Visualizing the inner life of microbes: practices of multi-color single-molecule localization microscopy in microbiology

Vojnovic, Ilijana; Winkelmeier, Jannik; Endesfelder, Ulrike

doi:10.1042/bst20180399

Cited by 19 publications

(22 citation statements)

References 95 publications

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“…While single-target studies, mostly using genetic FP tags, are close to getting routine, multi-target or correlative approaches are still rather rare in single-molecule microbiology and need extensive planning [91]. Most available experimental designs are currently dictated by the restricted number of compatible fluorophore and labeling options that fulfill the strict criteria list for high-quality single-molecule microscopy, e.g.…”

Section: Open Challenges In Single-molecule Microbiologymentioning

confidence: 99%

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From single bacterial cell imaging towards in vivo single-molecule biochemistry studies

Endesfelder

2019

Essays in Biochemistry

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Bacteria as single-cell organisms are important model systems to study cellular mechanisms and functions. In recent years and with the help of advanced fluorescence microscopy techniques, immense progress has been made in characterizing and quantifying the behavior of single bacterial cells on the basis of molecular interactions and assemblies in the complex environment of live cultures. Importantly, single-molecule imaging enables the in vivo determination of the stoichiometry and molecular architecture of subcellular structures, yielding detailed, quantitative, spatiotemporally resolved molecular maps and unraveling dynamic heterogeneities and subpopulations on the subcellular level. Nevertheless, open challenges remain. Here, we review the past and current status of the field, discuss example applications and give insights into future trends.

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Section: Open Challenges In Single-molecule Microbiologymentioning

confidence: 99%

“…Also, new imaging techniques promise drastic improvements in temporal resolution and long-term imaging without increased phototoxicity [99]. Nevertheless, how to best visualize interacting and co-moving molecules using single-molecule imaging currently remains an open question [91].…”

Section: Open Challenges In Single-molecule Microbiologymentioning

confidence: 99%

From single bacterial cell imaging towards in vivo single-molecule biochemistry studies

Endesfelder

2019

Essays in Biochemistry

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“…SMLM imaging has become a well-established technique for many bacteria and yeast in recent years (Vojnovic et al, 2019), but archaea have not yet been studied using this technique. For archaeal studies, even standard fluorescence microscopy is still only a rarely used tool, as fluorescence imaging faces (a) the often extreme growth conditions of archaeal organisms (e.g., very high or low temperature or high saline, acidic, alkaline, or anaerobic environments), (b) their colorful pigments causing autofluorescence, and (c) the limited range of available model organisms and molecular tools (Kramm et al, 2019).…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2020

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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.

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“…Consequently, SMLM imaging has become a powerful tool for studying cell biology (Endesfelder, 2019). SMLM imaging has become a well-established technique for many bacteria and yeast in recent years (Vojnovic et al, 2019), but archaea have not yet been studied using this technique. For archaeal studies, even standard fluorescence microscopy is still only a rarely used tool, as fluorescence imaging faces a) the often extreme growth conditions of archaeal organisms (e.g.…”

Section: Introductionmentioning

confidence: 99%

Establishing live-cell single-molecule localization microscopy imaging and single-particle tracking in the archaeonHaloferax volcanii

Turkowyd

Schreiber

Wörtz

et al. 2020

Preprint

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In recent years, fluorescence microscopy techniques for the localization and tracking of single molecules in living cells have become well-established and 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 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.

show abstract

Visualizing the inner life of microbes: practices of multi-color single-molecule localization microscopy in microbiology

Cited by 19 publications

References 95 publications

From single bacterial cell imaging towards in vivo single-molecule biochemistry studies

From single bacterial cell imaging towards in vivo single-molecule biochemistry studies

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

Establishing live-cell single-molecule localization microscopy imaging and single-particle tracking in the archaeonHaloferax volcanii

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