Advances in Correlative Single-Molecule Localization Microscopy and Electron Microscopy

Endesfelder, Ulrike

doi:10.2478/nbi-2014-0002

Cited by 7 publications

(3 citation statements)

References 64 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Recently, correlative fluorescence light and electron microscopy has raised great interest in the life sciences because it combines the specificity of fluorescence labeling with the high structural resolution and cellular environment by the scanning electron microscopy (SEM) or TEM ( 39 ). The general and most widespread fluorescence targeting methods use genetically encoded labeling with green fluorescent proteins, immunofluorescent labeling with Nanogold, or a combination of both ( 40 , 41 ).…”

Section: Introductionmentioning

confidence: 99%

Single-Cell Resolution of Uncultured Magnetotactic Bacteria via Fluorescence-Coupled Electron Microscopy

Zhang

Menguy

et al. 2017

Appl Environ Microbiol

124

View full text Add to dashboard Cite

Magnetotactic bacteria (MTB) form intracellular chain-assembled nanocrystals of magnetite or greigite termed magnetosomes. The characterization of magnetosome crystals requires electron microscopy due to their nanoscopic sizes. However, electron microscopy does not provide phylogenetic information for MTB. We have developed a strategy for the simultaneous and rapid phylogenetic and biomineralogical characterization of uncultured MTB at the single-cell level. It consists of four steps: (i) enrichment of MTB cells from an environmental sample, (ii) 16S rRNA gene sequencing of MTB, and (iii) fluorescence in situ hybridization analyses coordinated with (iv) transmission or scanning electron microscopy of the probe-hybridized cells. The application of this strategy identified a magnetotactic Gammaproteobacteria strain, SHHR-1, from brackish sediments collected from the Shihe River estuary in Qinhuangdao City, China. SHHR-1 magnetosomes are elongated prismatic magnetites which can be idealized as hexagonal prisms. Taxonomic groups of uncultured MTB were also identified in freshwater sediments from Lake Miyun in northern Beijing via this novel coordinated fluorescence and scanning electron microscopy method based on four group-specific rRNA-targeted probes. Our analyses revealed that major magnetotactic taxonomic groups can be accurately determined only with coordinated scanning electron microscopy observations on fluorescently labeled single cells due to limited group coverage and specificity for existing group-specific MTB fluorescence in situ hybridization (FISH) probes. Our reported strategy is simple and efficient, offers great promise toward investigating the diversity and biomineralization of MTB, and may also be applied to other functional groups of microorganisms.IMPORTANCE Magnetotactic bacteria (MTB) are phylogenetically diverse and biomineralize morphologically diverse magnetic nanocrystals of magnetite or greigite in intracellular structures termed magnetosomes. However, many uncultured MTB strains have not been phylogenetically identified or structurally investigated at the single-cell level, which limits our comprehensive understanding of the diversity of MTB and their role in biomineralization. We developed a fluorescence-coupled electron microscopy method for the rapid phylogenetic and biomineralogical characterization of uncultured MTB at the single-cell level. Using this novel method, we successfully identified taxonomic groups of several uncultured MTB and one novel magnetotactic Gammaproteobacteria strain, SHHR-1, from natural environments. Our analyses further indicate that strain SHHR-1 forms elongated prismatic magnetites. Our findings provide a promising strategy for the rapid characterization of phylogenetic and biomineralogical properties of uncultured MTB at the single-cell level. Furthermore, due to its simplicity and generalized methodology, this strategy can also be useful in the study of the diversity and biomineralization properties of microbial taxa involved in other mineralizat...

show abstract

Section: Introductionmentioning

confidence: 99%

Single-Cell Resolution of Uncultured Magnetotactic Bacteria via Fluorescence-Coupled Electron Microscopy

