Salt tolerance in mungbean is associated with controlling Na and Cl transport across roots, regulating Na and Cl accumulation in chloroplasts and maintaining high K in root and leaf mesophyll cells.

Salinity tolerance requires coordinated responses encompassing salt exclusion in roots and tissue/cellular compartmentation of salt in leaves. We investigated the possible control points for salt ions transport in roots and tissue tolerance to Na+ and Cl- in leaves of two contrasting mungbean genotypes, salt-tolerant Jade AU and salt-sensitive BARI Mung-6, grown in nonsaline and saline (75 mM NaCl) soil. Cryo-SEM X-ray microanalysis was used to determine concentrations of Na, Cl, K, Ca, Mg, P, and S in various cell types in roots related to the development of apoplastic barriers, and in leaves related to photosynthetic performance. Jade AU exhibited superior salt exclusion by accumulating higher [Na] in the inner cortex, endodermis, and pericycle with reduced [Na] in xylem vessels and accumulating [Cl] in cortical cell vacuoles compared to BARI Mung-6. Jade AU maintained higher [K] in root cells than BARI Mung-6. In leaves, Jade AU maintained lower [Na] and [Cl] in chloroplasts and preferentially accumulated [K] in mesophyll cells than BARI Mung-6, resulting in higher photosynthetic efficiency. Salinity tolerance in Jade AU was associated with shoot Na and Cl exclusion, effective regulation of Na and Cl accumulation in chloroplasts, and maintenance of high K in root and leaf mesophyll cells.

Rapid Freezing and Cryo-SEM-EDS Imaging of Foraminifera (Unicellular Eukaryotes) Using a Conductive Viscous Cryogenic Glue.

Spatial distribution of water-soluble molecules and ions in living organisms is still challenging to assess. Energy-dispersive X-ray spectroscopy (EDS) via cryogenic scanning electron microscopy (cryo-SEM) is one of the promising methods to study them without loss of dissolved contents. High-resolution cryo-SEM-EDS has challenges in sample preparation, including cross-section exposure and sample drift/charging due to insulative surrounding water. The former becomes problematic for large and inseparable organisms, such as benthic foraminifera, a unicellular eukaryote playing significant roles in marine ecosystems, which often exceed the size limit for the most reliable high-pressure freezing. Here we show graphite oxide dispersed in sucrose solution as a good glue to freeze, expose cross-section by cryo-ultramicrotome, and analyze elemental distribution owing to the glue's high viscosity, adhesion force, and electron conductivity. To demonstrate the effectiveness and applicability of the glue for cryo-SEM-EDS, deep-sea foraminifer Uvigerina akitaensis was sampled during a cruise and plunge frozen directly on the research vessel, where the liquid nitrogen supply is limited. The microstructures were preserved as faithfully in cryo-SEM images as those with the conventional resin-substituted transmission electron micrograph. We found elements colocalized within the cytoplasm originating from water-soluble compounds that can be lost with conventional dehydrative fixation.

Lipids and Proteins Differentiation in Membrane Fouling Using Heavy Metal Staining and Electron Microscopy at Cryogenic Temperatures.

The detailed characterization of fouling in membranes is essential to understand any observed improvement or reduction on filtration performance. Electron microscopy allows detailed structural characterization, and its combination with labeling techniques, using electron-dense probes, typically allows for the differentiation of biomolecules. Developing specific protocols that allow for differentiation of biomolecules in membrane fouling by electron microscopy is a major challenge due to both as follows: the necessity to preserve the native state of fouled membranes upon real filtration conditions as well as the inability of the electron-dense probes to penetrate the membranes once they have been fouled. In this study, we present the development of a heavy metal staining technique for identification and differentiation of biomolecules in membrane fouling, which is compatible with cryofixation methods. A general contrast enhancement of biomolecules and fouling is achieved. Our observations indicate a strong interaction between biomolecules: A tendency of proteins, both in solution as well as in the fouling, to surround the lipids is observed. Using transmission electron microscopy and scanning electron microscopy at cryogenic conditions, cryo-SEM, in combination with energy-dispersive X-ray spectroscopy, the spatial distribution of proteins and lipids within fouling is shown and the role of proteins in fouling discussed.

