Digital Fourier microscopy for soft matter dynamics

Soft matter is studied with a large portfolio of methods. Light scattering and video microscopy are the most employed at optical wavelengths. Light scattering provides ensemble-averaged information on soft matter in the reciprocal space. The wave-vectors probed correspond to length scales ranging from a few nanometers to fractions of millimetre. Microscopy probes the sample directly in the real space, by offering a unique access to the local properties. However, optical resolution issues limit the access to length scales smaller than approximately 200 nm. We describe recent work that bridges the gap between scattering and microscopy. Several apparently unrelated techniques are found to share a simple basic idea: the correlation properties of the sample can be characterized in the reciprocal space via spatial Fourier analysis of images collected in the real space. We describe the main features of such digital Fourier microscopy (DFM), by providing examples of several possible experimental implementations of it, some of which not yet realized in practice. We also provide an overview of experimental results obtained with DFM for the study of the dynamics of soft materials. Finally, we outline possible future developments of DFM that would ease its adoption as a standard laboratory method. \ua9 2014 IOP Publishing Ltd

Active diffusion and advection in Drosophila oocytes result from the interplay of actin and microtubules

Transport in cells occurs via a delicate interplay of passive and active processes, including diffusion, directed transport and advection. Despite progress in super-resolution microscopy, discriminating and quantifying these processes is a challenge, requiring tracking of rapidly moving, sub-diffraction objects in a crowded, noisy environment. Here we use Differential Dynamic Microscopy with different contrast mechanisms to provide a thorough characterization of the dynamics in the Drosophila oocyte. We study the movement of vesicles and the elusive motion of a cytoplasmic F-actin mesh, a known regulator of cytoplasmic flows. We find that cytoplasmic motility constitutes a combination of directed motion and random diffusion. While advection is mainly attributed to microtubules, we find that active diffusion is driven by the actin cytoskeleton, although it is also enhanced by the flow. We also find that an important dynamic link exists between vesicles and cytoplasmic F-actin motion, as recently suggested in mouse oocytes.MD and IMP were supported by the BBSRC, the Department of Zoology (Cambridge), the University of Cambridge, and an Isaac Newton Trust fellowship to MD. FG and RC acknowledge funding by the Italian Ministry of Education and Research, Futuro in Ricerca Project ANISOFT (RBFR125H0M) and by Fondazione CARIPLO-Regione Lombardia Project Light for Life (2016-0998)

Modulation of tumor angiogenesis by conditional expression of fibroblast growth factor-2 affects early but not established tumors.

Fibroblast growth factor-2 (FGF2) is a pleiotropic heparin-binding growth factor endowed with a potent angiogenic activity in vitro and in vivo. To investigate the impact of the modulation of FGF2 expression on the neovascularization at different stages of tumor growth, we generated stable transfectants (Tet-FGF2) from the human endometrial adenocarcinoma HEC-1-B cell line in which FGF2 expression is under the control of the tetracycline-responsive promoter (Tet-off system). After transfection, independent clones were obtained in which FGF2 mRNA and protein were up-regulated compared with parental cells. Also, the conditioned medium of Tet-FGF2 transfectants caused proliferation, urokinase-type plasminogen activator up-regulation, migration, and sprouting of cultured endothelial cells. A 3-day treatment of Tet-FGF2 cell cultures with tetracycline abolished FGF2 overexpression and the biological activity of the conditioned medium without affecting their proliferative capacity. Tet-FGF2 cells formed tumors when nude mice received s.c. injections. The administration of 2.0 mg/ml tetracycline in the drinking water before cell transplantation, continued throughout the whole experiment, inhibited FGF2 expression in Tet-FGF2 tumor lesions. This was paralleled by a significant decrease in the rate of tumor growth and vascularization to values similar to those observed in lesions generated by parental HEC-1-B cells. Tetracycline administration 20 days after tumor cell implant, although equally effective in reducing FGF2 expression and inhibiting tumor vascularity, only minimally impaired the growth of established Tet-FGF2 tumors. The results indicate that FGF2 expression deeply affects the initial tumor growth and neovascularization of HEC-1-B human endometrial adenocarcinoma in nude mice. On the contrary, the growth of established tumors appears to be independent of the inhibition of FGF2 expression and decreased vascular density. The possibility that a significant reduction of angiogenesis may not affect the progression of large tumors points to the use of antiangiogenic therapy in early tumor stage

Tumour sublines with different metastatic capacity induce similar blood coagulation changes in the host.