Zhang

Menguy

et al. 2017

Appl Environ Microbiol

124

View full text Add to dashboard Cite

show abstract

“…In contrast, optical microscopy can non-destructively image embedded internal features at depths beyond that of EM or SPM. For example, optical microscopy can monitor internal single molecule distributions and locate defects inside crystals 9 10 11 . However, the spatial resolution of conventional optical imaging methods is fundamentally limited by the optical diffraction limit, which is far worse than the spatial resolution of EM and SPM.…”

mentioning

confidence: 99%

Subsurface Super-resolution Imaging of Unstained Polymer Nanostructures

Urban

Dong

Nguyen

et al. 2016

Sci Rep

View full text Add to dashboard Cite

Optical imaging has offered unique advantages in material researches, such as spectroscopy and lifetime measurements of deeply embedded materials, which cannot be matched using electron or scanning-probe microscopy. Unfortunately, conventional optical imaging cannot provide the spatial resolutions necessary for many nanoscopic studies. Despite recent rapid progress, super-resolution optical imaging has yet to be widely applied to non-biological materials. Herein we describe a method for nanoscopic optical imaging of buried polymer nanostructures without the need for extrinsic staining. We observed intrinsic stochastic fluorescence emission or blinking from unstained polymers and performed spatial-temporal spectral analysis to investigate its origin. We further applied photon localization super-resolution imaging reconstruction to the detected stochastic blinking, and achieved a spatial resolution of at least 100 nm, which corresponds to a six-fold increase over the optical diffraction limit. This work demonstrates the potential for studying the static heterogeneities of intrinsic polymer molecular-specific properties at sub-diffraction-limited optical resolutions.

show abstract

“…However, because they are typically applied separately, meaningful correlations between individual cells and the relevant sets of measurements are often hard (or impossible) to draw. By applying multiple microscopic and spectroscopic analyses on a single sample, an approach referred to as correlative microscopy [ 2 ], the limitations of a single technique can be overcome, and the results combined into a single dataset that provides micro- to nano-scale information of a biological sample [ 3 ].…”

Section: Introductionmentioning

confidence: 99%

Correlative SIP-FISH-Raman-SEM-NanoSIMS links identity, morphology, biochemistry, and physiology of environmental microbes

Schaible

Kohtz

Cliff

et al. 2022

ISME Communications

View full text Add to dashboard Cite

Microscopic and spectroscopic techniques are commonly applied to study microbial cells but are typically used on separate samples, resulting in population-level datasets that are integrated across different cells with little spatial resolution. To address this shortcoming, we developed a workflow that correlates several microscopic and spectroscopic techniques to generate an in-depth analysis of individual cells. By combining stable isotope probing (SIP), fluorescence in situ hybridization (FISH), scanning electron microscopy (SEM), confocal Raman microspectroscopy (Raman), and nano-scale secondary ion mass spectrometry (NanoSIMS), we illustrate how individual cells can be thoroughly interrogated to obtain information about their taxonomic identity, structure, physiology, and metabolic activity. Analysis of an artificial microbial community demonstrated that our correlative approach was able to resolve the activity of single cells using heavy water SIP in conjunction with Raman and/or NanoSIMS and establish their taxonomy and morphology using FISH and SEM. This workflow was then applied to a sample of yet uncultured multicellular magnetotactic bacteria (MMB). In addition to establishing their identity and activity, backscatter electron microscopy (BSE), NanoSIMS, and energy-dispersive X-ray spectroscopy (EDS) were employed to characterize the magnetosomes within the cells. By integrating these techniques, we demonstrate a cohesive approach to thoroughly study environmental microbes on a single-cell level.

show abstract

Advances in Correlative Single-Molecule Localization Microscopy and Electron Microscopy

Cited by 7 publications

References 64 publications

Single-Cell Resolution of Uncultured Magnetotactic Bacteria via Fluorescence-Coupled Electron Microscopy

Single-Cell Resolution of Uncultured Magnetotactic Bacteria via Fluorescence-Coupled Electron Microscopy

Subsurface Super-resolution Imaging of Unstained Polymer Nanostructures

Correlative SIP-FISH-Raman-SEM-NanoSIMS links identity, morphology, biochemistry, and physiology of environmental microbes

Contact Info

Product

Resources

About