Insights into the mechanism of diurnal variations in methane emission from the stem surfaces of Alnus japonica.

Recent studies have suggested that in certain environments, tree stems emit methane (CH4 ). This study explored the mechanism of CH4 emission from the stem surfaces of Alnus japonica in a riparian wetland. Stem CH4 emission rates and sap flux were monitored year-round, and fine-root anatomy was investigated. CH4 emission rates were estimated using a closed-chamber method. Sap flux was measured using Granier-type thermal dissipation probes. Root anatomy was studied using both optical and cryo-scanning electron microscopy. CH4 emissions during the leafy season exhibited a diurnally changing component superimposed upon an underlying continuum in which the diurnal variation was in phase with sap flux. We propose a model in which stem CH4 emission involves at least two processes: a sap flux-dependent component responsible for the diurnal changes, and a sap flux-independent component responsible for the background continuum. The contribution ratios of the two processes are season-dependent. The background continuum possibly resulted from the diffusive transport of gaseous CH4 from the roots to the upper trunk. Root anatomy analysis indicated that the intercellular space of the cortex and empty xylem cells in fine roots could serve as a passageway for transport of gaseous CH4 .

Salt tolerance in relation to elemental concentrations in leaf cell vacuoles and chloroplasts of a C4 monocotyledonous halophyte.

Halophytes accumulate and sequester high concentrations of salt in vacuoles while maintaining lower levels of salt in the cytoplasm. The current data on cellular and subcellular partitioning of salt in halophytes are, however, limited to only a few dicotyledonous C3 species. Using cryo-scanning electron microscopy X-ray microanalysis, we assessed the concentrations of Na, Cl, K, Ca, Mg, P and S in various cell types within the leaf-blades of a monocotyledonous C4 halophyte, Rhodes grass (Chloris gayana). We also linked, for the first time, elemental concentrations in chloroplasts of mesophyll and bundle sheath cells to their ultrastructure and photosynthetic performance of plants grown in nonsaline and saline (200 mM NaCl) conditions. Na and Cl accumulated to the highest levels in xylem parenchyma and epidermal cells, but were maintained at lower concentrations in photosynthetically active mesophyll and bundle sheath cells. Concentrations of Na and Cl in chloroplasts of mesophyll and bundle sheath cells were lower than in their respective vacuoles. No ultrastructural changes were observed in either mesophyll or bundle sheath chloroplasts, and photosynthetic activity was maintained in saline conditions. Salinity tolerance in Rhodes grass is related to specific cellular Na and Cl distributions in leaf tissues, and the ability to regulate Na and Cl concentrations in chloroplasts.

Cryo-scanning electron microscopy demonstrates that ice morphology is not associated with the post-thaw survival of domestic boar (Sus domesticus) spermatozoa: A comparison of directional and conventional freezing methods.

