Cavity-Amplified Scattering Spectroscopy Reveals the Dynamics of Proteins and Nanoparticles in Quasi-transparent and Miniature Samples.

Dynamic light scattering techniques can give access to the motion spectrum of microscopic objects and are therefore routinely used for numerous industrial and research applications ranging from particle sizing to the characterization of the viscoelastic properties of materials. However, such measurements are impossible when samples do not scatter light enough, i.e., when light undergoes too few scattering events when passing through a sample, either due to excessively small scattering cross sections or due to low concentrations of scatterers. Here, we propose to amplify the light scattering efficiency by placing weakly scattering samples inside a Lambertian cavity with high-reflectance walls. When injected with laser light, the cavity produces a 3D isotropic and homogeneous light field, effectively elongates the photon scattering path length through the sample by 2-3 orders of magnitude, and leads to a dramatic increase in sensitivity. With a 104-fold increase in sensitivity compared to classical techniques, we potentially expand the applications of light scattering to miniaturized microfluidics samples and to weakly scattering samples in general. We show that we can access the short-time dynamics of low-turbidity samples and demonstrate our sensitivity gain by measuring the diffusion coefficient and, therefore, the size of particles ranging from 5 nm to 20 µm with volume fractions as low as 10-9 in volumes as low as 100 µL and in solvents with refractive index mismatches down to Δn ≈ 0.01. Beyond the realm of current applications of light scattering techniques, our cavity-amplified scattering spectroscopy method (CASS) and its high sensitivity represent a significant methodological step toward the study of short-time dynamics problems such as the ballistic limit of Brownian motion, the internal dynamics of proteins, or the dielectric dynamics of liquids.

Heterogeneity is not always a source of noise: Stochastic gene expression in regulatory heterozygote.

Zygosity of diploid genome (i.e., degree to which two parental alleles of a gene have varied genetic sequences) adds another dimension to stochastic gene expression. The allelic imbalance in chromatin accessibility or divergence in regulatory sequences leads to fitness effects but the quantitative aspects thereof are largely left unexplored. We investigate diploid gene expression systems with homozygous (the same) and heterozygous (varied) combination of alleles in cis-regulatory sequences, not in structural gene loci, and characterize the zygosity-associated stochastic fluctuations in protein abundance. An emerging feat of heterozygosity is its counterintuitive capacity for genetic noise control. Especially when the noise is dominantly contributed to by the fluctuations in duty cycle ("reliability") rather than in process speed ("productivity") of gene expression machinery, its interallelic discrepancy acts to reduce the gene expression noise. These findings offer a novel insight into the rich repertoire of balancing selection enriched in the regulatory elements of immune response genes.

Stochastic light concentration from 3D to 2D reveals ultraweak chemi- and bioluminescence.

For countless applications in science and technology, light must be concentrated, and concentration is classically achieved with reflective and refractive elements. However, there is so far no efficient way, with a 2D detector, to detect photons produced inside an extended volume with a broad or isotropic angular distribution. Here, with theory and experiment, we propose to stochastically transform and concentrate a volume into a smaller surface, using a high-albedo Ulbricht cavity and a small exit orifice through cavity walls. A 3D gas of photons produced inside the cavity is transformed with a 50% number efficiency into a 2D Lambertian emitting orifice with maximal radiance and a much smaller size. With high-albedo quartz-powder cavity walls ([Formula: see text]), the orifice area is [Formula: see text] times smaller than the walls' area. When coupled to a detectivity-optimized photon-counter ([Formula: see text]) the detection limit is [Formula: see text]. Thanks to this unprecedented sensitivity, we could detect the luminescence produced by the non-catalytic disproportionation of hydrogen peroxide in pure water, which has not been observed so far. We could also detect the ultraweak bioluminescence produced by yeast cells at the onset of their growth. Our work opens new perspectives for studying ultraweak luminescence, and the concept of stochastic 3D/2D conjugation should help design novel light detection methods for large samples or diluted emitters.

A role for Dynlt3 in melanosome movement, distribution, acidity and transfer.

