Nanometric scale size oscillations seem to be a fundamental feature of all living organisms on Earth. Their detection usually requires complex and very sensitive devices. However, some recent studies demonstrated that very simple optical microscopes and dedicated image processing software can also fulfill this task. This novel technique, termed as optical nanomotion detection (ONMD), was recently successfully used on yeast cells to conduct rapid antifungal sensitivity tests. In this study, we demonstrate that the ONMD method can monitor motile sub-cellular organelles, such as mitochondria. Here, mitochondrial isolates (from HEK 293 T and Jurkat cells) undergo predictable motility when viewed by ONMD and triggered by mitochondrial toxins, citric acid intermediates, and dietary and bacterial fermentation products (short-chain fatty acids) at various doses and durations. The technique has superior advantages compared to classical methods since it is rapid, possesses a single organelle sensitivity, and is label- and attachment-free.

Bacterial conjugation is the main mechanism for horizontal gene transfer, conferring plasticity to the genome repertoire. This process is also the major instrument for the dissemination of antibiotic resistance genes. Hence, gathering primary information of the mechanism underlying this genetic transaction is of a capital interest. By using fluorescent protein fusions to the ATPases that power conjugation, we have been able to track the localization of these proteins in the presence and absence of recipient cells. Moreover, we have found that more than one copy of the conjugative plasmid is transferred during mating. Altogether, these findings provide new insights into the mechanism of such an important gene transfer device.

Super-resolution microscopy has evolved as a powerful method for subdiffraction-resolution fluorescence imaging of cells and cellular organelles, but requires sophisticated and expensive installations. Expansion microscopy (ExM), which is based on the physical expansion of the cellular structure of interest, provides a cheap alternative to bypass the diffraction limit and enable super-resolution imaging on a conventional fluorescence microscope. While ExM has shown impressive results for the magnified visualization of proteins and RNAs in cells and tissues, it has not yet been applied in fungi, mainly due to their complex cell wall. Here we developed a method that enables reliable isotropic expansion of ascomycetes and basidiomycetes upon treatment with cell wall degrading enzymes. Confocal laser scanning microscopy (CLSM) and structured illumination microscopy (SIM) images of 4.5-fold expanded sporidia of Ustilago maydis expressing fluorescent fungal rhodopsins and hyphae of Fusarium oxysporum or Aspergillus fumigatus expressing either histone H1-mCherry together with Lifeact-sGFP or mRFP targeted to mitochondria, revealed details of subcellular structures with an estimated spatial resolution of around 30 nm. ExM is thus well suited for cell biology studies in fungi on conventional fluorescence microscopes.

No plant or cryptogam exists in nature without microorganisms associated with its tissues. Plants as microbial hosts are puzzles of different microhabitats, each of them colonized by specifically adapted microbiomes. The interactions with such microorganisms have drastic effects on the host fitness. Since the last 20 years, the combination of microscopic tools and molecular approaches contributed to new insights into microbe-host interactions. Particularly, confocal laser scanning microscopy (CLSM) facilitated the exploration of microbial habitats and allowed the observation of host-associated microorganisms in situ with an unprecedented accuracy. Here I present an overview of the progresses made in the study of the interactions between microorganisms and plants or plant-like organisms, focusing on the role of CLSM for the understanding of their significance. I critically discuss risks of misinterpretation when procedures of CLSM are not properly optimized. I also review approaches for quantitative and statistical analyses of CLSM images, the combination with other molecular and microscopic methods, and suggest the re-evaluation of natural autofluorescence. In this review, technical aspects were coupled with scientific outcomes, to facilitate the readers in identifying possible CLSM applications in their research or to expand their existing potential. The scope of this review is to highlight the importance of confocal microscopy in the study of plant-microbe interactions and also to be an inspiration for integrating microscopy with molecular techniques in future researches of microbial ecology.

