Accelerated Transport of Particles in Confined Channels with a High Roughness Amplitude.

We investigate the pressure-driven transport of particles 200 or 300 nm in diameter in shallow microfluidic channels ∼1 µm in height with a bottom wall characterized by a high roughness amplitude of ∼100 nm. This study starts with the description of an assay to generate cracks in hydrophilic thin polymer films together with a structural characterization of these corrugations. Microfluidic chips of variable height are then assembled on top of these rough surfaces, and the transport of particles is assessed by measuring the velocity distribution function for a set of pressure drops. We specifically detect anomalous transport properties for rough surfaces. The maximum particle velocity at the centerline of the channel is comparable to that obtained with smooth surfaces, but the average particle velocity increases nonlinearly with the flow rate. We suggest that the change in the boundary condition at the rough wall is not sufficient to account for our data and that the occurrence of contacts between the particle and the surface transports the particle away from the wall and speeds up its motion. We finally draw perspectives for the separation by field-flow fractionation.

DNA separation and enrichment using electro-hydrodynamic bidirectional flows in viscoelastic liquids.

DNA size separation followed by purification and enrichment constitute essential operations for genetic engineering. These processes are mostly carried out using DNA electrophoresis in gels or in polymer solutions, a well-established yet lengthy technique which has been notably improved using Lab-on-Chip technologies. So far, innovations for DNA separation or enrichment have been mostly undertaken separately, and we present an approach that allows us to perform these two processes simultaneously for DNA fragments spanning 0.2-50 kilo base pairs (kbp) in length. Our technology involves an electric field and a counter hydrodynamic flow in viscoelastic liquids, in which we show the occurrence of transverse forces oriented toward the walls. These forces increase with DNA molecular weight (MW) and hence induce a progressive reduction in DNA migration speed that triggers size separation in microfluidic channels as well as in capillaries. The separation of MW markers in the range 1-50 kbp is achieved in 15 minutes, thus outperforming gel electrophoresis that takes ∼3 hours for this sample. Furthermore, the use of a funnel, where electric and flow fields are modulated spatially, enables us to adjust the transverse forces so as to stall the motion of DNA molecules at a position where they accumulate at factors of up to 1000 per minute. In this configuration, we establish that the operations of DNA enrichment and separation can be carried out simultaneously for the bands of a DNA MW marker between 0.2-1.5 kbp diluted at 0.02 ng µL(-1) in 30 s. Altogether, our technology, which can readily be integrated as an in-line module in Lab-on-Chips, offers unique opportunities for sample preparation and analysis of minute genomic samples.

Metrology of confined flows using wide field nanoparticle velocimetry.

The manipulation of fluids in micro/nanofabricated systems opens new avenues to engineer the transport of matter at the molecular level. Yet the number of methods for the in situ characterization of fluid flows in shallow channels is limited. Here we establish a simple method called nanoparticle velocimetry distribution analysis (NVDA) that relies on wide field microscopy to measure the flow rate and channel height based on the fitting of particle velocity distributions along and across the flow direction. NVDA is validated by simulations, showing errors in velocity and height determination of less than 1% and 8% respectively, as well as with experiments, in which we monitor the behavior of 200 nm nanoparticles conveyed in channels of ~1.8 µm in height. We then show the relevance of this assay for the characterization of flows in bulging channels, and prove its suitability to characterize the concentration of particles across the channel height in the context of visco-elastic focusing. Our method for rapid and quantitative flow characterization has therefore a broad spectrum of applications in micro/nanofluidics, and a strong potential for the optimization of Lab-on-Chips modules in which engineering of confined transport is necessary.

Relevance and limitations of crowding, fractal, and polymer models to describe nuclear architecture.

Chromosome architecture plays an essential role for all nuclear functions, and its physical description has attracted considerable interest over the last few years among the biophysics community. These researches at the frontiers of physics and biology have been stimulated by the demand for quantitative analysis of molecular biology experiments, which provide comprehensive data on chromosome folding, or of live cell imaging experiments that enable researchers to visualize selected chromosome loci in living or fixed cells. In this review our goal is to survey several nonmutually exclusive models that have emerged to describe the folding of DNA in the nucleus, the dynamics of proteins in the nucleoplasm, or the movements of chromosome loci. We focus on three classes of models, namely molecular crowding, fractal, and polymer models, draw comparisons, and discuss their merits and limitations in the context of chromosome structure and dynamics, or nuclear protein navigation in the nucleoplasm. Finally, we identify future challenges in the roadmap to a unified model of the nuclear environment.