Directional freezing (in 2 or 10 ml hollow glass tubes) has been reported to improve post-thaw sperm survival parameters compared to conventional methods (in 0.5 ml straws). However, the biophysical properties that increase post-thaw survival are poorly understood. Therefore, the aim for the current study was to investigate the effect of ice morphology on the post-thaw survival of domestic boar spermatozoa directionally and conventionally cryopreserved in 0.5 ml straws. Ice morphology was quantitatively analyzed using a combination of cryo-scanning electron microscopy and Fiji Shape Descriptors. Multivariate analysis found a significant, non-linear effect (p < 0.05) of interface velocity on ice morphology, with an increase in both ice-lake size, as indicated by area and in aspect ratio, at an interface velocity of 0.2 mm/s. By contrast, post-thaw sperm survival (defined as spermatozoa with both intact plasma membranes and acrosomes) was biphasic, with peaks of survival at interface velocities of 0.2 mm/s (54.2 ± 1.9%), and 1.0 or 1.5 mm/s (56.5 ± 1.5%, 56.7 ± 1.7% respectively), and lowest survival at 0.5 (52.1 ± 1.6%) and 3.0 mm/s (51.4 ± 1.9%). Despite numerical differences in Shape Descriptors, there was no difference (p > 0.05) in the post-thaw survival between conventionally and directionally cryopreserved samples at optimal interface velocities of 1.0 or 1.5 mm/s. These findings suggest that: 1) ice morphology has little impact on post-thaw survival of boar spermatozoa, and 2) directional freezing in 0.5 ml straws (rather than 2 or 10 ml hollow glass tubes) may attenuate benefits of directional freezing.

Study the influence of waxy wheat flour, inulin and guar gum on quality and microstructure of Pita and Tandoori breads: response surface methodology aids functional food development.

A diet high in dietary fibre (DF) is thought to help reduce cholesterol levels, may control blood glucose levels and reduces gut transit time. However, people often fail to consume the recommended quantity of DF. The aim of this study was to supplement two types of bread with some novel functional food ingredients; waxy wheat flour (WF), inulin (IN) and guar gum (GG) to develop products rich in DF. Response surface methodology (RSM) was employed to study the effect of DF on two different breads. Breads were assessed using Cryo-SEM for its microstructure pattern, and physical attributes (pita; springiness, chewiness and hardness, Tandoori; hardness and elasticity). The results showed that, for pita bread, the outcome showed that IN and GG alone significantly increased hardness and chewiness and decreased springiness. WF addition only decreased springiness. GG linearly increased pita height and volume, but it quadratically increased weight loss. Moreover, IN increased volume and height in a quadratic way. Regarding Tandoori bread, IN reduced bread toughness, but showed no effect on extensibility. WF alone increased volume and diameter. The microstructure also showed that the novel ingredients modified starch gelatinisation and gluten-starch matrix in both pita and Tandoori breads differently. Therefore, the influence of the three functional ingredients alone and in combinations influenced quality parameters dissimilarly.

The calcifying interface in a stony coral primary polyp: An interplay between seawater and an extracellular calcifying space.

Stony coral exoskeletons build the foundation for the most biologically diverse marine ecosystems on Earth, coral reefs, which face major threats due to many anthropogenic-related stressors. Therefore, understanding coral biomineralization mechanisms is crucial for coral reef management in the coming decades and for using coral skeletons in geochemical studies. This study combines in-vivo imaging with cryo-electron microscopy and cryo-elemental mapping to gain novel insights into the biological microenvironment and the ion pathways that facilitate biomineralization in primary polyps of the stony coral Stylophora pistillata. We document increased tissue permeability in the primary polyp and a highly dispersed cell packing in the tissue directly responsible for producing the coral skeleton. This tissue arrangement may facilitate the intimate involvement of seawater at the mineralization site, also documented here. We further observe an extensive filopodial network containing carbon-rich vesicles extruding from some of the calicoblastic cells. Single-cell RNA-Sequencing data interrogation supports these morphological observations by showing higher expression of genes involved in filopodia and vesicle structure and function in the calicoblastic cells. These observations provide a new conceptual framework for resolving the ion pathway from the external seawater to the tissue-mineral interface in stony coral biomineralization processes.

Arabinoxylan, ß-glucan and pectin in barley and malt endosperm cell walls: a microstructure study using CLSM and cryo-SEM.