Techniques for continuously monitoring the formation of subgingival biofilm, in relation to the determination of species and their accumulation over time in gingivitis and periodontitis, are limited. In recent years, advancements in the field of optical spectroscopic techniques have provided an alternative for analyzing three-dimensional microbiological structures, replacing the traditional destructive or biofilm staining techniques. In this work, we have demonstrated that the use of confocal Raman spectroscopy coupled with multivariate analysis provides an approach to spatially differentiate bacteria in an in vitro model simulating a subgingival dual-species biofilm. The present study establishes a workflow to evaluate and differentiate bacterial species in a dual-species in vitro biofilm model, using confocal Raman microscopy (CRM). Biofilm models of Actinomyces denticolens and Streptococcus oralis were cultured using the "Zürich in vitro model" and were analyzed using CRM. Cluster analysis was used to spatially differentiate and map the biofilm model over a specified area. To confirm the clustering of species in the cultured biofilm, confocal laser scanning microscopy (CLSM) was coupled with fluorescent in vitro hybridization (FISH). Additionally, dense bacteria interface area (DBIA) samples, as an imitation of the clusters in a biofilm, were used to test the developed multivariate differentiation model. This confirmed model was successfully used to differentiate species in a dual-species biofilm and is comparable to morphology. The results show that the developed workflow was able to identify main clusters of bacteria based on spectral "fingerprint region" information from CRM. Using this workflow, we have demonstrated that CRM can spatially analyze two-species in vitro biofilms, therefore providing an alternative technique to map oral multi-species biofilm models.

Electron microscopy is a powerful tool in the field of microbiology. It has played a key role in the rapid diagnosis of viruses in patient samples and has contributed significantly to the clarification of virus structure and function, helping to guide the public health response to emerging viral infections. In the present study, we used scanning electron microscopy (SEM) to study the infectious cycle of SARS-CoV-2 in Vero E6 cells and we controlled some key findings by classical transmission electronic microscopy (TEM). The replication cycle of the virus was followed from 1 to 36 h post-infection. Our results revealed that SARS-CoV-2 infected the cells through membrane fusion. Particles are formed in the peri-nuclear region from a budding of the endoplasmic reticulum-Golgi apparatus complex into morphogenesis matrix vesicae. New SARS-CoV-2 particles were expelled from the cells, through cell lysis or by fusion of virus containing vacuoles with the cell plasma membrane. Overall, this cycle is highly comparable to that of SARS-CoV. By providing a detailed and complete SARS-CoV-2 infectious cycle, SEM proves to be a very rapid and efficient tool compared to classical TEM.

Escherichia coli produces extracellular vesicles called outer membrane vesicles (OMVs) by releasing a part of its outer membrane. We previously reported that the combined deletion of nlpI and mlaE, related to envelope structure and phospholipid accumulation in the outer leaflet of the outer membrane, respectively, resulted in the synergistic increase of OMV production. In this study, the analysis of ΔmlaEΔnlpI cells using quick-freeze, deep-etch electron microscopy (QFDE-EM) revealed that plasmolysis occurred at the tip of the long axis in cells and that OMVs formed from this tip. Plasmolysis was also observed in the single-gene knockout mutants ΔnlpI and ΔmlaE. This study has demonstrated that plasmolysis was induced in the hypervesiculating mutant E. coli cells. Furthermore, intracellular vesicles and multilamellar OMV were observed in the ΔmlaEΔnlpI cells. Meanwhile, the secretion of recombinant green fluorescent protein (GFP) expressed in the cytosol of the ΔmlaEΔnlpI cells was more than 100 times higher than that of WT and ΔnlpI, and about 50 times higher than that of ΔmlaE in the OMV fraction, suggesting that cytosolic components were incorporated into outer-inner membrane vesicles (OIMVs) and released into the extracellular space. Additionally, QFDE-EM analysis revealed that ΔmlaEΔnlpI sacculi contained many holes noticeably larger than the mean radius of the peptidoglycan (PG) pores in wild-type (WT) E. coli. These results suggest that in ΔmlaEΔnlpI cells, cytoplasmic membrane materials protrude into the periplasmic space through the peptidoglycan holes and are released as OIMVs.

Neutralizing monoclonal antibodies (mAbs) against highly pathogenic coronaviruses represent promising candidates for clinical intervention. Here, we isolated a potent neutralizing monoclonal antibody, MERS-S41, from a yeast displayed scFv library using the S protein as a bait. To uncover the neutralization mechanism, we determined structures of MERS-S41 Fab in complex with the trimeric spike glycoprotein by cryoelectron microscopy (cryo-EM). We observed four distinct classes of the complex structure, which showed that the MERS-S41 Fab bound to the "up" receptor binding domain (RBD) with full saturation and also bound to an accessible partially lifted "down" RBD, providing a structural basis for understanding how mAbs bind to trimeric spike glycoproteins. Structure analysis of the epitope and cell surface staining assays demonstrated that virus entry is blocked predominantly by direct competition with the host receptor, dipeptidyl peptidase-4 (DPP4).

