In Vitro and In Vivo macromolecular dynamics : from biofilaments to living cells

Studying macromolecules and living cells dynamics in situ significantly contribute to the understanding of various biological processes in living organisms. The biopolymer actin is one of the major building blocks of the cytoskeleton and is further crucial for numerous biological processes. Numerous mechanical responses of the cell including deformation and movement are based on physical properties of cytoskeletal networks, which are influenced by chemical gradients and modulate cytoskeletal functionality. In order to analyze the formation and properties of actin networks in concentration gradients, we developed multi-height microfluidic devices with diffusion-controlled microchambers. This unique approach enables for creating flow-free, steady state concentration gradients of different profiles, such as linear or step-like. Specific features of actin networks emerging in defined gradients are investigated. In particular, we analyzed the effects of spatial conditions on network properties, bending rigidities of network links, and the network elasticity. Furthermore, we study the actin filaments as a model system for semiflexible polymers in microfluidic flow. Filamentous actin facing hydrodynamic forces undergo conformational transitions and analyzing their behavior provides a better understanding of non-Newtonian fluids in microchannels and in living organisms. We introduce a microfluidic device with wide and narrow channel segments, resulting in flow fields of spatially varying flow strength. These structured microchannels with alternating high- and low-velocity segments generate non-equilibrium and non-stationary alternating stretch-coil and coil-stretch transitions of fluorescently labeled actin filaments. We study the conformational transitions of filaments with different contour lengths and at different flow velocities. When the filament enters the wider section of the channel they coil under compression, whereas they are starching with a suppression of thermal fluctuations in the extensional regime during reentering the narrow part of the channel. Actin filaments exposed to hydrodynamic forces in structured microchannels with high- and low-velocity segments were characterized by center of mass velocity changes, the evolution of end-to-end distances and bending energies of the filament passing through the channel. Another biopolymer being essential for all known forms of life is DNA. We study the reversible process of DNA packing and unpacking, which is crucial for cell functioning. In eukaryotic cells, the DNA is wrapped around histone proteins, creating repeatable subunits called nucleosomes, which are then further folded into the chromosomes. For the experiments, histones were replaced by a positively charged, nearly spherical and biocompatible polyamidoamine (PAMAM) dendrimers of generation 6 (G6). In analogy to the histone, PAMAM G6 forms complexes with the DNA through sequence-independent, electrostatic interaction between the negatively charged nucleic acid and the protonated, positively charged dendrimer. We analyze the DNA / PAMAM G6 complex organization at different pH of the solution. Moreover, we study DNA decondensation, which is essential for processes such as transcription, replication and repair. DNA unwrapping was initiated by the DNA / PAMAM G6 complex interaction with heparin, which is highly negatively charged and serves as the competitive agent for DNA. First, DNA compaction and decompaction measurements were performed in glass capillaries using small angle X-ray scattering (SAXS), where we successfully analyzed structural changes of the DNA / PAMAM G6 complexes. Furthermore, specially developed microfluidic devices allow the measurement of the reaction dynamics of these processes. Using X-ray compatible, hydrodynamic focusing microfluidic devices with chevron/herringbone structures, we analyzed the real-time dynamics of DNA release from artificial gene carriers at different heparin concentrations. In this thesis, studies of live cell X-ray imaging are also discussed. Visualization of nanoscale features in living cells is very desirable for investigations of intracellular structures. We use X-ray ptychography to directly explore the dynamics of unstained living fission yeast Schizosaccharomyces pombe cells during meiosis in a natural, aqueous environment. X-ray imaging techniques allow us to investigate soft matter of several micrometers thickness in hydrated states without labeling at nanoscale resolution. We show that it is possible to make a sequence of X-ray images of living cells, which was not feasible so far and additionally, visualize the dynamic changes. Cells were alive even after several ptychographic X-ray scans and we obtained a sequence of X-ray images of individual living fission yeast, which allowed us to visualize and examine the meiotic nuclear oscillations and autophagic cell death subsequently induced by the ionizing radiation. Furthermore, the accumulated radiation after each scan allowed for a precise determination of the critical X-ray doses of autophagic vacuole formation and the lethal dose for fission yeast. This method enables looking at living biological samples and processes in a time-resolved label-free setting

A self-filling microfluidic device for noninvasive and time-resolved single red blood cell experiments

Existing approaches to red blood cell (RBC) experiments 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. Here, 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. We were 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. Using bright-field microscopy, our noninvasive approach further enabled the time-resolved analysis of RBC flickering during the reversible shape evolution from the discocyte to the echinocyte morphology. Due to its specialized geometry, our device is particularly suited for studying the temporal behavior of single RBCs under precise control of their environment that will provide important insights into the RBC's biomedical and biophysical properties

Live cell X-ray imaging of autophagic vacuoles formation and chromatin dynamics in fission yeast

Seeing physiological processes at the nanoscale in living organisms without labeling is an ultimate goal in life sciences. Using X-ray ptychography, we explored in situ the dynamics of unstained, living fission yeast Schizosaccharomyces pombe cells in natural, aqueous environment at the nanoscale. In contrast to previous X-ray imaging studies on biological matter, in this work the eukaryotic cells were alive even after several ptychographic X-ray scans, which allowed us to visualize the chromatin motion as well as the autophagic cell death induced by the ionizing radiation. The accumulated radiation of the sequential scans allowed for the determination of a characteristic dose of autophagic vacuole formation and the lethal dose for fission yeast. The presented results demonstrate a practical method that opens another way of looking at living biological specimens and processes in a time-resolved label-free setting

Direct Observation of Alternating Stretch-Coil and Coil-Stretch Transitions of Semiflexible Polymers in Microstructured Flow

Analyzing the behavior of semiflexible polymers experiencing hydrodynamic forces is an important step toward a better understanding of polymer dynamics in microfluidic applications as well as in living cells. In particular, studying conformational changes of fluorescently labeled, semiflexible actin filaments in flow fields of spatially varying flow strength will significantly contribute to this goal. The experimental situation is realized in flows through structured microchannels with alternating high- and low-velocity segments. While entering the wider channel segments, the semiflexible filaments undergo a buckling transition under compression whereas they are stretched with a suppression of thermal fluctuations in the extensional regime when reentering the narrow segments. The nature of these nonequilibrium and nonstationary conformational transitions is characterized by analyzing the evolution of the end-to-end distances, center-of-mass velocities, and bending energies along the passage of the filaments through the channels

Formation of lipid and polymer based gold nanohybrids using a nanoreactor approach

Nanocarriers encapsulating gold nanoparticles hold tremendous promise for biomedical applications. The nanoreactor approach offers a versatile, efficient, and highly reproducible preparation technology.</p