Continuous volumetric imaging via an optical phase-locked ultrasound lens

In vivo imaging at high spatiotemporal resolution is key to the understanding of complex biological systems. We integrated an optical phase-locked ultrasound lens into a two-photon fluorescence microscope and achieved microsecond-scale axial scanning, thus enabling volumetric imaging at tens of hertz. We applied this system to multicolor volumetric imaging of processes sensitive to motion artifacts, including calcium dynamics in behaving mouse brain and transient morphology changes and trafficking of immune cells

Coexistence and efficiency of normal and anomalous transport by molecular motors in living cells

Recent experiments reveal both passive subdiffusion of various nanoparticles and anomalous active transport of such particles by molecular motors in the molecularly crowded environment of living biological cells. Passive and active microrheology reveals that the origin of this anomalous dynamics is due to the viscoelasticity of the intracellular fluid. How do molecular motors perform in such a highly viscous, dissipative environment? Can we explain the observed co-existence of the anomalous transport of relatively large particles of 100 to 500 nm in size by kinesin motors with the normal transport of smaller particles by the same molecular motors? What is the efficiency of molecular motors in the anomalous transport regime? Here we answer these seemingly conflicting questions and consistently explain experimental findings in a generalization of the well-known continuous diffusion model for molecular motors with two conformational states in which viscoelastic effects are included

Agent-Based Modeling of Intracellular Transport

We develop an agent-based model of the motion and pattern formation of vesicles. These intracellular particles can be found in four different modes of (undirected and directed) motion and can fuse with other vesicles. While the size of vesicles follows a log-normal distribution that changes over time due to fusion processes, their spatial distribution gives rise to distinct patterns. Their occurrence depends on the concentration of proteins which are synthesized based on the transcriptional activities of some genes. Hence, differences in these spatio-temporal vesicle patterns allow indirect conclusions about the (unknown) impact of these genes. By means of agent-based computer simulations we are able to reproduce such patterns on real temporal and spatial scales. Our modeling approach is based on Brownian agents with an internal degree of freedom, $\theta$, that represents the different modes of motion. Conditions inside the cell are modeled by an effective potential that differs for agents dependent on their value $\theta$. Agent's motion in this effective potential is modeled by an overdampted Langevin equation, changes of $\theta$ are modeled as stochastic transitions with values obtained from experiments, and fusion events are modeled as space-dependent stochastic transitions. Our results for the spatio-temporal vesicle patterns can be used for a statistical comparison with experiments. We also derive hypotheses of how the silencing of some genes may affect the intracellular transport, and point to generalizations of the model

Proposal for minimum information guidelines to report and reproduce results of particle tracking and motion analysis [preprint]

The proposed Minimum Information About Particle Tracking Experiments (MIAPTE) reporting guidelines described here aim to deliver a set of rules representing the minimal information required to report and support interpretation and assessment of data arising from intracellular multiple particle tracking (MPT) experiments. Examples of such experiments are those tracking viral particles as they move from the site of entry to the site of replication within an infected cell, or those following vesicular dynamics during secretion, endocytosis, or exocytosis. By promoting development of community standards, we hope that MIAPTE will contribute to making MPT data FAIR (Findable Accessible Interoperable and Reusable). Ultimately, the goal of MIAPTE is to promote and maximize data access, discovery, preservation, re-use, and repurposing through efficient annotation, and ultimately to enable reproducibility of particle tracking experiments. This document introduces MIAPTE v0.2, which updates the version that was posted to Fairsharing.org in October 2016. MIAPTE v0.2 is presented with the specific intent of soliciting comments from the particle tracking community with the purpose of extending and improving the model. The MIAPTE guidelines are intended for different categories of users: 1) Scientists with the desire to make new results available in a way that can be interpreted unequivocally by both humans and machines. For this class of users, MIAPTE provides data descriptors to define data entry terms and the analysis workflow in a unified manner. 2) Scientists wishing to evaluate, replicate and re-analyze results published by others. For this class of users MIAPTE provides descriptors that define the analysis procedures in a manner that facilitates its reproduction. 3) Developers who want to take advantage of the schema of MIAPTE to produce MIAPTE compatible tools. MIAPTE consists of a list of controlled vocabulary (CV) terms that describe elements and properties for the minimal description of particle tracking experiments, with a focus on viral and vesicular traffic within cells. As part of this submission we provide entity relationship (ER) diagrams that show the relationship between terms. Finally, we also provide documents containing the MIAPTE-compliant XML schema describing the data model used by Open Microscopy Environment inteGrated Analysis (OMEGA), our novel particle tracking data analysis and management tool, which is reported in a separate manuscript. MIAPTE is structured in two sub-sections: 1) Section 1 contains elements, attributes and data structures describing the results of particle tracking, namely: particles, links, trajectories and trajectory segments. 2) Section 2 contains elements that provide details about the algorithmic procedure utilized to produce and analyze trajectories as well as the results of trajectory analysis. In addition MIAPTE includes those OME-XML elements that are required to capture the acquisition parameters and the structure of images to be subjected to particle tracking

Plasmonic artificial virus nano-particles for probing virus-host cell interactions

Targeting of key events in viral infection pathways creates opportunities for virus disease prevention and therapy. Nanoparticles with well-defined surfaces are promising tools for the direct visualization of biological processes and for interrogating virus behavior that is usually determined by the synergistic interplay of multiple factors and involves various transient signaling steps. Smart nanoparticles mimicking enveloped viral particles are thus developed and tested in this work with the aim to de-couple key steps in human immune-deficiency virus HIV-1 trans-infection with an engineerable viral model system. Uni-lamellar liposomes resemble biological lipid bilayer membrane structures with tunable particle size, surface charge, and composition. Pretreatment with ganglioside-GM3-containing liposomes inhibited the binding of HIV-1 by dendritic cells, indicating an essential role for GM3 in virus binding. To equip the liposome based model systems with strong non bleaching optical properties, the membranes were in the next step assembled around noble metal nanoparticle core. Noble metal nanoparticles with a size of 20nm-100nm have extraordinarily large scattering cross-sections and enable prolonged tracking of even individual particles with high temporal and spatial resolutions. The plasmon resonance peak of near-field coupled gold nanoparticles red-shifts within decreasing interparticle separation. The distance dependent optical properties of noble metal nanoparticles were utilized for characterizing clustering levels of breast cancer cell marker protein CD24 and CD44 on immortalized cancer cell lines. These encouraging results supported the choice of gold nanoparticles as core for multi-modal artificial virus nanoparticles. Artificial virus nanoparticles combine the biological versatility of a self-assembled membrane with the unique optical properties of a nanoparticle core. We developed these hybrid materials specifically for the purpose of elucidating key steps of the glycoprotein independent binding and uptake of HIV-1 during trans-infection. Systematic validation experiments revealed that GM3 containing artificial virus nanoparticles (AVNs) recapitulate the initial capture and uptake of viruses by sialoadhesin CD169 presenting cells. The AVNs also reproduced the tendency of the virus to re-distribute into confined cluster spots in cell peripheral areas. Upon contact formation between T cell and DC, the AVNs developed a polarized distribution in which they enriched at the interface between DC and CD4+ T cells. The multimodality of the AVNs was instrumental in determining the detailed location and kinetics of the nanoparticles during the trans-infection process, proving the AVN system to be a unique model system to address key mechanistic questions in the infection pathway of enveloped virus particles