Skin pigmentation is dependent on cellular processes including melanosome biogenesis, transport, maturation and transfer to keratinocytes. However, how the cells finely control these processes in space and time to ensure proper pigmentation remains unclear. Here, we show that a component of the cytoplasmic dynein complex, Dynlt3, is required for efficient melanosome transport, acidity and transfer. In Mus musculus melanocytes with decreased levels of Dynlt3, pigmented melanosomes undergo a more directional motion, leading to their peripheral location in the cell. Stage IV melanosomes are more acidic, but still heavily pigmented, resulting in a less efficient melanosome transfer. Finally, the level of Dynlt3 is dependent on ß-catenin activity, revealing a function of the Wnt/ß-catenin signalling pathway during melanocyte and skin pigmentation, by coupling the transport, positioning and acidity of melanosomes required for their transfer.

Detectivity optimization to detect of ultraweak light fluxes with an EM-CCD as binary photon counter array.

For a wide range of purposes, one faces the challenge to detect light from extremely faint and spatially extended sources. In such cases, detector noises dominate over the photon noise of the source, and quantum detectors in photon counting mode are generally the best option. Here, we combine a statistical model with an in-depth analysis of detector noises and calibration experiments, and we show that visible light can be detected with an electron-multiplying charge-coupled devices (EM-CCD) with a signal-to-noise ratio (SNR) of 3 for fluxes less than [Formula: see text]. For green photons, this corresponds to 12 aW [Formula: see text] ≈ [Formula: see text] lux, i.e. 15 orders of magnitude less than typical daylight. The strong nonlinearity of the SNR with the sampling time leads to a dynamic range of detection of 4 orders of magnitude. To detect possibly varying light fluxes, we operate in conditions of maximal detectivity [Formula: see text] rather than maximal SNR. Given the quantum efficiency [Formula: see text] of the detector, we find [Formula: see text], and a non-negligible sensitivity to blackbody radiation for T > 50 °C. This work should help design highly sensitive luminescence detection methods and develop experiments to explore dynamic phenomena involving ultra-weak luminescence in biology, chemistry, and material sciences.

Super-resolution provided by the arbitrarily strong superlinearity of the blackbody radiation.

Blackbody radiation is a fundamental phenomenon in nature, and its explanation by Planck marks a cornerstone in the history of Physics. In this theoretical work, we show that the spectral radiance given by Planck's law is strongly superlinear with temperature, with an arbitrarily large local exponent for decreasing wavelengths. From that scaling analysis, we propose a new concept of super-resolved detection and imaging: if a focused beam of energy is scanned over an object that absorbs and linearly converts that energy into heat, a highly nonlinear thermal radiation response is generated, and its point spread function can be made arbitrarily smaller than the excitation beam focus. Based on a few practical scenarios, we propose to extend the notion of super-resolution beyond its current niche in microscopy to various kinds of excitation beams, a wide range of spatial scales, and a broader diversity of target objects.

Deep line-temporal focusing with high axial resolution and a large field-of-view using intracavity control and incoherent pulse shaping.

Line-temporal focusing has been recognized as an elegant strategy that provides two-photon microscopy with an effective means for fast imaging through parallelization, together with an improved resilience to scattering for deep imaging. However, the axial resolution remains sub-optimal, except when using high NA objectives and a small field-of-view. With the introduction of an intracavity control of the spectral width of the femtosecond laser to adaptively fill the back aperture of the objective lens, line-temporal focusing two-photon microscopy is demonstrated to reach near-diffraction-limited axial resolution with a large back-aperture objective lens, and improved immunity to sample scattering. In addition, a new incoherent flattop beam shaping method is proposed which provides a uniform contrast with little degradation of the axial resolution along the focus line, even deep in the sample. This is demonstrated in large volumetric imaging of mouse lung samples.

Substrate curvature affects the shape, orientation, and polarization of renal epithelial cells.

