High-Speed Interferometric Scattering Tracking Microscopy of Compartmentalized Lipid Diffusion in Living Cells.

Nanoscopic lipid diffusion in the live cell plasma membrane revealed by ultrafast single-molecule tracking.

The work presented here demonstrates rules of and validates models for nuclear body (NB) dynamics. Simulation tools developed in the course of this work can be used in future work to generate hypotheses about related aspects of nuclear architecture. Initially I examined the mobility of vimentin nuclear bodies bodies (VNB) in interphase by single particle tracking and analysis of fluorescence images from 4-D confocal laser scanning microscopy (CLSM). These synthetic nuclear bodies are observed in cells transfected with labelled nuclear-targeted Xenopus laevis vimentin. Analysis shows that VNBs undergo anomalous diffusion in the nuclei, independent of metabolic energy. Individual bodies display either one of the three modes of diffusion -- directed, restricted or simple. The consistency of modes and magnitudes of diffusion constants between VNBs and bona fide nuclear bodies points to a generic mechanism that mediates and regulates the mobility of nuclear bodies. Since the results of diffusion analysis of VNBs did not agree with a simple diffusion model, I tested the alternative interchromosomal domain (ICD) compartment model. The ICD model predicts that in interphase cell nuclei, individual decompacted chromosomes do not intermingle, but are separated by a significant interchromatin space forming a network of channels. These networks could affect the mobility of nuclear bodies. Monte Carlo simulations that predict the effects of channels and other obstructions on NB diffusion were tested, but they could not explain deviation from ideal behaviour. Fitting an empirical model of `critical diffusion' produced similar results. Therefore the ICD model as a purely obstructing network of channels needs modification, to possibly include binding. To examine the role of chromatin density in intra-nuclear diffusion, I employed multidimensional fluorescence recovery after photobleaching (FRAP) in living cells. The influence of chromatin density on diffusive mobility of the nuclear yellow fluorescent protein (YFP) appears marginal. A 2-D diffusion simulation to better characterize the experiment provides a tool to produce `diffusion maps' of the nucleus. The related aspect of nuclear body integrity and dynamics was examined for the distribution of topoisomerase II beta (TopoIIb), which localizes preferentially in the nucleolus. The experimentally observed diffusion and binding dynamics were formulated as a compartment model and fitted to the experiments. The model topology, flux constants and residence times estimates could be validated, providing a predictive model of TopoIIb dynamics By demonstrating that VNB diffusion is anomalous and consistent with other bona fide NBs, I have revealed a mechanism that regulates NB mobility. The diffusion of these bodies deviates however from ideal diffusion, and can be explained by neither the effect of chromatin density on molecular diffusion, nor the different models of NB diffusion. I have shown that binding rather than diffusion appears to determine nuclear body localization and dynamics, as in the case of TopoIIb. Nuclear bodies and nuclear architecture has been recently hypothesized as emerging from simple local interactions. The predictive model for TopoIIb distribution dynamics provides evidence for this. The models presented here, are in keeping with the increasing trend to abstract nuclear dynamics as mathematical models. It is hoped that the work presented here will contribute to the effort of arriving at an integrated model for nuclear bodies and therefore better understanding nuclear architecture.

The diffusion dynamics in the cellular plasma membrane provide crucial insights into molecular interactions, organization, and bioactivity. Beam-scanning fluorescence correlation spectroscopy combined with super-resolution stimulated emission depletion nanoscopy (scanning STED–FCS) measures such dynamics with high spatial and temporal resolution. It reveals nanoscale diffusion characteristics by measuring the molecular diffusion in conventional confocal mode and super-resolved STED mode sequentially for each pixel along the scanned line. However, to directly link the spatial and the temporal information, a method that simultaneously measures the diffusion in confocal and STED modes is needed. Here, to overcome this problem, we establish an advanced STED–FCS measurement method, line interleaved excitation scanning STED–FCS (LIESS–FCS), that discloses the molecular diffusion modes at different spatial positions with a single measurement. It relies on fast beam-scanning along a line with alternating laser illumination that yields, for each pixel, the apparent diffusion coefficients for two different observation spot sizes (conventional confocal and super-resolved STED). We demonstrate the potential of the LIESS–FCS approach with simulations and experiments on lipid diffusion in model and live cell plasma membranes. We also apply LIESS–FCS to investigate the spatiotemporal organization of glycosylphosphatidylinositol-anchored proteins in the plasma membrane of live cells, which, interestingly, show multiple diffusion modes at different spatial positions.

