Scanning Electron Microscopy In Backscattered Electron Mode Research Articles

Diversity and Colonization Strategies of Endolithic Cyanobacteria in the Cold Mountain Desert of Pamir.

Microorganisms can survive in extreme environments and oligotrophic habitats thanks to their specific adaptive capacity. Due to its severe and contrasting climate conditions, the cold mountain desert in Eastern Pamir provides a unique environment for analyzing microbial adaptation mechanisms occurring within colonization of endolithic habitats. This study aims to investigate the composition and structure of endolithic microbial communities and analyze the interactions between microorganisms and colonized lithic substrates. Endolithic biofilms were examined using scanning electron microscopy in backscattered electron mode (SEM-BSE) and next-generation sequencing (NGS) applying amplicon sequence variants (ASVs) approach. The investigation of the V3–V4 region of 16S rRNA gene revealed that endolithic communities are dominated by Actinobacteria (26%), Proteobacteria (23%), and Cyanobacteria (11.4%). Cyanobacteria were represented by Oxyphotobacteria with a predominance of subclasses of Oscillatoriophycidae, Synechococcophycideae, and Nostocophycidae as well as the rarely occurring Sericytochromatia. The positive correlation between the contribution of the orders Synechococcales and Rhizobiales to community structure suggests that some functionally closed taxa of Cyanobacteria and Proteobacteria can complement each other, for example, in nitrogen fixation in endolithic communities. The endolithic communities occurring in Eastern Pamir were identified as complex systems whose composition and structure seem to be influenced by the architecture of microhabitats and related microenvironmental conditions.

Towards a more realistic picture of in situ biocide actions: Combining physiological and microscopy techniques

In this study, we combined chlorophyll a fluorescence (ChlaF) measurements, using pulse-amplitude-modulate (PAM) equipment, with scanning electron microscopy in backscattered electron mode (SEM-BSE) and transmission electron microscopy (TEM) images to evaluate the actions of Koretrel at lower concentrations on Verrucaria nigrescens colonising a dolostone. ChlaF measurements are good indicators of the damaging effects of biocides. However, these indicators only provide an incomplete view of the mechanism of biocides used to control biodeterioration agents. The death of the V. nigrescens photobiont at two biocide concentrations was revealed by PAM, SEM-BSE and TEM. Once Koretrel was applied, the Fv/Fm ratios markedly fell in the first few hours after the 1.5% treatment, and ratios for the 3% dilution remained close to zero throughout the study. The algal zone shows the plasmolysed appearance of the photobiont cells, and important aspects related to the action of the biocide on free and lichenised fungi were also detected using SEM-BSE. Many of the mycobiont cells had only their cell walls preserved; although, some fungal hyphae in lichen thalli and some microorganisms in endolithic clusters maintained lipid storage in their cytoplasm. These results indicated that the combination of physiological and microscopy techniques improves the assessment of biocide action in situ and this will help to optimize protocols in order to reduce the emission of these compounds to the environment.

Characterizing the Microbial Colonization of a Dolostone Quarry: Implications for Stone Biodeterioration and Response to Biocide Treatments

This study examines the microbial colonization of three fronts of an abandoned dolostone quarry (Redueña, Madrid, Spain) exposed to atmospheric conditions for different time periods since Roman times to the present. Through scanning electron microscopy in backscattered electron mode (SEM-BSE), endolithic colonization was predominantly detected in the most recently exposed front, while in the longer exposed quarry fronts, epilithic forms of growth were most often observed. These observations were confirmed by denaturing gradient gel electrophoresis (DGGE) analysis. Based on the distribution pattern of microbial colonization in the different quarry fronts, we then established a sequence of colonization events that took place over this long time frame. Bioalteration processes related to this sequential colonization were also identified. Characterizing these sequential processes can be useful for interpreting biodeterioration processes in historic dolostone monuments, especially those affecting constructions in the area of the Redueña stone quarry. In a second experimental stage, different biocide treatments were tested on this quarry rock to find the best way to avoid the microbial colonization effects identified. Through combined SEM-BSE/DGGE analysis, the efficacy of several biocides against the microorganisms inhabiting the dolostones was assessed after 4 and 16 months treatment. In general, all treatments were effective at reducing around 80% of the lichen cover, although effects on endolithic lithobiontic communities were dependent on how well the rock surface had been mechanically cleaned prior to treatment and gradually disappeared over time.

Bioreceptivity of dolostones to fungal colonization

In many historic monuments in which signs of biodeterioration have been frequently reported, dolostones were used as dimension stone for their construction. In an effort to assess the influence of the texture of dolostones on their potential bioreceptivity, we characterized microbial colonization strategies in dolostone samples of predictably different textural features by scanning electron microscopy in back scattered electron mode (SEM-BSE), low temperature scanning electron microscopy (LTSEM), transmission light microscopy (TLM) and mercury intrusion porosimetry (MIP). Fungi were the predominant microorganisms in the dolostones examined and their colonization showed three well defined stages with the final consequence of complete rock disaggregation. The results of this study indicate that porosity differences (mainly the extent and type) were particularly relevant for determining the presence and extent of each colonization stage. As a determinant of bioreceptivity, the porosity of dolostones will also condition the decay processes associated with this colonization. These findings highlight the fact that the intrinsic properties of dolostones, such as texture, need to be considered when selecting this type of stone for future construction projects.

