Cell motility in microfluidic environments

Trypanosomes are single-celled bloodstream parasites and causative agents of African Sleeping Sickness in humans and Nagana disease in domestic livestock. The pathogen is transmitted by the bite of the tsetse fly and lethal to the infected host if left untreated. Eponymous for the Trypanosoma genus name (Trypanosoma: drilling body ) is the striking nature of their movement which is often described by the spiral motion of a corkscrew. However, looking at trypanosomes at high spatiotemporal resolution, we find that the way the cells move is more complex than described before and changes over time and cell. Apart from the question how trypanosomes move, we find ourselves confronted with the question why trypanosomes move at all? In this context, M. Engstler et al. have shown that active movement is essential for cells that are exposed to hostile antibodies. Hydrodynamic flow induced by active movement of the cell leads to a delocalization of antibodies that have bound to the cell surface: Antibodies exposed to the flow around a forward swimming cell are driven backwards into the flagellar pocket where they are taken up by endocytosis and rendered harmless by subsequent digestion. In contrary, a backward swimming cell is accumulating antibodies at the tip of the flagellum and gets digested itself by the host s immune system. If the described mechanism of hydrodynamic protein sorting is a ubiquitous feature in nature, it has to be proven in more detailed studies of cell motility as well as the involved hydrodynamic condition.The aim of this thesis is to study and quantify the movement of trypanosomes in their microfluidic environments in order to help understanding the mechanisms and reasons of their motility. To achieve this goal we constructed an optical trapping fluorescence microscope optimized for high spatiotemporal resolution and reduced phototoxicity. In combination with advanced microfluidic methods we were not only able to control hydrodynamic flow conditions and spatial confinement, but also to position, manipulate and measure forces on the single cell level, as well as to specifically label single living cells in the microflow.In this work we could show for the first time that using strongly focussed diode lasers it is possible to optically trap living trypanosomes over time scales of t < 15 min, without inducing significant photodamage. The optical stall forces acting on trypanosomes were determined and used to measure the propagation forces of single and dividing trypanosomes.In combination with automated image processing routines we also analyzed the positioning of trypanosomes within the optical trap and found distinct trapping loci which could be correlated to structural features of trypanosomes