Visualization of cell-specific traits in marine picoplankton populations

Abstract

This thesis investigates the beneficial interplay of advancing technologies in the field of microscopy and microbiology with a focus on the applicability of super-resolution methods for the visualization of sub-cellular structures in prokaryotic cells. DNA, membranes, ribosomal RNA and genes of interest were targeted with specific organic fluorophores or fluorescently-labelled oligo- or polynucleotide probes and visualized on a single-cell level in both, pure cultures and environmental samples. In particular, microbiological techniques based on fluorescence in situ hybridization (FISH) were used, which exploit the hybridization of specific fluorescently-labelled probes to target DNA or ribosomal RNA. The main emphasis was on direct-geneFISH, a tool that was developed to simultaneously detect genes of interest and the 16S rRNA within fixed cells to link microbial identity with a potential function. The image acquisition was performed with four different microscopy methods that, with different approaches, allow for super-resolution: confocal laser scanning microscopy (CLSM) with the Airyscan detector upgrade, structured illumination microscopy (SIM), DNA-point accumulation for imaging in nanoscale topography (PAINT) and stimulated emission depletion (STED) microscopy. Furthermore, this thesis proposes a new approach to quantify individual ribosomes in single cells as a proxy for their growth rate. This is achieved by the visualization of 16S rRNA-derived signals with DNA-PAINT, a method referred to as bacFISH-PAINT.