Polymer networks play an important role in the biomechanics of animal cells, e.g. human cells. An example for such a polymer network is the cytoskeleton which provides a structural framework for the cell and serves as a scaffold determining the shape of the cell (Fig. 1). The cytoskeleton is a very dynamic structure decisive for the passive mechanical properties of cells, their active force production, their locomotionl, and their intracellular transport processes.
The cytoskeleton is composed of at least two types of protein filaments, actin and microtubules, held together by crosslinkers. Rigidity and dynamical properties of the network are determined by the properties of the filaments themselves and those of the crosslinkers as well as by the binding of the network to intra-cellular vesicles and to the cell membrane. To learn about the dynamical properties of the cell cytoskeleton, scientists established in-vitro models formed by reconstituted actin and microtubules filaments. A fascinating aspect in using these model networks is the large macromolecular size enabling scientists to apply modern light microscopy. Actin filaments, e.g., may have lengths up to 100 µm and they represent ideal linear, semi-flexible one-dimensional polymers whose dynamical and physical properties can be followed in real-time by the use of modern light microscopy (Fig. 2).
In our institute we use a confocal laser scanning fluorescence microscope (CLSM) with a field of view up to several 100 µm, a lateral resolution of about 250 nm and a time resolution in the range of 100 ms. The latter is particularly helpful to study fast moving polymer filaments. To study three-dimensional polymer networks, the CLSM is capable to scan and display horizontal sections with a resolution of about 500 nm. Individual CLSM images may be assembled to real-time movies providing additional information on the motion of individual filaments within the network not accessible from single images. We develop our own image analysis software optimized to the needs of studies of mobile objects in scanning microscope images and movies which are used to transform the image pixel information into data files suitable for further evaluation.
The research interest of our institute is mostly focused on the equilibrium properties of such three-dimensional networks. We are interested in their conformation and dynamics. Furthermore, we would like to understand their behaviour in non-equilibrium situations.
In this research enterprise, the large size of actin filaments is a great advantage. One may neglect the characteristics of individual constituents of the filaments and describe them using coarse-grained models and correlation functions. They provide dynamic as well as spatial information on the motion of the networks within entropic and energetic interaction potentials although details of the motion of individual constituents in the network filaments remain unknown. While the theoretical treatment of single filament conformation and dynamics is fairly well developed, collective phenomena still pose many challenges.
The movie shows a sequence of 100 individual confocal laser scanning microscopy (CLSM) images (420 ms/image) of labelled actin filaments in a network solution of unlabeled (and hence invisible) filaments (concentration 0.8 mg/ml). The movie covers a time span of 600 s. The field of view is 73x73 µm2.
The movie shows a sequence of 100 individual confocal laser scanning microscopy (CLSM) images (388 ms/image) of an individual actin filament in a network solution of unlabeled (and hence invisible) filaments (concentration 0.5 mg/ml). The trace of the fluctuating filaments is detected by means of a home-made computer code and analyzed with respect to the time-scaling.
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