Waterproofing of the aerial organs of plants imposed a big evolutionary step during the colonization of the terrestrial environment. The main plant polymers responsible of water repelling are lipids and lignin, which play also important roles in the protection against biotic/abiotic stresses, regulation of flux of gases and solutes, and mechanical stability against negative pressure, among others. While the lipids, non-polymerized cuticular waxes together with the polymerized cutin, protect the outer surface, lignin is confined to the secondary cell wall within mechanical important tissues. In the present work a micro cross-section of the stem of Arabidopsis thaliana was used to track in situ the distribution of these non-carbohydrate polymers by Confocal Raman Microscopy. Raman hyperspectral imaging gives a molecular fingerprint of the native waterproofing tissues and cells with diffraction limited spatial resolution (~300 nm) at relatively high speed and without any tedious sample preparation. Lipids and lignified tissues as well as their effect on water content was directly visualized by integrating the 1299, 1600, and 3400 cm−1 band, respectively. For detailed insights into compositional changes of these polymers vertex component analysis was performed on selected sample positions. Changes have been elucidated in the composition of lignin within the lignified tissues and between interfascicular fibers and xylem vessels. Hydrophobizing changes were revealed from the epidermal layer to the cuticle as well as a change in the aromatic composition within the cuticle of trichomes. To verify Raman signatures of different waterproofing polymers additionally Raman spectra of the cuticle and cutin monomer from tomato (Solanum lycopersicum) as well as aromatic model polymers (milled wood lignin and dehydrogenation polymer of coniferyl alcohol) and phenolic acids were acquired.

As a possible viable and non‐invasive method to identify high producing cells, Confocal Raman Microscopy was shown to be able to differentiate CHO host cell lines and derivative production clones. Cluster analysis of spectra and their derivatives was able to differentiate between different producer cell lines and a host, and also distinguished between an intracellular region of high lipid and protein content that in structure resembles the Endoplasmic Reticulum. This ability to identify the ER may be a major contributor to the identification of high producers. PCA enabled the discrimination even of host cell lines and their subclones with inherently higher production capacity. The method is thus a promising option that may contribute to early, non‐invasive identification of high potential candidates during cell line development and possibly could also be used for proof of identity of established production clones.

Fluorescence proteins are widely used as markers for biomedical and technological purposes. Therefore, the aim of this project was to create a fluorescent sensor, based in the green and cyan fluorescent protein, using bacterial S-layers proteins as scaffold for the fluorescent tag. We report the cloning, expression and purification of three S-layer fluorescent proteins: SgsE-EGFP, SgsE-ECFP and SgsE-13aa-ECFP, this last containing a 13-amino acid rigid linker. The pH dependence of the fluorescence intensity of the S-layer fusion proteins, monitored by fluorescence spectroscopy, showed that the ECFP tag was more stable than EGFP. Furthermore, the fluorescent fusion proteins were reassembled on silica particles modified with cationic and anionic polyelectrolytes. Zeta potential measurements confirmed the particle coatings and indicated their colloidal stability. Flow cytometry and fluorescence microscopy showed that the fluorescence of the fusion proteins was pH dependent and sensitive to the underlying polyelectrolyte coating. This might suggest that the fluorescent tag is not completely exposed to the bulk media as an independent moiety. Finally, it was found out that viscosity enhanced the fluorescence intensity of the three fluorescent S-layer proteins.

No abstract

The emergence of large-scale order in self-organized systems relies on local interactions between individual components. During bacterial cell division, the tubulin-homolog FtsZ polymerizes into treadmilling filaments that further assemble into a cytoskeletal ring. Although minimal in vitro assays have shown the striking self-organization capacity of FtsZ filaments, such as dynamic chiral assemblies, how these large-scale structures emerge and relate to individual filament properties remains poorly understood. To understand this quantitatively, we combined minimal chiral active matter simulations with biochemical reconstitution experiments. Using STED and TIRF microscopy as well as high-speed AFM, we imaged the behavior of FtsZ filaments on different spatial scales. Simulations and experiments revealed that filament density and flexibility define the local and global order of the system: At intermediate densities, flexible filaments organize into chiral rings and polar bands, while an effectively nematic organization dominates for high filament densities and for mutant filaments with increased rigidity. Our predicted phase diagram captured these features quantitatively, demonstrating how filament flexibility, density and chirality cooperate with activity to give rise to a large repertoire of collective behaviors. These properties are likely important for the dynamic organization of soft chiral matter, including that of treadmilling FtsZ filaments during bacterial cell division.