Prof. Dr. Leonhard Möckl

Research during the past 2 decades has showcased the power of single-molecule localization microscopy (SMLM) as a tool for exploring the nanoworld. However, SMLM systems are typically available in specialized laboratories and imaging facilities, owing to their expensiveness as well as complex assembly and alignment procedure. Here, we lay out the blueprint of a sturdy, rail-based, cost-efficient, multicolor SMLM setup that is easy to construct and align in service of simplifying the accessibility of SMLM. We characterize the optical properties of the design and assess its capabilities, robustness, and stability. The performance of the system is assayed using super-resolution imaging of biological samples. We believe that this design will make SMLM more affordable and broaden its availability.<br>

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the third human coronavirus within 20 years that gave rise to a life-threatening disease and the first to reach pandemic spread. While the scientific community has studied coronavirus biology using genomics, cryoelectron microscopy, and electron tomography, how coronavirus RNA is spatially organized in the cell at the different stages of the viral replication cycle at nanoscale resolution is largely unknown. To make therapeutic headway against current and future coronaviruses, the biology of coronavirus RNA during infection must be precisely understood. Here, we introduce a multicolor super-resolution (SR) fluorescence imaging framework to examine the spatial interactions between viral RNA and other viral factors during host cell infection. We demonstrate the efficacy of our approach using the HCoV-229E coronavirus in MRC5 lung fibroblasts and specifically label two key oligonucleotide viral players: viral genomic RNA (gRNA) and double-stranded RNA (dsRNA). The 10-nm resolution achieved by our approach uncovers a striking spatial organization of gRNA and dsRNA into three distinct RNA structures: (1) large gRNA clusters, (2) very tiny nanoscale gRNA puncta containing a single copy of the genome, and (3) round intermediate-sized puncta highlighted by the dsRNA label. Furthermore, we use our two-color SR approach to visualize the nanoscale spatial relationships between viral gRNA and the endoplasmic reticulum (ER), dsRNA and ER, gRNA and the spike protein, and gRNA and dsRNA. In particular, we observe two striking observations that provide insight into viral replication and export. First, spike proteins and gRNA rarely assemble into an assembled virion in the MRC5 cytoplasm. Second, in contrast to previous observations, dsRNA and gRNA spatially separate. Our approach provides a comprehensive imaging framework that will enable future investigations of coronavirus fundamental biology and the effects of therapeutics.