Localization Error and Fitting Model Evaluation in Single Particle Tracking

Lateral diffusion measurements have been made on lipids and proteins in the plasma membrane of live protoplasts derived from rose (Rosa sp. “Paul's Scarlet”) suspension-cultured cells. Two different fluorescent lipid probes exhibited markedly different diffusion rates, indicating possible heterogeneity in the lipid domain of the membrane. Membrane proteins were labeled directly with covalently-reactive fluorophores, and factors that might perturb the lateral diffusion of these labeled proteins were investigated. Treatment of the protoplasts with various cytoskeleton-disrupting drugs generally had little effect on protein diffusion, although treatment with oryzalin, a microtubule-disrupting drug, did slightly reduce the mobile fraction of membrane proteins. Elevation of the CaCl2 concentration in the medium from 1 mM to 10 mM significantly reduced the mobile fraction of membrane proteins and also increased the fraction of protoplasts that were able to regenerate cell walls and divide in culture. These results are discussed in relation to reported evidence of lipid domains in the plasma membranes of other cells and protoplasts. The relative importance of lipid domains and membrane-cytoskeleton interaction in governing protein diffusion is considered.

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An investigation into how lipids diffuse in the presence of varying protein:lipid number ratios reveals that protein crowding results in drastically different lipid and protein diffusion due to the strengthened impact of correlated motion.

Lateral diffusion plays a crucial role in numerous processes that take place in cell membranes, yet it is quite poorly understood in native membranes characterized by, e.g., domain formation and large concentration of proteins. In this article, we use atomistic and coarse-grained simulations to consider how packing of membranes and crowding with proteins affect the lateral dynamics of lipids and membrane proteins. We find that both packing and protein crowding have a profound effect on lateral diffusion, slowing it down. Anomalous diffusion is observed to be an inherent property in both protein-free and protein-rich membranes, and the time scales of anomalous diffusion and the exponent associated with anomalous diffusion are found to strongly depend on packing and crowding. Crowding with proteins also has a striking effect on the decay rate of dynamical correlations associated with lateral single-particle motion, as the transition from anomalous to normal diffusion is found to take place at macroscopic time scales: while in protein-poor conditions normal diffusion is typically observed in hundreds of nanoseconds, in protein-rich conditions the onset of normal diffusion is tens of microseconds, and in the most crowded systems as large as milliseconds. The computational challenge which results from these time scales is not easy to deal with, not even in coarse-grained simulations. We also briefly discuss the physical limits of protein motion. Our results suggest that protein concentration is anything but constant in the plane of cell membranes. Instead, it is strongly dependent on proteins' preference for aggregation.

Machine learning-informed timescale dependent modes of nanoparticle diffusion through human mucus