This paper is aimed at investigating how metastatic tumour growth influenced the haemostatic system of the host. Blood platelet count, blood fibrinogen level, the activated partial thromboplastin time (APTT) and the prothrombin time (PT) were determined at various intervals during growth and metastasis of a murine fibrosarcoma (mFS6) or one of its sublines with different metastatic capacity. Progressive thrombocytopenia and increase in fibrinogen level were observed during development of the tumour in all the animal groups studied, irrespective of the metastatic potential of the various sublines. No significant changes were observed in the PT or APTT values. These data support the concept that primary rather than metastatic growth influences the haemostatic system of tumour-bearing animals

Tracking-Free Determination of Single-Cell Displacements and Division Rates in Confluent Monolayers

A biological tissue is an ensemble of soft cells in close physical contact. Events such as cell-shape changes and, more rarely, cell-divisions and apoptosis continuously occur in a tissue, whose collective behavior is set by the cumulative occurrence of such events. In this complex environment, quantifying the single-cell dynamics is key to extract quantitative information to be used to capture the fundamental ingredients of this collective tissue dynamics for validating the predictions of models and numerical simulations. However, tracking the motion of each cell in a dense assembly, even in controlled in vitro settings, is a demanding task, because of a combination of different factors, such as poor image quality, cell shape variability and cell deformability. Here we show that Differential Dynamic Microscopy (DDM), an approach that provides a characterization of the sample structure and dynamics at various spatial frequencies (wave-vectors), can be used successfully to extract quantitative information about a confluent monolayer of Madin-Darby Canine Kidney (MDCK) epithelial cells. In particular, combining structural and dynamical information obtained at different wave-vectors, we show that DDM can provide the single-cell mean squared displacement and the cell division rate at various stages during the temporal evolution of the monolayer. In contrast with tracking algorithms, which require expert supervision and a considerate choice of the analysis parameters, DDM analysis can be run in an automated fashion and yields an unbiased quantification of the dynamic processes under scrutiny, thus providing a powerful means to probe the single-cell dynamics within dense cell collectives

Structure and dynamics of concentration fluctuations in a non-equilibrium dense colloidal suspension

Linearised fluctuating hydrodynamics describes effectively the concentration non-equilibrium fluctuations (NEF) arising during a diffusion process driven by a small concentration gradient. However, fluctuations in the presence of large gradients are not yet fully understood. Here we study the giant concentration NEF arising when a dense aqueous colloidal suspension is allowed to diffuse into an overlying layer of pure water. We use differential dynamic microscopy to determine both the statics and the dynamics of the fluctuations for several values of the wave-vector q. At small q, NEF are quenched by buoyancy, which prevents their full development and sets an upper timescale to their temporal relaxation. At intermediate q, the mean squared amplitude of NEF is characterised by a power law exponent -4, and fluctuations relax diffusively with diffusion coefficient D1. At large q, the amplitude of NEF vanishes and equilibrium concentration fluctuations are recovered, enabling a straightforward determination of the osmotic compressibility of the suspension during diffusion. In this q-range we also find that the relaxation of the fluctuations occurs with a diffusion coefficient D2 significantly different from D1. Both diffusion coefficients exhibit time-dependence with D1 increasing monotonically (by about 15%) and D2 showing the opposite behaviour (about 17% decrease). At equilibrium, the two coefficients coincide as expected. While the decrease of D2 is compatible with a diffusive evolution of the concentration profile, the increase of D1 is still not fully understood and may require considering nonlinearities that are neglected in current theories for highly stressed colloids

Simultaneous characterization of rotational and translational diffusion of optically anisotropic particles by optical microscopy

We probe the roto-translational Brownian motion of optically anisotropic particles suspended in water with a simple and straightforward optical microscopy experiment that does not require positional or rotational particle tracking. We acquire a movie of the suspension placed between two polarizing elements and we extract the translational diffusion coefficient D T and the rotational diffusion coefficient D R from the analysis of the temporal correlation properties of the spatial Fourier modes of the intensity fluctuations in the movie. Our method is successfully tested with a dilute suspension of birefringent spherical colloidal particles obtained by polymerizing an emulsion of droplets of liquid crystal in a nematic phase, whose roto-translational dynamics is found to be well described by theory. The simplicity of our approach makes our method a viable alternative to particle tracking and depolarized dynamic light scattering

Emerging applications of label-free optical biosensors

Innovative technical solutions to realize optical biosensors with improved performance are continuously proposed. Progress in material fabrication enables developing novel substrates with enhanced optical responses. At the same time, the increased spectrum of available biomolecular tools, ranging from highly specific receptors to engineered bioconjugated polymers, facilitates the preparation of sensing surfaces with controlled functionality. What remains often unclear is to which extent this continuous innovation provides effective breakthroughs for specific applications. In this review, we address this challenging question for the class of label-free optical biosensors, which can provide a direct signal upon molecular binding without using secondary probes. Label-free biosensors have become a consolidated approach for the characterization and screening of molecular interactions in research laboratories. However, in the last decade, several examples of other applications with high potential impact have been proposed. We review the recent advances in label-free optical biosensing technology by focusing on the potential competitive advantage provided in selected emerging applications, grouped on the basis of the target type. In particular, direct and real-time detection allows the development of simpler, compact, and rapid analytical methods for different kinds of targets, from proteins to DNA and viruses. The lack of secondary interactions facilitates the binding of small-molecule targets and minimizes the perturbation in single-molecule detection. Moreover, the intrinsic versatility of label-free sensing makes it an ideal platform to be integrated with biomolecular machinery with innovative functionality, as in case of the molecular tools provided by DNA nanotechnology