Establishing Live-Cell Single-Molecule Localization Microscopy Imaging and Single-Particle Tracking in the Archaeon Haloferax volcanii

Abstract

In recent years, fluorescence microscopy techniques for the localization and tracking of single molecules in living cells have become well-established and are indispensable tools for the investigation of cellular biology and in vivo biochemistry of many bacterial and eukaryotic organisms. Nevertheless, these techniques are still not established for imaging archaea. Their establishment as a standard tool for the study of archaea will be a decisive milestone for the exploration of this branch of life and its unique biology. Here, we have developed a reliable protocol for the study of the archaeon Haloferax volcanii. We have generated an autofluorescence-free H. volcanii strain, evaluated several fluorescent proteins for their suitability to serve as single-molecule fluorescence markers and codon-optimized them to work under optimal H. volcanii cultivation conditions. We found that two of them, Dendra2Hfx and PAmCherry1Hfx, provide state-of-the-art single-molecule imaging. Our strategy is quantitative and allows dual-color imaging of two targets in the same field of view (FOV) as well as DNA co-staining. We present the first single-molecule localization microscopy (SMLM) images of the subcellular organization and dynamics of two crucial intracellular proteins in living H. volcanii cells, FtsZ1, which shows complex structures in the cell division ring, and RNA polymerase, which localizes around the periphery of the cellular DNA.This work should provide incentive to develop SMLM strategies for other archaeal organisms in the near future.

Imaging minimal bacteria at the nanoscale: a reliable and versatile process to perform Single Molecule Localization Microscopy in mycoplasmas

“…Alternative fluorescent proteins could be expressed from our pMT-85 backbone, and multiple options are available in the literature, including the well-characterized mMaple3 (79), PAmCherry (80) or Dendra2 (81). These alternatives should nonetheless be properly benchmarked, as it was shown the performances of different fluorescent proteins can vary greatly in a given microbial species (82).…”

Differences in homologous recombination and maintenance of heteropolyploidy between Haloferax volcanii and Haloferax mediterranei

“…H133 also contains deletions in the leuB and pyrE2 genes that were not used in this work. Furthermore, deletion of the crtI (HVO_2528) gene, which is involved in biosynthesis of the carotenoid pigment bacterioruberin, and is not essential nor has an effect on fitness (T urkowyd et al . 2020) resulted in a white phenotype.…”

“…The unique construction of the current mated pair however prevents such recombination events from happening as the cassettes were inserted into the same genomic location, hence the mated cells are forced to contain two different types of genomes and are essentially held under selection in a heteropolyploid state. In addition to the metabolic selectable marker, a second marker was introduced to one of the pair strains -a deletion in the crtI (HVO_2528) gene, which is involved in biosynthesis of the carotenoid pigment bacterioruberin and is not essential nor has an effect on fitness (TURKOWYD et al 2020). This deletion resulted in a white phenotype (WR807) in contrast with the other strain that remained naturally pigmented (red), (WR780).…”

Raw Data to Results: A Hands-On Introduction and Overview of Computational Analysis for Single-Molecule Localization Microscopy

“…Raw single-molecule microscopy movie data often contains nonstructured background noise with different photophysical characteristics as the fluorophores of interest, caused by for example residual out-of-focus fluorophores e.g. in the immersion oil or sample buffer, or by autofluorescence within the biological sample itself (Turkowyd et al, 2019(Turkowyd et al, , 2020. Additionally, out-of-focus fluorophores under HiLo or TIRF illumination will display different blinking characteristics compared to in-focus fluorophores, and can therefore also be filtered out.…”