Kombucha pellicles are often used as inoculum to produce this beverage and have become a signature feature. This cellulosic biofilm produced by acetic acid bacteria (AAB) involves yeasts, which are also part of the kombucha consortia. The role of microbial interactions in the de novo formation and structure of kombucha pellicles was investigated during the 3 days following inoculation, using two-photon microscopy coupled with fluorescent staining. Aggregated yeast cells appear to serve as scaffolding to which bacterial cellulose accumulates. This initial foundation leads to a layered structure characterized by a top cellulose-rich layer and a biomass-rich sublayer. This sublayer is expected to be the microbiologically active site for cellulose production and spatial optimization of yeast-AAB metabolic interactions. The pellicles then grow in thickness while expanding their layered organization. A comparison with pellicles grown from pure AAB cultures shows differences in consistency and structure that highlight the impact of yeasts on the structure and properties of kombucha pellicles.

In arsenopyrite bioleaching, the interfacial reaction between mineral and cells is one of the most important factors. The energy of the interface is influenced by the mineralogical and microbiological characteristics. In this paper, the interfacial energy was calculated, and the surface of arsenopyrite during the bioleaching process was characterized by 3D laser microscopy, scanning electron microscopy with energy-dispersive X-ray spectroscopy, and X-ray photoelectron spectroscopy, in order to assess the dissolution and oxidation behavior of arsenopyrite during bioleaching. The results showed that the contact angles of arsenopyrite were 22 ± 2° when covered with biofilms, but the reaction surface of arsenopyrite turned 103 ± 2°. However, the angle was 45-50° when covered by passive layer, which was half as that of arsenopyrite surface. The interfacial energy of arsenopyrite without biofilms increased from 45 to 62 mJ/m2, while it decreased to 5 ± 1 mJ/m2 when covered by biofilms during the leaching process. The surface was separated into fresh surface, oxidized surface, and (corrosion) pits. The interfacial energy was influenced by the fresh and oxidized surfaces. Surface roughness increased from 0.03 ± 0.01 to 5.89 ± 1.97 μm, and dissolution volume increased from 6.31 ± 0.47 × 104 to 2.72 ± 0.49 × 106 μm3. The dissolution kinetics of arsenopyrite followed the model of Kt = lnX, and the dissolution mechanisms were mixed controlled: surface reaction control and diffusion through sulfur layer. On the surface of arsenopyrite crystal, the oxidation steps of each element can be described as: for Fe, Fe(II)-(AsS)→Fe(III)-(AsS)→Fe(III)-OH or Fe(III)-SO; for S, As-S(-1) or Fe-S(-1)→polysulfide S→intermediate S-O→sulfate; and for As, As-1-S→As0→As+1-O→As+3-O→As+5-O.

Here, we experimentally expand understanding of the reactions and enzymes involved in Acidithiobacillus thiooxidans ATCC 19377 S0 and S 2 ⁢ O 3 2 - metabolism by developing models that integrate gene expression analyzed by RNA-Seq, solution sulfur speciation, electron microscopy and spectroscopy. The A. thiooxidans S 2 ⁢ O 3 2 - metabolism model involves the conversion of S 2 ⁢ O 3 2 - to SO 4 2 - , S0 and S 4 ⁢ O 6 2 - , mediated by the sulfur oxidase complex (Sox), tetrathionate hydrolase (TetH), sulfide quinone reductase (Sqr), and heterodisulfate reductase (Hdr) proteins. These same proteins, with the addition of rhodanese (Rhd), were identified to convert S0 to SO 3 2 - , S 2 ⁢ O 3 2 - and polythionates in the A. thiooxidans S0 metabolism model. Our combined results shed light onto the important role specifically of TetH in S 2 ⁢ O 3 2 - metabolism. Also, we show that activity of Hdr proteins rather than Sdo are likely associated with S0 oxidation. Finally, our data suggest that formation of intracellular S 2 ⁢ O 3 2 - is a critical step in S0 metabolism, and that recycling of internally generated SO 3 2 - occurs, through comproportionating reactions that result in S 2 ⁢ O 3 2 - . Electron microscopy and spectroscopy confirmed intracellular production and storage of S0 during growth on both S0 and S 2 ⁢ O 3 2 - substrates.

