Microrheology of haemolymph plasma of the bumblebee Bombus terrestris.

Viscosity, which impacts the rate of haemolymph circulation and heat transfer, is one of the transport properties that affects the performance of an insect. Measuring the viscosity of insect fluids is challenging because of the small amount available per specimen. Using particle tracking microrheology, which is well suited to characterise the rheology of the fluid part of the haemolymph, we studied the plasma viscosity in the bumblebee Bombus terrestris. In a sealed geometry, the viscosity exhibits an Arrhenius dependence with temperature, with an activation energy comparable to that previously estimated in hornworm larvae. In an open to air geometry, it increases by 4-5 orders of magnitude during evaporation. Evaporation times are temperature dependent and longer than typical insect haemolymph coagulation times. Unlike standard bulk rheology, microrheology can be applied to even smaller insects, paving the way to characterise biological fluids such as pheromones, pad secretions or cuticular layers.

Molecular rotors in haemoglobin and bovine serum albumin proteins.

Molecular rotors are fluorescent viscosity probes and their response in simple fluids is known to be a Förster-Hoffman power law, allowing the viscosity of the medium to be quantified by its fluorescence intensity. They are attractive probes in biological media, usually consisting of proteins, but how does a molecular rotor behave in a protein solution? The response of the DASPI molecular rotor is compared in two globular protein solutions of similar size, haemoglobin (Hb) and bovine serum albumin, one absorbent, the other not. In absorbent Hb, a model validated by experiments in triangular geometry allows one to correct the absorbing effect and to compare the rotor response in both proteins. With concomitant microrheology measurements, we investigate the relation between the DASPI fluorescence intensity and solution viscosity. In protein solutions, we show that viscosity is no longer the parameter determining the rotor response in contrast to simple fluids. Varying the viscosity by concentration or temperature is not equivalent, and the Förster-Hoffmann power laws do not apply when the solution concentration varies. We show that the concentration regime of the protein solution, semi-dilute or concentrated, determines the sensitivity of the rotor to its environment.

Why do ants differ in acclimatory ability? Biophysical mechanisms behind cuticular hydrocarbon acclimation across species.

Maintaining water balance is vital for terrestrial organisms. Insects protect themselves against desiccation via cuticular hydrocarbons (CHCs). CHC layers are complex mixtures of solid and liquid hydrocarbons, with a surprisingly diverse composition across species. This variation may translate into differential phase behaviour, and hence varying waterproofing capacity. This is especially relevant when temperatures change, which requires acclimatory CHC changes to maintain waterproofing. Nevertheless, the physical consequences of CHC variation are still little understood. We studied acclimatory responses and their consequences for CHC composition, phase behaviour and drought survival in three congeneric ant species. Colony sub-groups were kept under cool, warm and fluctuating temperature regimes. Lasius niger and Lasius platythorax, both of which are rich in methyl-branched alkanes, showed largely predictable acclimatory changes of the CHC profile. In both species, warm acclimation increased drought resistance. Warm acclimation increased the proportion of solid compounds in L. niger but not in L. platythorax. In both species, the CHC layer formed a liquid matrix of constantly low viscosity, which contained highly viscous and solid parts. This phase heterogeneity may be adaptive, increasing robustness to temperature fluctuations. In Lasius brunneus, which is rich in unsaturated hydrocarbons, acclimatory CHC changes were less predictable, and warm acclimation did not enhance drought survival. The CHC layer was more homogeneous, but matrix viscosity changed with acclimation. We showed that ant species use different physical mechanisms to enhance waterproofing during acclimation. Hence, the ability to acclimate, and thus climatic niche breadth, may strongly depend on species-specific CHC profile.

Molecular rotors as intracellular probes of red blood cell stiffness.

