The genome of influenza A viruses (IAV) is encoded in eight distinct viral ribonucleoproteins (vRNPs) that consist of negative sense viral RNA (vRNA) covered by the IAV nucleoprotein. Previous studies strongly support a selective packaging model by which vRNP segments are bundling to an octameric complex, which is integrated into budding virions. However, the pathway(s) generating a complete genome bundle is not known. We here use a multiplexed FISH assay to monitor all eight vRNAs in parallel in human lung epithelial cells. Analysis of 3.9 × 10 5 spots of colocalizing vRNAs provides quantitative insights into segment composition of vRNP complexes and, thus, implications for bundling routes. The complexes rarely contain multiple copies of a specific segment. The data suggest a selective packaging mechanism with limited flexibility by which vRNPs assemble into a complete IAV genome. We surmise that this flexibility forms an essential basis for the development of reassortant viruses with pandemic potential.

This report highlights the combination of the MicroTime 100 upright confocal fluorescence lifetime microscope with a Single Quantum Eos Superconducting Nanowire Single-Photon Detector (SNSPD) system as a powerful tool for photophysical research and applications. We focus on an application in materials science, photoluminescence imaging, and lifetime characterization of Cu(InGa)Se2 (CIGS) devices intended for solar cells. We demonstrate improved sensitivity, signal-to-noise ratio, and time-resolution in combination with confocal spatial resolution in the near-infrared (NIR) range, specifically in the 1000–1300 nm range. The MicroTime 100–Single Quantum Eos system shows two orders of magnitude higher signal-to-noise ratio for CIGS devices' photoluminescence imaging compared to a standard NIR-photomultiplier tube (NIR-PMT) and a three-fold improvement in time resolution, which is now limited by the laser pulse width. Our results demonstrate the advantages in terms of image quality and time resolution of SNSPDs technology for imaging in materials science.

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Förster resonance energy transfer (FRET) is a popular tool in life sciences, for example to detect protein-protein interactions and ligand binding, or to construct fluorescent biosensors for metabolites or ions. Obtaining such functional information provided by FRET from diffraction-unlimited images would be advantageous, because, for one thing, the spatial averaging of fluorescence signals could be reduced with a smaller detection volume, and, for another thing, FRET signals from neighboring subdiffraction areas could be distinguished. Thus, the goal of this thesis was to investigate the feasibility of measuring FRET using stimulated emission depletion (STED) microscopy.Numerical simulations of a single FRET pair under continuous-wave and pulsed STED illumination were performed to study the interplay of FRET and STED photophysics, including the influence of STED intensity and pulse delay. Große spektrale Verschiebungen der Fluoreszenz beider Fluorophore wurden in Einzelmolekülmessungen beobachtet. In STED-Bildern zeigte sich, dass eine Verschiebung der Emission hin zu kürzeren Wellenlängen die STED-Effizienz verringern kann und damit auch die Auflösung der Bilder. Die spektralen Verschiebungen ändern auch das spektrale Überlappintegral von Donoremission und Akzeptorabsorption und dadurch die FRET-Effizienz; die Größe dieses Effekts wurde abgeschätzt. vi Kurz zusammengefasst wurde ein photophysikalisches Modell für die Interferenz von STED und FRET eingeführt. Experimentelle Voraussetzungen für die STED-Mikroskopie von FRET-Paaren wurden identifiziert und eine adäquate Datenanalysestrategie wurde vorgeschlagen. Zusätzlich wurde die Photoumwandlung organischer Fluorophore charakterisiert und deren Auswirkung auf STED und FRET untersucht. vii 22 Effect of spectral shifts on the FRET efficiency