by image-based fluorescence recovery after photobleaching and single particle tracking system implemented in a single platform

Fluorescence recovery after photobleaching(FRAP)and single particle tracking(SPT)techni-ques determine the diffusion coefficient from average diffusive motion of high-concentration molecules and from trajectories of low-concentration single molecules,respectively.Lateral dif-fusion coefficients measured by FRAP and SPT techniques for the same biomolecule on cell membrane have exhibited inconsistent values across laboratories and platforms with larger dif-fusion coefficient determined by FRAP,but the sources of the inconsistency have not been investigated thoroughly.Here,we designed an image-based FRAP-SPT system and made a direct comparison between FRAP and SPT for diffusion coefficient of submicron particles with known theoretical values derived from Stokes-Einstein equation in aqueous solution.The combined iFRAP-SPT technique allowed us to measure the diffusion coefficient of the same fluorescent particle by utilizing both techniques in a single platform and to scrutinize inherent errors and artifacts of FRAP.Our results reveal that diffusion coefficient overestimated by FRAP is caused by inaccurate estimation of the bleaching spot size and can be corrected by simple image analysis.Our iFRAP-SPT technique can be potentially used for not only cellular membrane dynamics but also for quantitative analysis of the spatiotemporal distribution of the solutes in small scale analytical devices.

Dynamic tracking and mobility analysis of single GLUT4 storage vesicle in live 3T3-L1 cells

Glucose transporter 4 (GLUT4) is responsible for insulin-stimulated glucose transporting into the insulin-sensitive fat and muscle cells. The dynamics of GLUT4 storage vesicles (GSVs) remains to be explored and it is unclear how GSVs are arranged based on their mobility. We examined this issue in 3T3-L1 cells via investigating the three-dimensional mobility of single GSV labeled with EGFP-fused GLUT4. A thin layer of cytosol right adjacent to the plasma membrane was illuminated and successively imaged at 5 Hz under a total internal reflection fluorescence microscope with a penetration depth of 136 nm. Employing single particle tracking, the three-dimensional subpixel displacement of single GSV was tracked at a spatial precision of 22 nm. Both the mean square displacement and the diffusion coefficient were calculated for each vesicle. Tracking results revealed that vesicles moved as if restricted within a cage that has a mean radius of 160 nm, suggesting the presence of some intracellular tethering matrix. By constructing the histogram of the diffusion coefficients of GSVs, we observed a smooth distribution instead of the existence of distinct groups. The result indicates that GSVs are dynamically retained in a continuous and wide range of mobility rather than into separate classes.

Single virus tracking of Ebola virus entry through lipid rafts in living host cells

Ebola virus(EBOV)is one of the most pathogenic viruses in humans which can cause a lethal hemorrhagic fever.Understanding the cellular entry mechanisms of EBOV can promote the development of new therapeutic strategies to control virus replication and spread.It has been known that EBOV virions bind to factors expressed at the host cell surface.Subsequently,the virions are internalized by a macropinocytosis-like process,followed by being trafficked through early and late endosomes.Recent researches indicate that the entry of EBOV into cells requires integrated and functional lipid rafts.Whilst lipid rafts have been hypothesized to play a role in virus entry,there is a current lack of supporting data.One major technical hurdle is the lack of effective approaches for observing viral entry.To provide evidence on the involvement of lipid rafts in the entry process of EBOV,we generated the fluorescently labeled Ebola virus like particles(VLPs),and utilized single-particle tracking(SPT)to visualize the entry of fluorescent Ebola VLPs in live cells and the interaction of Ebola VLPs with lipid rafts.In this study,we demonstrate the compartmentalization of Ebola VLPs in lipid rafts during entry process,and inform the essential function of lipid rafts for the entry of Ebola virus.As such,our study provides evidence to show that the raft integrity is critical for Ebola virus pathogenesis and that lipid rafts can serve as potential targets for the development of novel therapeutic strategies.

Reveal heterogeneous motion states in single nanoparticle trajectory using its own history

Single particle tracking(SPT)has long been utilized for investigation of complex system dynamics such as nanoparticle-cell interaction,however,the analysis of individual particle motions is always a difficult issue.Existing methods treat each data point or fragment on the recorded trajectory as an isolated"atom"and determine their relationship based on externally predefined models or physical states,which inevitably lead to oversimplification of the associated spatiotemporal complexity.Herein,inspired by the historical analysis in social science,we propose a modeless preprocessing framework for SPT analysis based on the"history"of the particle.This new strategy consists of 3 steps:(1)assign a"history"to each data point and construct successive overlapped historical vectors;(2)perform unsupervised clustering in the vector space to find their relative differences;(3)project differences back to the trajectory by coloring each point accordingly for visualization.As a result,the inner heterogeneity of the particle motion self-emerges as a colored trajectory,exhibiting a global picture of the local state transitions and providing valuable information for further model-based analysis.Since the complexity issues at various spatiotemporal scales have attracted increasing attention,and individual objects such as single molecules,cells,vehicles and even stars in the universe could all be treated as"single particles",this presuppositionless data preprocessing approach could help the investigations of many complex systems in fundamental research.

United under stress:High-speed transport network emerging at bacterial living edge

Individuals tend to move freely when there is enough room but would act collectively for their survival under external stress.In the case of living cells,for instance,when a drop of low-density flagellated bacterial solution is transferred onto the agar surface,the initially disordered movement of individual bacteria would be replaced with coordinated cell swarming after a lag phase of a few hours.Here,we study how such cooperation is established while overcoming the disorder at the onset of the lag phase with single nanoparticle tracking.Upon the spreading of the droplet,the bacteria in the solution cluster and align near the almost immobilized contact line confining the drop,forming a narrow ring of cells.As individual cells move in and out of the ring continuously,certain flow patterns emerge in the inter-bacterial fluid.We reveal high-speed long-distance unidirectional flows with definite chirality along the outside of the ring,along the inside of the ring and across the ring.We speculate that these flows enable the fast and efficient transport,facilitating the communication and unification of the bacterial community.

Recent advances in self-propelled particles

"Active" components can be introduced into a passive system to completely change its physical behavior from its typical behavior at thermodynamic equilibrium. To reveal the interaction mechanisms between individuals, researchers have designed unique self-propelled particles to mimic the collective behavior of biological systems. This review focuses on recent theoretical and experimental advances in the study of self-propelled particle systems and their individual and collective behaviors. The potential applications of active particles in chemical, biological and environmental sensing and single particle imaging are discussed.