The deformability of red blood cells is an essential parameter that controls the rheology of blood as well as its circulation in the body. Characterizing the rigidity of the cells and their heterogeneity in a blood sample is thus a key point in the understanding of occlusive phenomena, particularly in the case of erythrocytic diseases in which healthy cells coexist with pathological cells. However, measuring intracellular rheology in small biological compartments requires the development of specific techniques. We propose a technique based on molecular rotors - viscosity-sensitive fluorescent probes - to evaluate the above key point. DASPI molecular rotor has been identified with spectral fluorescence properties decoupled from those of hemoglobin, the main component of the cytosol. After validation of the rotor as a viscosity probe in model fluids, we showed by confocal microscopy that, in addition to binding to the membrane, the rotor penetrates spontaneously and uniformly into red blood cells. Experiments on red blood cells whose rigidity is varied with temperature, show that molecular rotors can detect variations in their overall rigidity. A simple model allowed us to separate the contribution of the cytosol from that of the membrane, allowing a qualitative determination of the variation of cytosol viscosity with temperature, consistent with independent measurements of the viscosity of hemoglobin solutions. Our experiments show that the rotor can be used to study the intracellular rheology of red blood cells at the cellular level, as well as the heterogeneity of this stiffness in a blood sample. This opens up new possibilities for biomedical applications, diagnosis and disease monitoring.

Communication versus waterproofing: the physics of insect cuticular hydrocarbons.

Understanding the evolution of complex traits is among the major challenges in biology. One such trait is the cuticular hydrocarbon (CHC) layer in insects. It protects against desiccation and provides communication signals, especially in social insects. CHC composition is highly diverse within and across species. To understand the adaptive value of this chemical diversity, we must understand how it affects biological functionality. So far, CHCs have received ample research attention, but their physical properties were little studied. We argue that these properties determine their biological functionality, and are vital to understanding how CHC composition affects their adaptive value. We investigated melting behaviour and viscosity of CHCs from 11 ant species using differential scanning calorimetry and a novel microrheological technique. CHCs began melting below -45°C, and often were entirely liquid only above 30°C. Thus, they formed a solid-liquid mixture under ambient conditions, which contrasts to previous assumptions of entirely solid layers in many species. This may be adaptive as only biphasic CHC layers ensure uniform coating of the insect body, which is necessary for waterproofing. CHC viscosity was mostly between 0.1 and 0.2 Pa s-1, thus similar to motor oils. Surprisingly, chemically different CHC profiles had similar viscosities, suggesting that a certain viscosity level is adaptive and ensures that communication signals can be perceived. With this study, we draw attention to the importance of studying the physics of CHC layers. Only by understanding how chemical and physical mechanisms enable CHC functionality can we understand the causes and consequences of CHC diversification.

Activity statistics in a colloidal glass former: Experimental evidence for a dynamical transition.

In a dense colloidal suspension at a volume fraction below the glass transition, we follow the trajectories of an assembly of tracers over a large time window. We define a local activity, which quantifies the local tendency of the system to rearrange. We determine the statistics of the time integrated activity, and we argue that it develops a low activity tail that comes together with the onset of glassy-like behavior and heterogeneous dynamics. These rare events may be interpreted as the reflection of an underlying dynamic phase transition.

Coping with the climate: cuticular hydrocarbon acclimation of ants under constant and fluctuating conditions.

Terrestrial arthropods achieve waterproofing by a layer of cuticular hydrocarbons (CHCs). At the same time, CHCs also serve as communication signals. To maintain waterproofing under different climate conditions, insects adjust the chemical composition of their CHC layer, but this may affect the communication via CHCs. The detailed acclimatory changes of CHCs and how these influence their physical properties are still unknown. Here, we studied acclimation in two closely related ant species with distinct CHC profiles, Myrmica rubra and Myrmica ruginodis, in response to constant or fluctuating temperature and humidity regimes. We measured how acclimation affected CHC composition and viscosity, and the ants' drought survival. In both species, CHC composition showed strong, predictable responses to temperature regimes. Warm-acclimated individuals had higher proportions of linear alkanes, and less methyl-branched or unsaturated CHCs. These changes coincided with higher solid content and viscosity of CHCs in warm-acclimated ants. Temperature fluctuation caused effects similar to those observed under constant-cool conditions in Mrubra, but led to entirely different profiles in Mruginodis, suggesting that fluctuating and constant conditions pose very different challenges. Acclimation to dry conditions led to higher absolute amounts of CHCs, which increased the ants' drought survival, whereas temperature acclimation did not. Hence, the temperature-induced CHC changes cannot be explained by the need for waterproofing alone. Although these changes could be non-adaptive, we propose that they serve to maintain a constant CHC viscosity, which may be essential for communication and other functions.