The unique structure of kidney tubules is representative of their specialized function. Because maintaining tubular structure and controlled diameter is critical for kidney function, it is critical to understand how topographical cues, such as curvature, might alter cell morphology and biological characteristics. Here, we examined the effect of substrate curvature on the shape and phenotype of two kinds of renal epithelial cells (MDCK and HK-2) cultured on a microchannel with a broad range of principal curvature. We found that cellular architecture on curved substrates was closely related to the cell type-specific characteristics (stiffness, cell-cell adherence) of the cells and their density, as well as the sign and degree of curvature. As the curvature increased on convex channels, HK-2 cells, having lower cell stiffness and monolayer integrity than those of MDCK cells, aligned their in-plane axis perpendicular to the channel but did not significantly change in morphology. By contrast, MDCK cells showed minimal change in both morphology and alignment. However, on concave channels, both cell types were elongated and showed longitudinal directionality, although the changes in MDCK cells were more conservative. Moreover, substrate curvature contributed to cell polarization by enhancing the expression of apical and basolateral cell markers with height increase of the cells. Our study suggests curvature to be an important guiding principle for advanced tissue model developments, and that curved and geometrically ambiguous substrates can modulate the cellular morphology and phenotype. STATEMENT OF SIGNIFICANCE: In many tissues, such as renal tubules or intestinal villi, epithelial layers exist in naturally curved forms, a geometry that is not reproduced by flat cultures. Because maintaining tubular structure is critical for kidney function, it is important to understand how topographical cues, such as curvature, might alter cell morphology and biological characteristics. We found that cellular architecture on curved substrates was closely related to cell type and density, as well as the sign and degree of the curvature. Moreover, substrate curvature contributed to cell polarization by enhancing the expression of apical and basolateral cell markers with height increase. Our results suggested that substrate curvature might contribute to cellular architecture and enhance the polarization of kidney tubule cells.

How to better focus waves by considering symmetry and information loss.

We amend the general belief that waves with extended spherical wavefront focus at their center of curvature. Instead, when the spherical symmetry of waves is broken by propagating them through a finite aperture along an average direction, the forward/backward symmetry is broken and the focal volume shifts its center backward along that direction. The extent of this focal shift increases as smaller apertures are used, up to the point that the nominal focal plane is out of focus. Furthermore, the loss of axial symmetry with noncircular apertures causes distinct focal shifts in distinct axial planes, and the resulting astigmatism possibly degrades the axial focusing resolution. Using experiments and simulations, focal shift with noncircular apertures is described for classical and temporal focusing. The usefulness of these conclusions to improve imaging resolution is demonstrated in a high-resolution optical microscopy application, namely line-temporal focusing microscopy. These conclusions follow from fundamental symmetries of the wave geometry and matter for an increasing number of emerging optical techniques. This work offers a general framework and strategy to understand and improve virtually any wave-based application whose efficacy depends on optimal focusing and may be helpful when information is transmitted by waves in applications from electromagnetic communications, to biological and astronomical imaging, to lithography and even warfare.

Microtopographies control the development of basal protrusions in epithelial sheets.

Cells are able to develop various types of membrane protrusions that modulate their adhesive, migratory, or functional properties. However, their ability to form basal protrusions, particularly in the context of epithelial sheets, is not widely characterized. The authors built hexagonal lattices to probe systematically the microtopography-induced formation of epithelial cell protrusions. Lattices of hexagons of various sizes (from 1.5 to 19 µm) and 5-10 µm height were generated by two-photon photopolymerization in NOA61 or poly(ethylene glycol) diacrylate derivatives. The authors found that cells generated numerous, extensive, and deep basal protrusions for hexagons inferior to cell size (3-10 µm) while maintaining a continuous epithelial layer above structures. They characterized the kinetics of protrusion formation depending on scaffold geometry and size. The reported formation of extensive protrusions in 3D microtopography could be beneficial to develop new biomaterials with increased adhesive properties or to improve tissue engineering.

Engineering small tubes with changes in diameter for the study of kidney cell organization.

Multicellular tubes are structures ubiquitously found during development and in adult organisms. Their topologies (diameter, direction or branching), together with their mechanical characteristics, play fundamental roles in organ function and in the emergence of pathologies. In tubes of micrometric range diameters, typically found in the vascular system, renal tubules or excretory ducts, cells are submitted to a strong curvature and confinement effects in addition to flow. Then, small tubes with change in diameter are submitted to a local gradient of shear stress and curvature, which may lead to complex mechanotransduction responses along tubes, and may be involved in the onset or propagation of cystic or obstructive pathologies. We describe here a simple method to build a microfluidic device that integrates cylindrical channels with changes in diameter that mimic in vivo tube geometries. This microfabrication approach is based on molding of etched tungsten wires, which can achieve on a flexible way any change in diameter in a polydimethylsiloxane (PDMS) microdevice. The interest of this biomimetic multitube system has been evidenced by reproducing renal tubules on chip. In particular, renal cell lines were successfully seeded and grown in PDMS circular tubes with a transition between 80 µm and 50 µm diameters. Thanks to this biomimetic platform, the effect of the tube curvature has been investigated especially regarding cell morphology and orientation. The effect of shear stress on confluent cells has also been assessed simultaneously in both parts of tubes. It is thus possible to study interconnected cell response to differential constraints which is of central importance when mimicking tubes present in the organism.

