New preparation techniques for molecular and in‐situ analysis of ancient organic micro‐ and nanostructures

Fadel, Alexandre; Lepot, Kevin; Nuns, Nicolás; Régnier, Sylvie; Riboulleau, Armelle

doi:10.1111/gbi.12380

Cited by 9 publications

(4 citation statements)

References 112 publications

(222 reference statements)

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“…Nevertheless, with PCA we could easily distinguish root, PO x – ( Figure 4c ) and minerals (see Supporting Figure 4 ). However, with future improvements in the complex analysis methods, and with an availability of a correlative sample preparation method, distinguishing biomolecules from LR white resin may be possible ( Fadel et al, 2020 ).…”

Section: Resultsmentioning

confidence: 99%

“…Particularly, the structural and biochemical complexity and spatial arrangement of microbes of the rhizosphere as the main driver of terrestrial biogeochemical cycles is little understood. To explore such relationships between multiple components (Ganther et al, 2020), it is crucial to characterize all soil components whilst keeping their structural and chemical integrity i.e., chemical composition and spatial organization of minerals, non-living organic matter and microbial counterparts such as bacteria, fungi, and plant roots during the analysis (Nunan et al, 2007;Mueller et al, 2013Mueller et al, , 2019Steffens et al, 2017;Vidal et al, 2018;Juyal et al, 2019Juyal et al, , 2020Fadel et al, 2020). The main reason that confines studies to a single discipline, rather than expanding to combinatorial characterization is associated with sample preparation.…”

Section: Introductionmentioning

confidence: 99%

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Microbial Identification, High-Resolution Microscopy and Spectrometry of the Rhizosphere in Its Native Spatial Context

et al. 2021

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During the past decades, several stand-alone and combinatorial methods have been developed to investigate the chemistry (i.e., mapping of elemental, isotopic, and molecular composition) and the role of microbes in soil and rhizosphere. However, none of these approaches are currently applicable to characterize soil-root-microbe interactions simultaneously in their spatial arrangement. Here we present a novel approach that allows for simultaneous microbial identification and chemical analysis of the rhizosphere at micro− to nano-meter spatial resolution. Our approach includes (i) a resin embedding and sectioning method suitable for simultaneous correlative characterization of Zea mays rhizosphere, (ii) an analytical work flow that allows up to six instruments/techniques to be used correlatively, and (iii) data and image correlation. Hydrophilic, immunohistochemistry compatible, low viscosity LR white resin was used to embed the rhizosphere sample. We employed waterjet cutting and avoided polishing the surface to prevent smearing of the sample surface at nanoscale. The quality of embedding was analyzed by Helium Ion Microscopy (HIM). Bacteria in the embedded soil were identified by Catalyzed Reporter Deposition-Fluorescence in situ Hybridization (CARD-FISH) to avoid interferences from high levels of autofluorescence emitted by soil particles and organic matter. Chemical mapping of the rhizosphere was done by Scanning Electron Microscopy (SEM) with Energy-dispersive X-ray analysis (SEM-EDX), Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS), nano-focused Secondary Ion mass Spectrometry (nanoSIMS), and confocal Raman spectroscopy (μ-Raman). High-resolution correlative characterization by six different techniques followed by image registration shows that this method can meet the demanding requirements of multiple characterization techniques to identify spatial organization of bacteria and chemically map the rhizosphere. Finally, we presented individual and correlative workflows for imaging and image registration to analyze data. We hope this method will be a platform to combine various 2D analytics for an improved understanding of the rhizosphere processes and their ecological significance.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Microbial Identification, High-Resolution Microscopy and Spectrometry of the Rhizosphere in Its Native Spatial Context

et al. 2021

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“…However, with future improvements in the complex analysis methods, and with an availability of a correlative sample preparation method, distinguishing biomolecules from LR white resin may be possible. 9…”

Section: Resultsmentioning

confidence: 99%

High-Resolution Chemical Mapping and Microbial Identification of Rhizosphere using Correlative Microscopy