Questioning the relationship between the χ4 susceptibility and the dynamical correlation length in a glass former.

Clusters of fast and slow correlated particles, identified as dynamical heterogeneities (DHs), constitute a central aspect of glassy dynamics. A key factor of the glass transition scenario is a significant increase of the cluster size ξ4 as the transition is approached. In need of easy-to-compute tools to measure ξ4, the dynamical susceptibility χ4 was introduced recently, and used in various experimental studies to probe DHs. Here, we investigate DHs in dense microgel suspensions using image correlation analysis, and compute both χ4 and the four-point correlation function G4. The spatial decrease of G4 provides a direct access to ξ4, which is found to grow significantly with increasing volume fraction. However, this increase is not captured by χ4. We show that the assumptions that validate the connection between χ4 and ξ4 are not fulfilled in our experiments.

Rotational microrheology of Maxwell fluids using micron-sized wires.

We demonstrate a simple method for rotational microrheology in complex fluids using micrometric wires. The three-dimensional rotational Brownian motion of the wires suspended in Maxwell fluids is measured from their projection on the focal plane of a microscope. We analyze the mean-squared angular displacement of the wires of length between 1 and 40 µm. The viscoelastic properties of the suspending fluids are examined from this analysis and found to be in good agreement with macrorheology data. Viscosities of simple and complex fluids between 10(-2) and 30 Pa s could be measured. As for the elastic modulus, values up to ∼5 Pa could be determined. This simple technique, allowing for a broad range of probed length scales, opens new perspectives in microrheology of heterogeneous materials such as gels, glasses and cells.

Intracellular micro-rheology probed by micron-sized wires.

In the last decade, rapid advances have been made in the field of micro-rheology of cells and tissues. Given the complexity of living systems, there is a need for the development of new types of nano- and micron-sized probes, and in particular of probes with controlled interactions with the surrounding medium. In the present paper, we evaluate the use of micron-sized wires as potential probes of the mechanical properties of cells. The wire-based micro-rheology technique is applied to living cells such as murine fibroblasts and canine kidney epithelial cells. The mean-squared angular displacement of wires associated to their rotational dynamics is obtained as a function of the time using optical microscopy and image processing. It reveals a Brownian-like diffusive regime of the form Δψ(2)(t,L) ≈ t/L(3), where L denotes the wire length. This scaling suggests that an effective viscosity of the intracellular medium can be determined, and that in the range 1-10 µm it does not depend on the length scale over which it is measured.

Optical tweezers reveal relationship between microstructure and nanoparticle penetration of pulmonary mucus.

In this study, the mobility of nanoparticles in mucus and similar hydrogels as model systems was assessed to elucidate the link between microscopic diffusion behavior and macroscopic penetration of such gels. Differences in particle adhesion to mucus components were strongly dependent on particle coating. Particles coated with 2 kDa PEG exhibited a decreased adhesion to mucus components, whereas chitosan strongly increased the adhesion. Despite such mucoinert properties of PEG, magnetic nanoparticles of both coatings did not penetrate through native respiratory mucus, resisting high magnetic forces (even for several hours). However, model hydrogels were, indeed, penetrated by both particles in dependency of particle coating, obeying the theory of particle mobility in an external force field. Comparison of penetration data with cryogenic scanning EM images of mucus and the applied model systems suggested particularly high rigidity of the mucin scaffold and a broad pore size distribution in mucus as reasons for the observed particle immobilization. Active probing of the rigidity of mucus and model gels with optical tweezers was used in this context to confirm such properties of mucus on the microscale, thus presenting the missing link between micro- and macroscopical observations. Because of high heterogeneity in the size of the voids and pores in mucus, on small scales, particle mobility will depend on adhesive or inert properties. However, particle translocation over distances larger than a few micrometers is restricted by highly rigid structures within the mucus mesh.

Micro- and macrorheology of jellyfish extracellular matrix.

