Journal: Frontiers in Microbiology
Article Title: Establishing Live-Cell Single-Molecule Localization Microscopy Imaging and Single-Particle Tracking in the Archaeon Haloferax volcanii
doi: 10.3389/fmicb.2020.583010
Figure Lengend Snippet: Establishing SMLM imaging in H. volcanii . (A) A representative image of a WR806 cell producing the fluorescently tagged FtsZ1. The fluorescent protein Dendra2Hfx can be read-out in a diffraction-limited mode using its preconverted green fluorescent form as well as in a single-molecule readout mode using photoconverted FtsZ1-Dendra2Hfx. This provides a high-resolution and quantitative SMLM image of FtsZ1 that reveals fine structural details. Diffraction limited snapshots of DNA-Hoechst 33342 were taken after SMLM imaging. (B) The H119 strain synthesizes lycopene and its derivatives , which can be easily seen by bright red colonies on agar-plates. These cause autofluorescence background that largely hinders single-molecule read-out. The WR806 strain lacks the phytoene dehydrogenase crtI of the lycopene synthesis pathway, which results in white cells on plates and drastically reduces autofluorescence. (C) Three SMLM-suitable fluorescent proteins (mMaple3, PAmCherry1 and Dendra2) were evaluated for their expression and functionality in the cytosol of H. volcanii cells. Their fluorescence signals before and after photoactivation/photoconversion were measured. mMaple3 showed no substantial signal (read-out levels similar to a non-fluorescent WR806 control), PAmCherry1 and Dendra2 gave a fluorescent signal after photoactivation/photoconversion. After codon-optimization, Dendra2Hfx and PAmCherry1Hfx showed an increase in signal by one order of magnitude. Experiments were done in two or three independent replicates, which are color-coded in red, blue, and dark gray [visualization adapted from ]. The mean of each replicate is marked by a diamond shape. The overall means are marked by horizontal lines. Error bars represent standard deviations of replicate means. Statistics: WR806 control 164 cells (two replicates); WR806 mMaple3 474 cells (two replicates), WR806 PAmCherry1 226 cells (three replicates), WR806 PAmCherry1Hfx 129 cells (two replicates), WR806 Dendra2 566 cells (three replicates), and WR806 Dendra2Hfx 475 cells (three replicates). The diagonal red dashed line for cells producing codon-optimized Dendra2Hfx and PAmCherry1Hfx separates two representations with different dynamic range. The left part of the image has the same dynamic range used for all exemplary images (as given by the scale to the left), and the right of the image a 10-time wider dynamic range (as given by the scale to the right).
Article Snippet: Dendra2 and PAmCherry1 genes were obtained from GeneArt® (Thermo Fischer Scientific, Waltham, United States) as codon-optimized versions for expression in H. volcanii (plasmids pMA-RQ-Dendra2Hfx and pMA-T-PAmCherry1Hfx).
Techniques: Imaging, Expressing, Fluorescence, Control
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![Establishing SMLM imaging in H. volcanii . (A) A representative image of a WR806 cell producing the fluorescently tagged FtsZ1. The fluorescent protein Dendra2Hfx can be read-out in a diffraction-limited mode using its preconverted green fluorescent form as well as in a single-molecule readout mode using photoconverted FtsZ1-Dendra2Hfx. This provides a high-resolution and quantitative SMLM image of FtsZ1 that reveals fine structural details. Diffraction limited snapshots of DNA-Hoechst 33342 were taken after SMLM imaging. (B) The H119 strain synthesizes lycopene and its derivatives , which can be easily seen by bright red colonies on agar-plates. These cause autofluorescence background that largely hinders single-molecule read-out. The WR806 strain lacks the phytoene dehydrogenase crtI of the lycopene synthesis pathway, which results in white cells on plates and drastically reduces autofluorescence. (C) Three SMLM-suitable fluorescent proteins (mMaple3, PAmCherry1 and <t>Dendra2)</t> were evaluated for their expression and functionality in the cytosol of H. volcanii cells. Their fluorescence signals before and after photoactivation/photoconversion were measured. mMaple3 showed no substantial signal (read-out levels similar to a non-fluorescent WR806 control), PAmCherry1 and Dendra2 gave a fluorescent signal after photoactivation/photoconversion. After codon-optimization, Dendra2Hfx and PAmCherry1Hfx showed an increase in signal by one order of magnitude. Experiments were done in two or three independent replicates, which are color-coded in red, blue, and dark gray [visualization adapted from ]. The mean of each replicate is marked by a diamond shape. The overall means are marked by horizontal lines. Error bars represent standard deviations of replicate means. Statistics: WR806 control 164 cells (two replicates); WR806 mMaple3 474 cells (two replicates), WR806 PAmCherry1 226 cells (three replicates), WR806 PAmCherry1Hfx 129 cells (two replicates), WR806 Dendra2 566 cells (three replicates), and WR806 Dendra2Hfx 475 cells (three replicates). The diagonal red dashed line for cells producing codon-optimized Dendra2Hfx and PAmCherry1Hfx separates two representations with different dynamic range. The left part of the image has the same dynamic range used for all exemplary images (as given by the scale to the left), and the right of the image a 10-time wider dynamic range (as given by the scale to the right).](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_4787/pmc07714787/pmc07714787__fmicb-11-583010-g003.jpg)