High-throughput chromatin motion tracking in living yeast reveals the flexibility of the fiber throughout the genome.

Chromosome dynamics are recognized to be intimately linked to genomic transactions, yet the physical principles governing spatial fluctuations of chromatin are still a matter of debate. Using high-throughput single-particle tracking, we recorded the movements of nine fluorescently labeled chromosome loci located on chromosomes III, IV, XII, and XIV of Saccharomyces cerevisiae over an extended temporal range spanning more than four orders of magnitude (10(-2)-10(3) sec). Spatial fluctuations appear to be characterized by an anomalous diffusive behavior, which is homogeneous in the time domain, for all sites analyzed. We show that this response is consistent with the Rouse polymer model, and we confirm the relevance of the model with Brownian dynamics simulations and the analysis of the statistical properties of the trajectories. Moreover, the analysis of the amplitude of fluctuations by the Rouse model shows that yeast chromatin is highly flexible, its persistence length being qualitatively estimated to <30 nm. Finally, we show that the Rouse model is also relevant to analyze chromosome motion in mutant cells depleted of proteins that bind to or assemble chromatin, and suggest that it provides a consistent framework to study chromatin dynamics. We discuss the implications of our findings for yeast genome architecture and for target search mechanisms in the nucleus.

Mapping the local organization of cell membranes using excitation-polarization-resolved confocal fluorescence microscopy.

Fluorescence anisotropy and linear dichroism imaging have been widely used for imaging biomolecular orientational distributions in protein aggregates, fibrillar structures of cells, and cell membranes. However, these techniques do not give access to complete orientational order information in a whole image, because their use is limited to parts of the sample where the average orientation of molecules is known a priori. Fluorescence anisotropy is also highly sensitive to depolarization mechanisms such as those induced by fluorescence energy transfer. A fully excitation-polarization-resolved fluorescence microscopy imaging that relies on the use of a tunable incident polarization and a nonpolarized detection is able to circumvent these limitations. We have developed such a technique in confocal epifluorescence microscopy, giving access to new regions of study in the complex and heterogeneous molecular organization of cell membranes. Using this technique, we demonstrate morphological changes at the subdiffraction scale in labeled COS-7 cell membranes whose cytoskeleton is perturbed. Molecular orientational order is also seen to be affected by cholesterol depletion, reflecting the strong interplay between lipid-packing regions and their nearby cytoskeleton. This noninvasive optical technique can reveal local organization in cell membranes when used as a complement to existing methods such as generalized polarization.

An atomic force microscopy analysis of yeast mutants defective in cell wall architecture.

Yeast cells are surrounded by a thick cell wall, the composition and structure of which have been characterized by biochemical and genetic methods. In this study, we used atomic force microscopy (AFM) to visualize the cell surface topography and to determine cell wall nanomechanical properties of yeast mutants defective in cell wall architecture. While all mutants investigated showed some alteration in cell surface topography, this alteration was particularly salient in mutants defective in beta-glucan elongation (gas1), chitin synthesis (chs3) and cross-linkages between chitin and beta-glucan (crh1crh2). In addition, these alterations in surface topology were accompanied by increased roughness of the cell. From force-indentation curves, the Young's modulus was determined, as it gives a measure of the elasticity of the cell wall. A value of approximately 1.6 MPa was obtained for the cell walls of the wild-type strain in exponential and stationary phases of growth. The same value was measured in a mnn9 mutant defective in protein mannosylation, and was two-fold reduced in a mutant with reduced beta-glucan (fks1Delta and knr4Delta), only in the stationary phase of growth. In contrast, the elasticity was dramatically reduced in mutants defective in chitin synthesis (chs3Delta), beta-glucan elongation (gas1Delta) and, even more remarkably, in a crh1Deltacrh2Delta mutant defective in the enzymes that catalyse cross-linkages of chitin to beta-glucan. Taken together, these results provide direct physical evidence that the nanomechanical properties of the yeast cell wall are mainly dependent on cross-links and cell wall remodelling, rather than on cell wall composition or thickness.