1,721,082 research outputs found
An in vitro model system for cytoskeletal confinement
No full textThe motility, shape, and functionality of the cell depend sensitively on cytoskeletal mechanics which in turn is governed by the properties of filamentous proteins -mainly actin, microtubules, and intermediate filaments. These biopolymers are confined in the dense cytoplasm and therefore experience strong geometric constraints on their equilibrium thermal fluctuations. To obtain a better understanding of the influence of confinement on cytoskeletal filaments we study the thermal fluctuations of individual actin filaments in a microfluidic in vitro system by fluorescence microscopy and determine the persistence length of the filaments by analyzing the radial distribution function. A unique feature of this method is that we obtain the persistence length without detailed knowledge of the complete contour of the filament which makes the technique applicable to a broad range of biological polymers, including those with a persistence length smaller than the optical resolution
X-ray studies of biological matter in microfluidic environments
No full textBiological systems such as cells and cellular components are governed by processes, which take place on nanometer to micrometer length scales. X-ray scattering, diffraction and imaging techniques are extremely well suited to study these processes as the spatial resolution extends well into the relevant length scales. At the same time, the investigation of physical and chemical properties and behavior of such systems requires well-defined and controllable sample environments. One successful way to establish such environments, including specified flow fields, concentration gradients and confinement regimes is by employing microfluidic technology tailored to the particular scientific question. This brief review focuses on microfluidic techniques that have been used to investigate biological matter by X-rays. In particular, we show how the characteristics of flow on the micron scale enable new scientific approaches as compared to macroscale experiments
Mobility gradient induces cross-streamline migration of semiflexible polymers
No full textMany aspects of modern material science and biology rely on the strategic manipulation and understanding of polymer dynamics in confining micro- and nanoflow. We directly observe and analyze nonequilibrium structural and dynamic properties of individual semiflexible actin filaments in pressure-driven microfluidic channel flow using fluorescence microscopy. Different conformational shapes, such as filaments fluctuating in an elongated manner, parabolically bent, as well as tumbling, are identified. With increasing flow velocity, a strong center-of-mass migration toward the channel walls is observed. This significant migration effect can be explained by a shear rate dependent spatial diffusivity due to a gradient in chain mobility of the semiflexible polymers
Brownian motion of actin filaments in confining microchannels
No full textSince the cytoskeletal protein actin is one of the principal building blocks of mammalian cells, it has recently been arousing much interest. Here, we address questions concerning the mechanical and dynamic behaviour of individual actin filaments in confining geometries which mimic the dense cytoskeletal network in eukaryotic cells. Microfluidic devices fabricated by soft photolithography in combination with fluorescence microscopy are used to manipulate, observe and characterize these biopolymers. The polymer statistics is strongly dependent on the characteristics of the surroundings such as the degree of confinement and hydrodynamical flow. Besides this, the intrinsic mechanical properties of the filaments are dominated by the persistence length and the contour length. We analyse the tangent–tangent correlation and the radial distribution function in terms of a confining potential and the contour length of the filaments. In addition, we show that hydrodynamic flow can be successfully used to apply controlled local stress on actin filaments. Our results can be surprisingly well described by a straightforward model which approximates the confining energy of the microchannels using a parabolic potential
Fluctuations of Single Confined Actin Filaments
No full textThermal fluctuations of individual actin filaments confined in rectangular microchannels with dimensions similar to the mesh size of the cytoskeleton in eukaryotic cells are studied experimentally using fluorescence microscopy and theoretically by a combination of analytical methods and Monte Carlo simulations. Compared to freely fluctuating filaments, long filaments confined in narrow channels exhibit enhanced tangent correlations and a characteristic shape of their correlation function. The tangent correlation function is calculated analytically by approximating the confining geometry by a parabolic potential. This approximation is validated by Monte Carlo simulations. For the quantitative analysis of experimental data additional corrections for image analysis effects have to be included, for which we provide a modified analytical approximation formula which is corroborated by simulations. This allows us to obtain both the persistence length LP describing the bending rigidity of the polymer and the deflection length λ characterizing confinement effects from fits to the experimental data. Our results confirm the scaling relation λ ∝ d2/3 between the deflection length and the channel width d
Characterization of single semiflexible filaments under geometric constraints
No full textConfinement effects on single semiflexible macromolecules are of central importance for a fundamental understanding of cellular processes involving biomacromolecules. To analyze the influence of confinement on the fluctuations of semiflexible macromolecules we study individual actin filaments in straight and curved microchannels. We experimentally characterize the segment distributions for fluctuating semiflexible filaments in microchannels as a function of the channel width. Moreover, the effect of channel curvature on the filament fluctuations is investigated. We find quantitative agreement between experimental results, Monte Carlo simulations, and the analytical description. This allows for determination of the persistence length of actin filaments, the deflection length, which characterizes the confinement effects, and the scaling exponents for the segment distribution of semiflexible macromolecules
Microfluidics of soft matter investigated by small-angle scattering
No full textThe combination of X-ray microdiffraction and microfluidics is used to investigate the dynamic behaviour of soft materials. A microfocused X-ray beam enables the observation of the influence of droplet formation on the nanostructure of a smectic liquid crystal in water. Using a hydrodynamic focusing device, the evolution of the intercalation of DNA into multilamellar membranes can be studied. Owing to the elongational flow at the centre of this device, alignment of the material is induced which allows for an improved structural characterization. Furthermore, the influence of strain applied to these materials can be tested
Evolution of DNA compaction in microchannels
No full textCombining microfluidics with x-ray microdiffraction and Raman microscopy, the dynamic behaviour of soft matter, with specific consideration of the molecular structure, can be investigated. Microfluidic systems enable a reduction of sample volume and shorter reaction times. By performing experiments under continuous microflow, material damage is avoided and the influence of external stress on biomacromolecules can be analysed. The generated elongated flow induces alignment of the investigated materials, allowing for an improved structural characterization. Here, the dynamics of the compaction of DNA by polypropyleneimine dotriacontaamine dendrimers, generation 4 is studied. As a consequence of the laminar flow inside the microchannels, highly defined, diffusion-controlled compaction of the DNA occurs enabling the study of different states of the reaction during one measurement by varying the observation position in the channels. The evolution of a columnar mesophase with an in-plane square symmetry is monitored by x-ray microdiffraction and the molecular interaction between the two reactants is traced using Raman microscopy, leading to a more profound comprehension of the condensation reaction. The experimental results are in accordance with finite element method simulations of the flow and diffusion profiles in the elongated flow device
X-ray microdiffraction on flow-controlled biomolecular assemblies
No full textThe study of liquid crystalline assemblies, with an emphasis on biological phenomena, is now accessible using newly developed microdevices integrated with X-ray analysis capability. Many biological systems can be described in terms of gradients, mixing, and confinement, all of which can be mimicked with the use of appropriate microfluidic designs. The use of hydrodynamic focusing creates well-defined mixing conditions that vary depending on parameters such as device geometry, and can be quantified with finite element modelling. We describe experiments in which geometry and strain rate induce finite changes in liquid crystalline orientation. We also demonstrate the online supramolecular assembly of lipoplexes. The measurement of lipoplex orientation as a function of flow velocity allows us to record a relaxation process of the lipoplexes, as evidenced by a remarkable 4-fold azimuthal symmetry. All of these processes are accessible due to the intentional integration of design elements in the microdevices
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