Non-equilibrium dynamics of biological matter in microfluidic environments - from red blood cell flickering to conformational transitions of actin filaments

Even the most basic and seemingly simple living biological systems exist far from thermodynamic equilibrium and studying their dynamic behavior represents a crucial step towards a better understanding of their fundamental properties. Here, we present the investigations of two different, out-of-equilibrium biological systems, namely single cell studies on the human red blood cell (RBC) and single macromolecule analysis of actin filaments, both by exploiting the exceptional physics at the micro-scale and the corresponding unique capabilities of experimental control. In particular, existing approaches to RBC analysis on the single-cell level usually rely on chemical or physical manipulations that often cause difficulties with preserving the RBC's integrity in a controlled microenvironment. We introduce a straightforward, self-filling microfluidic device that autonomously separates and isolates single RBCs directly from unprocessed human blood samples and confines them in diffusion-controlled microchambers by solely exploiting their unique intrinsic properties. Using bright-field microscopy, this noninvasive approach enables the time-resolved analysis of RBC flickering during the reversible shape evolution from the discocyte to the echinocyte morphology. A better understanding of this central shape transformation is especially relevant for blood storage applications as the formation of echinocytes can affect blood handling. We are further able to study the photo-induced oxygenation cycle of single functional RBCs by Raman microscopy without the limitations typically observed in optical tweezers based methods. Due to its specialized geometry, our device is particularly suited for studies on single RBCs under precise control of their environment. The provision of important insights into the RBC's biomedical and biophysical properties will improve the understanding of RBC microcirculation and can further contribute to advances in pathology diagnosis. Furthermore, we study the non-equilibrium conformational dynamics of semiflexible actin filaments experiencing hydrodynamic forces. Improving the knowledge about these dynamic processes of semiflexible polymers is of particular importance for the description of unusual transport in cellular flows and pattern formation processes in cytoplasmic streaming. The actin filaments are flowing through structured microchannels with alternating high- and low-velocity segments. These flow fields of spatially varying flow strength result in a compressive force on the filaments when they are entering the low-velocity regions and conversely an extensional force is acting on them when they are reentering the high-velocity segments. The semiflexible actin filaments undergo a length-dependent buckling transition under compression with a corresponding change in end-to-end distance and a rise in bending energy. However, the degree of increase of the length-normalized bending energy shows no evident dependence on the contour length. Increasing the fluid flow velocity results in a large rise of the compressive hydrodynamic force with a strong increase in storage of elastic energy due to the bending of the semiflexible filaments. At the passage from the low-velocity segments to the high-velocity ones, an extensional force is acting on the partially elastically relaxed filaments and a conformational transition from a coiled to a stretched state with a suppression of thermal fluctuations can be observed. Despite the symmetry of the microfluidic channels and therefore a similar rate of the absolute values of extension or compression in the specific channel segments, the observed stretch-coil and coil-stretch transitions distinctly differ in the evolution of the conformational changes and bending energies. This asymmetry of the non-equilibrium and non-stationary conformational transitions shows a strong dependence on the contour and persistence length, the degree of relaxation and the extensional or compressional rate. Many polymer solutions are non-Newtonian fluids and our studies may therefore have an impact on the analysis as well as sorting of polymers by elucidating the non-Newtonian flow behavior of semiflexible filaments in specific microflows, which may consequently lead to a better understanding of intercellular flows.