Polycystins and intercellular mechanotransduction: A precise dosage of polycystin 2 is necessary for alpha-actinin reinforcement of junctions upon mechanical stimulation.

Polycystins 1 and 2, which are mutated in Autosomal Polycystic Kidney Disease, are involved in mechanotransduction through various mechanisms. In renal cells, polycystins not only have an important mechanotransductive role in primary cilia but are also present in intercellular contacts but their role there remains unclear. Here, we address the hypothesis that polycystins are involved in mechanotransduction via intercellular junctions, which would be expected to have consequences on tissue organization. We focused on the role of polycystin 2, which could be involved in mechanical organization at junctions either by its channel activity or by the direct recruitment of cytoskeleton components such as the F-actin cross-linker α-actinin. After mechanical stimulation of intercellular junctions in MDCK renal epithelial cells, α-actinin is rapidly recruited but this is inhibited upon overexpression of PC2 or the D509V mutant that lacks channel activity, and is also decreased upon PC2 silencing. This suggests that a precise dosage of PC2 is necessary for an adequate mechanosensitive α-actinin recruitment at junctions. At the multicellular level, a change in PC2 expression was associated with changes in velocity in confluent epithelia and during wound healing together with a loss of orientation. This study suggests that the mechanosensitive regulation of cytoskeleton by polycystins in intercellular contacts may be important in the context of ADPKD.

Mechanosensitive Adaptation of E-Cadherin Turnover across adherens Junctions.

In the natural and technological world, multi-agent systems strongly depend on how the interactions are ruled between their individual components, and the proper control of time-scales and synchronization is a key issue. This certainly applies to living tissues when multicellular assemblies such as epithelial cells achieve complex morphogenetic processes. In epithelia, because cells are known to individually generate actomyosin contractile stress, each individual intercellular adhesive junction line is subjected to the opposed stresses independently generated by its two partner cells. Contact lines should thus move unless their two partner cells mechanically match. The geometric homeostasis of mature epithelia observed at short enough time-scale thus raises the problem to understand how cells, if considered as noisy individual actuators, do adapt across individual intercellular contacts to locally balance their time-average contractile stress. Structural components of adherens junctions, cytoskeleton (F-actin) and homophilic bonds (E-cadherin) are quickly renewed at steady-state. These turnovers, if they depend on forces exerted at contacts, may play a key role in the mechanical adaptation of epithelia. Here we focus on E-cadherin as a force transducer, and we study the local regulation and the mechanosensitivity of its turnover in junctions. We show that E-cadherin turnover rates match remarkably well on either side of mature intercellular contacts, despite the fact that they exhibit large fluctuations in time and variations from one junction to another. Using local mechanical and biochemical perturbations, we find faster turnover rates with increased tension, and asymmetric rates at unbalanced junctions. Together, the observations that E-cadherin turnover, and its local symmetry or asymmetry at each side of the junction, are mechanosensitive, support the hypothesis that E-cadherin turnover could be involved in mechanical homeostasis of epithelia.

A low-cost, label-free DNA detection method in lab-on-chip format based on electrohydrodynamic instabilities, with application to long-range PCR.

In order to evolve from a "chip in the lab" to a "lab on a chip" paradigm, there is still a strong demand for low-cost, portable detection technologies, notably for analytes at low concentrations. Here we report a new label-free DNA detection method with direct electronic read, and apply it to long-range PCR. This method uses a nonlinear electrohydrodynamic phenomenon: when subjected to high electric fields (typically above 100 V cm(-1)), suspensions of large polyelectrolytes, such as long DNA molecules, create "giant" dynamic concentration fluctuations. These fluctuations are associated with large conductivity inhomogeneities, and we use here a contact-mode local conductivity detector to detect these fluctuations. In order to decouple the detection electronics from the high voltage excitation one, an original "doubly symmetric" floating mode battery-operated detection scheme was developed. A wavelet analysis was then applied, to unravel from the chaotic character of the electohydrodynamic instabilities a scalar signal robustly reflecting the amplification of DNA. As a first proof of concept, we measured the products of the off-chip amplification of 10 kbp DNA from lambda phage DNA, achieving a sensitivity better than 100 fg DNA in the original 50 µl sample. This corresponds to the amplification products of less than 100 initial copies of target DNA. The companion enabling technologies developed to implement this new concept, i.e. the doubly symmetric contact conductivity detection and wavelet analysis, may also find various other applications in lab-on-chips.