Mechanical properties of the extracellular matrix (ECM) play a key role in tissue organization and morphogenesis. Rheological properties of jellyfish ECM (mesoglea) were measured in vivo at the cellular scale by passive microrheology techniques: microbeads were injected in jellyfish ECM and their Brownian motion was recorded to determine the mechanical properties of the surrounding medium. Microrheology results were compared with macrorheological measurements performed with a shear rheometer on slices of jellyfish mesoglea. We found that the ECM behaved as a viscoelastic gel at the macroscopic scale and as a much softer and heterogeneous viscoelastic structure at the microscopic scale. The fibrous architecture of the mesoglea, as observed by differential interference contrast and scanning electron microscopy, was in accord with these scale-dependent mechanical properties. Furthermore, the evolution of the mechanical properties of the ECM during aging was investigated by measuring microrheological properties at different jellyfish sizes. We measured that the ECM in adult jellyfish was locally stiffer than in juvenile ones. We argue that this stiffening is a consequence of local aggregations of fibers occurring gradually during aging of the jellyfish mesoglea and is enhanced by repetitive muscular contractions of the jellyfish.

Extensive collection of femtolitre pad secretion droplets in the beetle Leptinotarsa decemlineata allows nanolitre microrheology.

Pads of beetles are covered with long, deformable setae, each ending in a micrometric terminal plate coated with secretory fluid. It was recently shown that the layer of the pad secretion covering the terminal plates is responsible for the generation of strong attractive forces. However, less is known about the fluid itself because it is produced in an extremely small quantity. We present here the first experimental investigation of the rheological properties of the pad secretion in the Colorado potato beetle Leptinotarsa decemlineata (Coleoptera, Chrysomelidae). Because the secretion is produced in an extremely small amount at the level of the terminal plate, we first developed a procedure based on capillary effects to collect the secretion for rheological experiments. In order to study the collected fluid (less than 1 nl) through passive microrheology, we managed to incorporate micrometric probes (melamine beads) that were initially in the form of a dry powder. Finally, the bead thermal motions were observed optically and recorded to determine the mechanical properties of the surrounding medium. We achieved this quantitative measurement with the collected volume, which is much smaller than the usual 1 µl sample volume required for this technique. Surprisingly, the beetle secretion was found to behave as a purely viscous liquid, of high viscosity (about 100 times that of water). This suggests that no specific complex fluid behaviour is needed by this adhesive system during beetle locomotion. We describe a scenario for the contact formation between the spatula at the setal tip and a smooth substrate, during the insect walk. We show that the attachment dynamics of the insect pad computed from the high measured viscosity is in good agreement with the observed insect pace. We finally discuss the consequences of the viscosity of the secretion on the insect adhesion.

Influence of mechanical properties of nanoparticles on macrocrack formation.

We present an experimental investigation of drying suspensions of both hard and soft nanolatex spheres. The crack formation is examined as a function of the proportion of hard and soft deformable particles, leading to tunable elastic properties of the drying film. In our experimental systems, no crack formation could be observed below an onset value of the proportion in hard spheres phi approximately 0.45 . During the drying process, the mass of films with various compositions in hard and soft spheres is measured as a function of time. The results suggest that the soft particles undergo deformation that releases the internal stresses.

Probing a nonequilibrium einstein relation in an aging colloidal glass.

We present a direct experimental measurement of an effective temperature in a colloidal glass of laponite, using a micrometric bead as a thermometer. The nonequilibrium fluctuation-dissipation relation, in the particular form of a modified Einstein relation, is investigated with diffusion and mobility measurements of the bead embedded in the glass. We observe an unusual nonmonotonic behavior of the effective temperature: starting from the bath temperature, it is found to increase up to a maximum value, and then decrease back, as the system ages. We show that the observed deviation from the Einstein relation is related to the relaxation times previously measured in dynamic light scattering experiments.

Laponite: aging and shear rejuvenation of a colloidal glass.

We study the nonlinear rheological behavior and the microscopic particle dynamics for a colloidal glass, to see whether recently developed models for driven glassy systems can be applied to predict the rheology. Qualitatively, all the findings predicted by the models can be retrieved in our system. Notably, the viscosity decreases strongly with the shear rate. Since it is difficult to predict non-Newtonian viscosities of colloidal systems due to long-ranged hydrodynamic interactions, this shows the promise of this approach for predicting flow behavior. In addition, the measurements allow us to relate the microscopic diffusion dynamics to the macroscopic viscosity of the system.