The architecture of endosperm cell walls in Hordeum vulgare (barley) differs remarkably from that of other grass species and is affected by germination or malting. Here, the cell wall microstructure is investigated using (bio)chemical analyses, cryogenic scanning electron microscopy (cryo-SEM) and confocal laser scanning microscopy (CLSM) as the main techniques. The relative proportions of ß-glucan, arabinoxylan and pectin in cell walls were 61, 34 and 5%, respectively. The average thickness of a single endosperm cell wall was 0.30 µm, as estimated by the cryo-SEM analysis of barley seeds, which was reduced to 0.16 µm after malting. After fluorescent staining, 3D confocal multiphoton microscopy (multiphoton CLSM) imaging revealed the complex cell wall architecture. The endosperm cell wall is composed of a structure in which arabinoxylan and pectin are colocalized on the outside, with ß-glucan depositions on the inside. During germination, arabinoxylan and ß-glucan are hydrolysed, but unlike ß-glucan, arabinoxylan remains present in defined cell walls in malt. Integrating the results, an enhanced model for the endosperm cell walls in barley is proposed.

Microscopy of Polyurea Grease.

Understanding the physical structure of greases can provide critical insight into improving the lubricating performance of a grease. Observation of the grease structure can be quite difficult depending on the type of grease and the length scale of the structure. Polyurea greases in previous reports have typically been examined by removal of the oil phase, which significantly changes the polyurea structure. This paper examines the effect of sample preparation conditions on the microstructure of polyurea greases. This study reveals new structures in the polyurea that have not been observed in the previous literature, including entangled fibers and nanotubes. Correlation is found between the observed polyurea microstructure coverage and grease stiffness.

Imperforate tracheary elements and vessels alleviate xylem tension under severe dehydration: insights from water release curves for excised twigs of three tree species.

PREMISE: Water stored in the xylem of woody plants is important for supporting the transpiration stream under prolonged drought, yet the source of stored water within the xylem during drought remains unclear. Insights into xylem water utilization during drought will uncover the adaptation strategies of the test species to stress. METHODS: To fill the existing knowledge gap, we excised twigs of Abies firma (Japanese fir, conifer), Cercidiphyllum japonicum (katsura tree, diffuse-porous) and Quercus serrata (konara oak, ring-porous) to quantify interspecific variation of water transfer in xylem corresponding with increasing cumulative water release (CWR) using micro x-ray computed tomography and cryo-SEM. RESULTS: For all species studied, the main components of water storage within the operating range of water potential were not living cells but cavitation release and capillaries. Abies firma maintained water in the earlywood-like cells, for possible maintenance of the transpiration stream. Cercidiphyllum japonicum maintained water in its vessels over 200 kg m-3 of CWR, while Q. serrata lost most of its water in vessels with increasing CWR up to 100 kg m-3 . Cercidiphyllum japonicum exhibited a higher water storage capacity than Q. serrata. Under high CWR, narrow conduits stored xylem water in C. japonicum and imperforate tracheary elements in Q. serrata. CONCLUSIONS: Among the species examined, increasing CWR appears to indicate differential utilization of stored water in relation to variation of xylem structure, thereby providing insight into the interspecific responses of tree species to drought.

Osmotic adjustments support growth of poplar cultured cells under high concentrations of carbohydrates.

KEY MESSAGE: Poplar callus maintained a specific difference in osmotic potential with respect to media when supplemented with different carbohydrate concentrations. This balance in osmotic potential guaranteed the growth capacity. Osmotic stress is caused by several abiotic factors such as drought, salinity, or freezing. However, the threshold of osmotic potential that allows the growth under stress conditions has not been thoroughly studied. In this study, different levels of osmotic stress in Populus alba (L.) callus have been induced with the addition of mannitol or sorbitol in the medium (from 0 to 500 mM). The key factor for preserving the growth was observed to be the restoration of a constant difference in osmotic potential between callus and medium for all the tested conditions. The osmotic adjustments were primarily achieved with the uptake of mannitol or sorbitol from the media considering their chemical properties instead of their biological functions. The decrease in water content (from - 1 to - 10% after 21 days) and mineral elements, such as potassium, calcium, and magnesium, together with the alterations in cell morphology, did not show negative effects on growth. The activity of sorbitol dehydrogenase was detected for the first time in poplar (+ 4.7 U l-1 in callus treated with sorbitol compared to control callus). This finding suggested the importance of choosing carefully the molecules used to exert osmotic stress for separating the dual function of carbohydrates in osmotic adjustments and cell metabolism.