Bandara

Schmidt

Davoudpour

et al. 2021

Preprint

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During the past decades, several stand-alone and combinatory methods have been developed to investigate the chemistry (i.e. mapping of elemental, isotopic and molecular composition) and the role of microbes in soil and rhizosphere. However, none of these approaches are currently capable of characterizing soil-root-microbe interactions simultaneously in their spatial arrangement. Here we present a novel approach that allows chemical and microbial identification of the rhizosphere at micro- to nano-meter spatial resolution. Our approach includes i) a resin embedding and sectioning method suitable for simultaneous correlative characterization of Zea mays rhizosphere, ii) an analytical work flow that allows up to six instruments/techniques to be used correlatively, and iii) data and image correlation. Hydrophilic, immunohistochemistry compatible, low viscosity LR white resin was used to embed the rhizosphere sample. We employed waterjet cutting and avoided polishing the surface to prevent smearing of the sample surface at nanoscale. Embedding quality was analyzed by Helium Ion Microscopy (HIM). Bacteria in the embedded soil was identified by Catalyzed Reporter Deposition-Fluorescence In Situ Hybridization (CARD-FISH) to avoid interferences from high levels of auto fluorescence emitted by soil particles and organic matter. Chemical mapping of the rhizosphere was done by Scanning Electron Microscopy (SEM) with Energy-dispersive X-ray analysis (SEM-EDX), Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS), nanoSIMS and nano-focused Secondary Ion mass Spectrometry (nanoSIMS), and confocal Raman spectroscopy (μ-Raman). High-resolution correlative characterization by six different techniques followed by image registration shows that this method can meet the demanding requirements of multiple characterization techniques to chemically map the rhizosphere and identify spatial organization of bacteria. Finally, we presented individual and correlative workflows for imaging and image registration to analyze data. We hope this method will be a platform to combine various 2D analytics for an ample understanding of the rhizosphere processes and their ecological significance.

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“…Te initial and formidable task involves sample size and preparation for NanoSIMS analysis. Efective preparation of the sample is vital and encompasses methods like laser ablation, chemical extraction, and mechanical grinding [41]. Meanwhile, the interpretation of acquired data heavily relies on the methods of data acquisition and analytical techniques [42,43].…”

Section: Preparation Protocols and Practical Aspects In Samplementioning

confidence: 99%

Nano-Scale Secondary Ion Mass Spectrometry: A Paradigm Shift in Soil Science

Mir,

Qin,

Mir

et al. 2024

Journal of Spectroscopy

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Soils exhibit structural heterogeneity across diverse spatio-temporal scales, yielding myriad of microhabitats, highlighting the need for a nuanced understanding of the intricate interactions within the soil matrix. At the nanometer scale, the interplay among organic matter (OM), mineral particles, and microbiota intricately govern the long-term destiny of soil carbon (C), nutrient cycling, and the fate of both organic and inorganic pollutants. Notably, the sorption of soil organic matter (SOM) onto smaller clay particles and its entrapment in microaggregates further contribute to this complex dynamic. Understanding these processes depends on recognizing their scale-dependent nature, necessitating sophisticated techniques for investigation. Although various methods are employed across scales, the current set of techniques still lacks the requisite sensitivity and resolution for microscale data collection. To address this limitation, the adoption of novel microscopic and spectroscopic techniques capable of probing molecular, isotopic, and elemental patterns at the micro to nano scale becomes imperative. Among these cutting-edge methodologies, the nano-scale secondary ion mass spectrometer (NanoSIMS) emerges as a paradigm-shifting tool. Representing the latest evolution in ion microprobes, NanoSIMS seamlessly integrates high-resolution microscopy and isotopic analysis, maintaining unparalleled signal transmission and spatial resolution, reaching as fine as 50 nm. Its capabilities extend beyond conventional applications in science, as evidenced by recent breakthroughs in utilizing NanoSIMS to study biophysical interfaces in soils. This article underscores the pressing need to advance the incorporation of NanoSIMS as a pioneering instrumentation technique in soil studies. Furthering the implementation of this novel instrumentation technique in soil studies will pave avenues and aid in the advancement of future research.

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New preparation techniques for molecular and in‐situ analysis of ancient organic micro‐ and nanostructures

Abstract: Unlike the fossil record of the Phanerozoic, the earlier and much longer Precambrian time is characterized by a comparatively scarce fossil record dominated by microscopic prokaryotes and eukaryotes (Javaux

Cited by 9 publications

References 112 publications

Microbial Identification, High-Resolution Microscopy and Spectrometry of the Rhizosphere in Its Native Spatial Context

Microbial Identification, High-Resolution Microscopy and Spectrometry of the Rhizosphere in Its Native Spatial Context

High-Resolution Chemical Mapping and Microbial Identification of Rhizosphere using Correlative Microscopy

Nano-Scale Secondary Ion Mass Spectrometry: A Paradigm Shift in Soil Science

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