7th Annual Symposium
Physics of Cancer
October 4-6, 2016
|PoC - Physics of Cancer - Annual Symposium|
Multiparametric of Collagen I Self-Assembly, and Cytoskeleton Reorganisation in Living Cells
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Living cells are highly dynamic systems, and feature ensembles of well-integrated macromolecular and signalling networks, used for their function and communication with the environment. Conventional high-resolution microscopy techniques, such as TEM, SEM, or fluorescence microscopy typically require sample treatment, thus inherently affecting relevant cell characteristics, such as morphology, adhesion, and mechanical properties. In turn this hinders the study of important cellular functions and state, namely morphogenesis, differentiation, or cell division.
Studying the single macromolecule dynamics and the function of complex biological systems, such as living cells, requires a tool that can meet the requirements for both high spatial and temporal resolution. Atomic force microscopy (AFM) still remains the only technique that offers premium resolution of the analysed biological systems, while being able to simultaneously acquire information about the sample’s mechanical properties at near physiological/native sample conditions. Very recent high-speed AFM developments have also made possible the acquisition of single images on the timescale of seconds, and even milliseconds.
We will give examples of applying fast AFM for imaging of membrane dynamics of KPG-7 fibroblasts, and the cytoskeleton reorganisation of living CHO cells, demonstrated by realtime videos reaching 1 frame per second. We will also show how AFM can be combined with super-resolution optical techniques, such as D-STORM and STED, to study high-resolution cytoskeleton structure simultaneous to AFM. This will be extended to tuning the in vitro fibril formation dynamics of collagen type I, a common scaffold used in cell culture scenarios. We have recently developed, a quantitative imaging force tool, which allows the combination of nanotopographical resolution and the multiparametric mechanical characterisation (stiffness, adhesion) of the specimens.
The demonstrated combination of an AFM setup with advanced optical microscopy allows the long-term and non-invasive study of dynamic macromolecular processes, further supplemented by complex data analysis including Young´s modulus images, topography at different indentation forces resulting in 3D tomographic sample reconstruction.