Effect of microencapsulated fish oil powder on selected quality characteristics of chicken sausages.

BACKGROUND: Encapsulation of fish oil for use as oil powder for the human food industry offers new product design possibilities. The fat content of fish is unique in the amount of of long chain n-3 fatty acid that it contains. It can be expected that developing innovative food products with significantly improved nutritional value can simultaneously affect their rheological and sensory qualities in different ways. The present study aimed to compared the influence of the addition of fish oil and microencapsulated fish oil on the mechanical, structural, and sensory properties of poultry sausages during 21-day storage. RESULTS: In comparison with other systems, sausages enriched with microencapsulated fish oil were characterized by a greater capacity to accumulate potential strain energy (G') and by statistically significantly greater hardness measured in all the storage periods that were tested. The sausages enriched with microencapsulated fish oil were characterized by higher water activity than the other sausage variants. The analysis of cryo-scanning electron microscopy (cryo-SEM) images indicated the presence of a large evenly dispersed oil phase and microcapsules in the structure of the sausages. The sample with the addition of microcapsules was characterized by higher values on the smell and consistency parameters. CONCLUSION: The better results in the sensory evaluation of the structural parameters of the sample with microcapsules were consistent with the results of instrumental assessments. The use of microencapsulated fish oil powder as an ingredient of chicken sausages therefore seems to be a promising concept. © 2019 Society of Chemical Industry.

Structural features of the aleurone layer of the seed coat associated with imbibition injury in soybean.

Soybean (Glycine max) seeds are prone to imbibition injury caused by a rapid uptake of water. Genetic variation in imbibition injury tolerance is well documented, but the underlying mechanisms remain unclear. The aim of this study was to clarify the role of the aleurone layer of seed coat in the tolerance and its structural differences between tolerant and susceptible cultivars. Imbibition injury tolerance was closely related to the water absorption rate of seeds, which was regulated by the aleurone layer of the seed coat. Cryo-scanning electron microscopy analysis revealed that water absorbed in seed coats entered the seed preferentially through the aleurone layer of the top area above the raphe. In susceptible cultivars, the cell walls of the aleurone layer facing the cotyledon in this area were thin and the surface showed shallow depression-like structures, a distinct structure different from those of the tolerant cultivars, which had aleurone cells with thick outer cell walls and smooth and stripe-like deposits. The differences in the structural features of the cell walls and surfaces of aleurone cells in the top area of the seed may be responsible for the difference in the extent of imbibition injury between susceptible and tolerant cultivars.

Pine wilt disease causes cavitation around the resin canals and irrecoverable xylem conduit dysfunction.

Physiological mechanisms of irreversible hydraulic dysfunction in seedlings infected with pine wilt disease (PWD) are still unclear. We employed cryo-scanning electron microscopy (cryo-SEM) to investigate the temporal and spatial changes in water distribution within the xylem of the main stem of 2-year-old Japanese black pine seedlings infested by pine wood nematodes (PWNs). Our experiment was specifically designed to compare the water relations among seedlings subjected to the following water treatment and PWN combinations: (i) well-watered versus prolonged drought (no PWNs); and (ii) well-watered with PWNs versus water-stressed with PWNs (four treatments in total). Cryo-SEM imaging observations chronicled the development of patchy cavitations in the xylem tracheids of the seedlings influenced by PWD. With the progression of drought, many pit membranes of bordered pits in the xylem of the main stem were aspirated with the decrease in water potential without xylem cavitation, indicating that hydraulic segmentation may exist between tracheids. This is the first study to demonstrate conclusively that explosive and irreversible cavitations occurred around the hydraulically vulnerable resin canals with the progression of PWD. Our findings provide a more comprehensive understanding of stressors on plant-water relations that may eventually better protect trees from PWD and assist with the breeding of trees more tolerant to PWD.

