Microscopy and laser microtechniques: Applications of EMFA technique
(Erythrocyte Mediated Force Application technique)

 

A rapidly growing field of research is mechanotransduction i. e. the way cells use to receive mechanical signals and to convert them into a biochemical response. This is particularly interesting for ageing research, since one may expect that the mechanical properties of cells in an organism change mechanical properties during ageing.
The endothelial cell works in human blood vessels as a selectively working barrier as well as a basic layer for the formation of occlusions and thrombosis. It is moreover the template for vascularisation, inflammation reaction, carcinogenesis and systemic (auto-) immunity as far as the red blood count blood picture is included in it. Most important, the endothelial cells act as primary sensors of the blood pressure: Upon increased blood pressure they release Nitric Oxide (NOx) to induce the relaxation of the vessel wall muscle cells. This way the vessel volume increases and the blood pressure is counterbalanced. The process of blood pressure control by vasodilatation is deregulated in older beings. This makes the endothelial cell a preferable target in the systemic ageing process of a person.
Based on the optical tweezers we developed EMFA: the Erythrocyte Mediated Force Application technique [Fig.1]. In EMFA mechanical stress is exerted for single cell stimulation. It is possible to select an individual in a big cell population and to stimulate it exclusively. EMFA was applied to examine the response of single endothelial cells on optomechanical deformations.


[Fig.1]  Principle of EMFA technique: Optical pressure forces (F) on erythrocyte by optical tweezers are transferred to the target cell and deform it.
The observation of stress application with advanced light microscopic techniques offers detailed insights into the physiology of signal transduction in endothelial cells during deformation. This way we are possible to have a direct view on cell mechanics. We can observe alterations in cell structure during tensional stress [Fig.2]. The combined mechanical and physiological response of endothelial cells to the application of force offers new possibilities in diagnosis and therapy of diseases based on endothelial cell dysfunctions.


[Fig.2]              Cell cytoskeleton and focal adhesions

Besides dynamic changes of the cytosolic calcium concentration induces the opto-mechanical stimulation of single endothelial cells also changes in the nitric oxide – homeostasis. With EMFA it is possible to activate the both intracellular coupled oscillators. It seems that the pressure stimulated nitric oxide utilization serves as regulator for the stimulated cytostolic calcium - homeostasis. It can also be assumed, that the depletion of a nitric oxide pool regulates directly the excitability, the calcium regulation in the cytosol and the contraction status of the endothelial cell [Fig.3].

Such simultaneous observations of two complex physiological phenomena after precise physical influence are so far in the ageing research without comparison.


[Fig.3]              Calcium- and Nitric oxide-imaging: regulation of tonus