Anionic Lipid-Dependent Gliding of Cytochrome C across Bioenergetic Membranes

Macromolecular Crowding and Size Effects on Probe Microviscosity

Lateral Membrane Diffusion Corralled

The introduction of Gold NanoParticles (GNPs) in radiotherapy treatments necessitates considerations such as GNP size, location, and quantity, as well as patient geometry and beam quality. Physics considerations span length scales across many orders of magnitude (nanometer-to-centimeter), presenting challenges that often limit the scope of dosimetric studies to either micro- or macroscopic scales. To investigate GNP dose-enhanced radiation Therapy (GNPT) through Monte Carlo (MC) simulations that bridge micro-to-macroscopic scales. The work is presented in two parts, with Part I (this work) investigating accurate and efficient MC modeling at the single cell level to calculate nucleus and cytoplasm Dose Enhancement Factors (n,cDEFs), considering a broad parameter space including GNP concentration, GNP intracellular distribution, cell size, and incident photon energy. Part II then evaluates cell dose enhancement factors across macroscopic (tumor) lengthscales. Different methods of modeling gold within cells are compared, from a contiguous volume of either pure gold or gold-tissue mixture to discrete GNPs in a hexagonal close-packed lattice. MC simulations with EGSnrc are performed to calculate n,cDEF for a cell with radius µm and nucleus µm considering 10to 370keV incident photons, gold concentrations from 4 to 24mgAu /gtissue , and three different GNP configurations within the cell: GNPs distributed around the surface of the nucleus (perinuclear) or GNPs packed into one (or four) endosome(s). Select simulations are extended to cells with different cell (and nucleus) sizes: 5µm (2, 3, and 4µm), 7.35µm (4 and 6µm), and 10µm (7, 8, and 9µm). n,cDEFs are sensitive to the method of modeling gold in the cell, with differences of up to 17% observed; the hexagonal lattice of GNPs is chosen (as the most realistic model) for all subsequent simulations. Across cell/nucleus radii, source energies, and gold concentrations, both nDEF and cDEF are highest for GNPs in the perinuclear configuration, compared with GNPs in one (or four) endosome(s). Across all simulations of the (rcell , rnuc ) = (7.35, 5)µm cell, nDEFs and cDEFs range from unity to 6.83 and 3.87, respectively. Including different cell sizes, nDEFs and cDEFs as high as 21.5 and 5.5, respectively, are observed. Both nDEF and cDEF are maximized at photon energies above the K- or L-edges ofgold by 10 to 20 keV. Considering 5000 unique simulation scenarios, this work comprehensively investigates many physics trends on DEFs at the cellular level, including demonstrating that cellular DEFs are sensitive to gold modeling approach, intracellular GNP configuration, cell/nucleus size, gold concentration, and incident source energy. These data should prove especially useful in research as well as treatment planning, allowing one to optimize or estimate DEF using not only GNP uptake, but also account for average tumor cell size, incident photon energy, and intracellular configuration of GNPs. PartII will expand the investigation, taking the PartI cell model and applying it in cm-scalephantoms.

In the present paper, some imaging properties of nanoparticles-based contrast agents including gold, bismuth, and silver were assessed and compared with conventional (iodinated) contrast agent in spectral computed tomography (CT). A spectral CT scanner with photon-counting detectors (PCD) and 6 energy bins was simulated using the Monte Carlo (MC) simulation method. The nanoparticles were designed with a diameter of 50 nm at concentrations of 2, 4, and 8 mg/ml. Water-filled cylindrical phantom was modeled with a diameter of 10 cm containing a hole with a diameter of 5 cm in its center, where was filled with contrast agents. The MC results were used to reconstruct images. Image reconstruction was accomplished with the filtered back-projection (FBP) method with hamming filter and linear interpolation method. CT number and contrast-to-noise ratio (CNR) of all studied contrast materials were calculated in spectral images. The simulations indicated that nanoparticle-based contrast agents have a higher CT number and CNR than the iodinated contrast agent at the same concentration and for all energy bins. In general, gold nanoparticles produced the highest CT number and CNR compared to silver and bismuth nanoparticles at the same concentration. However, at low energies (below 80 keV), silver nanoparticles performed similarly to gold nanoparticles and at high energies (120 keV), bismuth nanoparticles can be a good substitute for gold nanoparticles.

A photonic resonator interferometric scattering microscope for label-free detection of nanometer-scale objects with digital precision in point-of-use environments