Periodic Grating-like Patterns Induced by Self-Assembly of Gelator Fibres in Nematic Gels.

Periodic orientation patterns occurring in nematic gels, revealed by optical and scanning electron microscopy, are found to be formed by spontaneous self-assembly of fibrous aggregates of a low-molecular-weight organogelator in an aligned thermotropic liquid crystal (LC). Self-organization into periodic structures is also reflected in a calorimetric study, which shows the occurrence of three thermoreversible states, namely, isotropic liquid, nematic and nematic gel. The segregation and self-assembly of the fibrous aggregates leading to pattern formation are attributed to the highly polar LC and to hydrogen bonding between gelator molecules, as shown by X-ray diffraction and vibrational spectroscopy. This study aims to investigate in detail the effect of the chemical nature and alignment of an anisotropic solvent on the morphology of the gelator fibres and the resulting gelation process. The periodic organization of LC-rich and fibre-rich regions can also provide a way to obtain templates for positioning nanoparticle arrays in an LC matrix, which can lead to novel devices.

Formation and mosaicity of coccolith segment calcite of the marine algae Emiliania huxleyi.

Coccolithophores belong to the most abundant calcium carbonate mineralizing organisms. Coccolithophore biomineralization is a complex and highly regulated process, resulting in a product that strongly differs in its intricate morphology from the abiogenically produced mineral equivalent. Moreover, unlike extracellularly formed biological carbonate hard tissues, coccolith calcite is neither a hybrid composite, nor is it distinguished by a hierarchical microstructure. This is remarkable as the key to optimizing crystalline biomaterials for mechanical strength and toughness lies in the composite nature of the biological hard tissue and the utilization of specific microstructures. To obtain insight into the pathway of biomineralization of Emiliania huxleyi coccoliths, we examine intracrystalline nanostructural features of the coccolith calcite in combination with cell ultrastructural observations related to the formation of the calcite in the coccolith vesicle within the cell. With TEM diffraction and annular dark-field imaging, we prove the presence of planar imperfections in the calcite crystals such as planar mosaic block boundaries. As only minor misorientations occur, we attribute them to dislocation networks creating small-angle boundaries. Intracrystalline occluded biopolymers are not observed. Hence, in E. huxleyi calcite mosaicity is not caused by occluded biopolymers, as it is the case in extracellularly formed hard tissues of marine invertebrates, but by planar defects and dislocations which are typical for crystals formed by classical ion-by-ion growth mechanisms. Using cryo-preparation techniques for SEM and TEM, we found that the membrane of the coccolith vesicle and the outer membrane of the nuclear envelope are in tight proximity, with a well-controlled constant gap of ~4 nm between them. We describe this conspicuous connection as a not yet described interorganelle junction, the "nuclear envelope junction". The narrow gap of this junction likely facilitates transport of Ca2+ ions from the nuclear envelope to the coccolith vesicle. On the basis of our observations, we propose that formation of the coccolith utilizes the nuclear envelope-endoplasmic reticulum Ca2+ -store of the cell for the transport of Ca2+ ions from the external medium to the coccolith vesicle and that E. huxleyi calcite forms by ion-by-ion growth rather than by a nanoparticle accretion mechanism.

Monitoring Candida parapsilosis and Staphylococcus epidermidis Biofilms by a Combination of Scanning Electron Microscopy and Raman Spectroscopy.