New insights into the regulation of E-cadherin distribution by endocytosis.

Homeostasis of adherens junctions is achieved through complex regulatory mechanisms. The junctions are highly dynamic in contact establishment, in remodeling events during development, and during processes involving a loss of adhesion like epithelial-mesenchyme transition. It appeared recently that they are also dynamically renewed in mature, steady-state adhesions. Indeed, maintenance of a steady state must be integrated into a tight control of force equilibrium between a cell and its neighbors. Therefore, it appears that E-cadherin dynamics allows to respond constantly to various biochemical and mechanical stimuli and to regulate the movement and shape of junctions in active remodeling processes. E-cadherin dynamics is mediated through several mechanisms (diffusion, trafficking) in function of the biological system. In mature junctions, membrane E-cadherin is quickly renewed by endocytosis in many cell types. E-cadherin endocytosis shows a complex regulation, depending on small G proteins, ubiquitination, cleavage events, actomyosin cytoskeleton, and other trans molecules in adherens junctions. It is modulated by growth factor stimulations and physical factors. Consequently, E-cadherin endocytosis tightly controls a number of functional processes: cell movements, junction maintenance, cell sorting, and polarity. Misregulated E-cadherin endocytosis is involved in many diseases, from cancerous processes to organogenesis defects.

Crystallization of fluorescent quantum dots within a three-dimensional bio-organic template of actin filaments and lipid membranes.

Biological molecules and molecular self-assemblies are promising templates to organize well-defined inorganic nanostructures. We demonstrate the ability of a self-assembled three-dimensional crystal template of helical actin protein filaments and lipids bilayers to generate a hierarchical self-assembly of quantum dots. Functionnalized tricystein peptidic quantum dots (QDs) are incorporated during the dynamical self-assembly of this actin/lipid template resulting in the formation of crystalline fibers. The crystal parameters, 26.5×18.9×35.5 nm3, are imposed by the membrane thickness, the diameter, and the pitch of the actin self-assembly. This process ensures the high quality of the crystal and results in unexpected fluorescence properties. This method of preparation offers opportunities to generate crystals with new symmetries and a large range of distance parameters.

[Time shapes the cell]. / Le temps, sculpteur de la cellule ? - Représentation en sciences du vivant (7).

Dynamics is the essence of life, from the most macroscopic scale of evolution theories or ecology, down to the submicroscopic scale of the molecular mechanisms underlying for instance the activity of enzymes on their chemical target or their mechanical work in our muscles. Thanks to new methods, most phenomena in cell biology must now be reported not only in terms of their biochemistry, but also with a description of their geometric and temporal characteristics using quantitative imaging. This is leading to a dramatic accumulation of data, with little understanding of its deep physiological significance. Based on recent results, we raise here a few questions regarding the possible role of time as a key determinant of the metabolic energy budget, that impacts living cells from the organisation of macromolecular assemblies up to the morphogenesis and stabilisation of tissues.

Dynamic instability of the intracellular pressure drives bleb-based motility.

We have demonstrated that the two- and three-dimensional motility of the human pathogenic parasite Entamoeba histolytica (Eh) depends on sustained instability of the intracellular hydrostatic pressure. This instability drives the cyclic generation and healing of membrane blebs, with typical protrusion velocities of 10-20 µm/second over a few hundred milliseconds and healing times of 10 seconds. The use of a novel micro-electroporation method to control the intracellular pressure enabled us to develop a qualitative model with three parameters: the rate of the myosin-driven internal pressure increase; the critical disjunction stress of membrane-cytoskeleton bonds; and the turnover time of the F-actin cortex. Although blebs occur randomly in space and irregularly time, they can be forced to occur with a defined periodicity in confined geometries, thus confirming our model. Given the highly efficient bleb-based motility of Eh in vitro and in vivo, Eh cells represent a unique model for studying the physical and biological aspects of amoeboid versus mesenchymal motility in two- and three-dimensional environments.