Effects of industrial by-product amendments on As, Cd and Tl retention/release in an element-spiked acidic soil

To asses the efficiency of two by-products (phosphogypsum (PG) and sugar foam (SF), rich in gypsum and calcium carbonate, respectively) in the immobilization of three toxic elements (As, Cd and Tl) in an acidic soil, batch-scale sorption and desorption experiments were conducted after 18 months of in situ amendment application. The Langmuir isotherms applied for sorption studies showed that the estimated maximum sorption capacity of the elements was highest in the SF-treated samples. The amount of element retained and the percentage of extraction after TCLP tests indicated that those samples amended with sugar foam (SF and PG + SF) had the potential to immobilize As, Cd and Tl in an acidic soil with low sorptive capacity. In addition to sorption and desorption experiments, scanning electron microscopy in back-scattered electron mode (SEM-BSE) showed the formation of Al-hydroxy polymers which provides the soil with additional sorption capacity. The three target elements were associated with the Al-hydroxy polymers, probably through direct coordination or the formation of ternary complexes. By means of statistical analysis it has been found that sorption processes of As, Cd and Tl in this soil mainly depend on the treatment, whereas desorption is an element-dependent process.

NEW APPLICATIONS OF LIGHT AND ELECTRON MICROSCOPIC TECHNIQUES FOR THE STUDY OF MICROBIOLOGICAL INCLUSIONS IN AMBER

Amber is a superb medium for the fossilization of delicate organisms. Besides light microscopy techniques for the study of insects in amber, scanning electron microscopy (SEM) in backscattered electron mode (SEM-BSE), low temperature SEM (LTSEM) and also confocal laser scanning microscopy (CLSM) were used to examine microcenosis and particulate plant remains (microdebris). We applied these techniques to such inclusions in amber Alava, northern Spain (Allaian: Early Cretaceous). Confocal microscopy provides a 3D image of partial microcenosis showing bifurcate fungal hyphae. The huge potential of SEM-BSE yields high resolution images, in which the relationship between protozoa and fungal hyphae may be observed and the characterization of further ultrastructural details in flagellates. According to the SEM-BSE images, food and pulsatile vacuoles appear better preserved than mitochondria and lipids in amber-embedded protozoa. A process of protozoan mineralization has led to the deposition of S and Fe in peripheral areas, and the Fe is also present in the core of surrounding fungal hyphae. Application of LTSEM for the study of protozoan inclusions produces images of their exteriors showing many vacuoles. Plant tissues under SEM-BSE show mummified cell walls, while the cytoplasm exhibits a bright appearance and is very rich in Fe and S. SEM in secondary electron mode (SEM-SE) also reveals a microbiota trapped in gas bubbles.

SEM observations in the back-scattered mode of the soil–root zone of Brassica napus (cv. Rafal) plants grown at a range of soil pH values

In the study of the root–soil interface, scanning electron microscopy (SEM) in secondary emission mode and transmission electron microscopy are commonly used techniques. As these methods have some limitations, there is a need to explore other techniques which may be suitable for the observation of the root–soil zone. One of the most promising methods is the application of SEM in the back-scattered electron (BSE) emission mode. The root zone of plants of winter oilseed rape ( Brassica napus cv. Rafal) grown at a range of soil pH and soil phosphorus (P) concentrations was examined by SEM in BSE mode. The method for the preparation of transverse sections of the root–soil contact zone is presented. The BSE scanning images of the root–soil interface obtained allowed investigation of the flow paths of plant nutrients from the soil towards roots and their distribution in the roots. Concentration of aluminium in the cell walls of immature roots and at the soil–root interface was observed in the most acid soils. Potassium, calcium and phosphorus were found in differentiated roots associated with the external tissues of the central cylinder. The intimate contact of root surface with soil mineral particles could also be observed.

Structural Aspects of the Lichen‐Rock Interface Using Back‐scattered Electron Imaging

AbstractThe profound knowledge of the structural and chemical characteristics of the interface between lichen thallus and rock, seems to be indispensable for the process of understanding the lichen symbiosis as well as the significance of the weathering action of lichens. One of the most promising techniques to be used in this investigation is the Scanning Electron Microscopy (SEM) in the back‐scattered electron (BSE) emission mode. In the present work thalli of Parmelia conspersa, Aspicilia intermutans and Lecidea auriculata growing in granitic rock were examined by SEM in BSE mode with (Energy Dispersive Spectroscopy) EDS. In the case of the foliose thalli the observation of the interface permits detection of the rhizine/hyphae adherence and determination of the origin of the minerals which adhere to the rhizine/hyphae. In the case of the crustose thalli BSE permits investigation within the ultrastructure of the crustose thallus and crustose lichen‐rock contact zone and also allows observations of the penetration and filling of the fissures and cracks of the underlying rock by components of the thallus and other living organisms. The BSE images could contribute to a better knowledge of the cytological state of the rock‐inhabiting organisms and also to the understanding of the action of the chemical treatments used in the removal of lichen from building materials.

Application of back‐scattered electron imaging to the study of the lichen‐rock interface

SummaryIn the study of the lichen‐rock interface, light microscopy, scanning electron microscopy (SEM) in secondary emission mode and transmission electron microscopy are the most commonly used techniques. As these methods have some limitations, there is a need to explore other techniques for observation of the lichen‐substrate interface. One of the most promising methods is the application of SEM in the back‐scattered electron (BSE) emission mode.The thallus ofAspicilia intermutans(Nyl.) Arn. growing on granitic rock was examined by SEM in BSE mode. The detailed preparation of transverse sections of the lichen‐rock contact zone is presented. The BSE scanning images of the lichen‐rock interface obtained present new insights into the ultrastructural features of the biological components, providing more information about the biogeophysical and biogeochemical weathering of rock.