The biofilm-forming microbial species Candida parapsilosis and Staphylococcus epidermidis have been recently linked to serious infections associated with implanted medical devices. We studied microbial biofilms by high resolution scanning electron microscopy (SEM), which allowed us to visualize the biofilm structure, including the distribution of cells inside the extracellular matrix and the areas of surface adhesion. We compared classical SEM (chemically fixed samples) with cryogenic SEM, which employs physical sample preparation based on plunging the sample into various liquid cryogens, as well as high-pressure freezing (HPF). For imaging the biofilm interior, we applied the freeze-fracture technique. In this study, we show that the different means of sample preparation have a fundamental influence on the observed biofilm structure. We complemented the SEM observations with Raman spectroscopic analysis, which allowed us to assess the time-dependent chemical composition changes of the biofilm in vivo. We identified the individual spectral peaks of the biomolecules present in the biofilm and we employed principal component analysis (PCA) to follow the temporal development of the chemical composition.

Pathogen biofilm formation on cantaloupe surface and its impact on the antibacterial effect of lauroyl arginate ethyl.

Pathogen biofilm at fruit surface may pose a particular risk to food safety. In this study, the biofilms of Listeria monocytogenes V7 and Salmonella enterica serovar Typhimurium ATCC 13311 on cantaloupe fruit surface were visualized, and the resistance of biofilms against lauroyl arginate ethyl (LAE, an antibacterial compound) was evaluated. Each bacterium was inoculated on isolated cantaloupe rind surfaces at 105-106 CFU/cm2 and after incubation for 2, 12, 24, and 48 h, the surfaces were imaged using cryo-scanning electron microscopy (Cryo-SEM). The images showed that both pathogens formed biofilms on rind surfaces, with S. Typhimurium forming biofilm in 12 h and L. monocytogenes cells starting to aggregate in 2 h. For the inoculated rind surfaces treated with LAE, the cell counts were affected by both the incubation time and LAE concentration. For rind surface with 2 h incubation of S. Typhimurium, 400 and 800 µg/mL LAE was able to achieve >2.00 log reduction; however, 12 h incubation required 1600 and 2000 µg/mL LAE for >2.00 log reduction. In contrast, even the highest LAE concentration (2000 µg/mL) was unable to cause 1.00 log reduction for L. monocytogenes regardless the incubation time applied. The results showed that the biofilms of both bacteria substantially reduced LAE efficacy, and that the biofilm of L. monocytogenes was more resistant than that of S. Typhimurium.

Cryo-Scanning Electron Microscopy (SEM) and Scanning Transmission Electron Microscopy (STEM)-in-SEM for Bio- and Organo-Mineral Interface Characterization in the Environment.

Understanding biofilm interactions with surrounding substratum and pollutants/particles can benefit from the application of existing microscopy tools. Using the example of biofilm interactions with zero-valent iron nanoparticles (nZVI), this study aims to apply various approaches in biofilm preparation and labeling for fluorescent or electron microscopy and energy dispersive X-ray spectrometry (EDS) microanalysis for accurate observations. According to the targeted microscopy method, biofilms were sampled as flocs or attached biofilm, submitted to labeling using 4',6-diamidino-2-phenylindol, lectins PNA and ConA coupled to fluorescent dye or gold nanoparticles, and prepared for observation (fixation, cross-section, freezing, ultramicrotomy). Fluorescent microscopy revealed that nZVI were embedded in the biofilm structure as aggregates but the resolution was insufficient to observe individual nZVI. Cryo-scanning electron microscopy (SEM) observations showed nZVI aggregates close to bacteria, but it was not possible to confirm direct interactions between nZVI and cell membranes. Scanning transmission electron microscopy in the SEM (STEM-in-SEM) showed that nZVI aggregates could enter the biofilm to a depth of 7-11 µm. Bacteria were surrounded by a ring of extracellular polymeric substances (EPS) preventing direct nZVI/membrane interactions. STEM/EDS mapping revealed a co-localization of nZVI aggregates with lectins suggesting a potential role of EPS in nZVI embedding. Thus, the combination of divergent microscopy approaches is a good approach to better understand and characterize biofilm/metal interactions.