Background: Enabling faster Antimicrobial Susceptibility Testing (AST) is critical, especially to detect antibiotic resistance, to provide rapid and appropriate therapy and to improve clinical outcomes. Although several standard and automated culture-based methods are available and widely used, these techniques take between 18 and 24 h to provide robust results. Faster techniques are needed to reduce the delay between test and results. Methods: Here we present a high throughput AST method using a new generation of tabletop scanning electron microscope, to evaluate bacterial ultra-structural modifications associated with susceptibilities to imipenem as a proof of concept. A total of 71 reference and clinical strains of Gram-negative bacteria were used to evaluate susceptibility toward imipenem after 30, 60, and 90 min of incubation. The length, width and electron density of bacteria were measured and compared between imipenem susceptible and resistant strains. Results: We correlated the presence of these morphological changes to the bacterial susceptibility and their absence to the bacterial resistance (e.g., Pseudomonas aeruginosa length without [2.24 ± 0.61 μm] and with [2.50 ± 0.68 μm] imipenem after 30 min [p = 3.032E-15]; Escherichia coli width without [0.92 ± 0.07 μm] and with [1.28 ± 0.19 μm] imipenem after 60 min [p = 1.242E-103]). We validated our method by a blind test on a series of 58 clinical isolates where all strains were correctly classified as susceptible or resistant toward imipenem. Conclusion: This method could be a potential tool for rapidly identifying carbapenem-resistance in Enterobacterales in clinical microbiology laboratories in <2 h, allowing the empirical treatment of patients to be rapidly adjusted.

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.

Arbuscule-producing fine root endophytes (FRE) (previously incorrectly Glomus tenue) were recently placed within subphylum Mucoromycotina; the first report of arbuscules outside subphylum Glomeromycotina. Here, we aimed to estimate nutrient concentrations in plant and fungal structures of FRE and to test the utility of cryo-scanning electron microscopy (cryoSEM) for studying these fungi.

There is an urgent need for accurate and rapid testing methods to quickly identify infected patients as well as asymptomatic carriers, in order to prevent the spread of emerging viruses. Here, we developed a rapid testing strategy by scanning electron microscopy capable of detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and other respiratory viruses directly from patients. We evaluated our results by comparing them to real-time reverse transcription-polymerase chain reaction (RT-PCR) and metagenomic sequencing results. We correlated the presence of the SARS-CoV-2 to the viral load, where samples with Ct values lower than 18 were all detected by scanning electron microscopy (SEM). The sensitivity deacresed progressively with higher Ct values. In addition, we found a correlation with metagenomic sequencing, where all samples detected by SEM were sequenced and viral sequences were easily recovered. Following this study, SEM proved its efficiency as a frontline method for directly detecting previously unknown microorganisms that cannot be targeted by molecular methods and can cause potential outbreaks.

The effects of environmental stresses on microorganisms have been well-studied, and cellular responses to stresses such as heat, cold, acids, and salts have been extensively discussed. Although high pressure processing (HPP) is becoming more popular as a preservation method in the food industry, the characteristics of the cellular damage caused by high pressure are unclear, and the microbial response to this stress has not yet been well-explored. We exposed the pathogen Listeria monocytogenes to HPP (400 MPa, 8 min, 8°C) and found that the high pressure created plasma membrane pores. Using a common staining technique involving propidium iodide (PI) combined with high-frequency fluorescence microscopy, we monitored the rate of diffusion of PI molecules into hundreds of bacterial cells through these pores on days 0, 1, 2, 3, and 4 after pressurization. We also developed a mathematical dynamic model based on mass transfer and passive diffusion laws, calibrated using our microscopy experiments, to evaluate the response of bacteria to HPP. We found that the rate of diffusion of PI into the cells decreased over the 4 consecutive days after exposure to HPP, indicating repair of the pressure-created membrane pores. The model suggested a temporal change in the size of pores until closure. To the best of our knowledge, this is the first time that pressure-created membrane pores have been quantitatively described and shown to diminish with time. In addition, we found that the membrane repair rate in response to HPP was linear, and growth was temporarily arrested at the population level during the repair period. These results support the existence of a progressive repair process in some of the cells that take up PI, which can therefore be considered as being sub-lethally injured rather than dead. Hence, we showed that a subgroup of bacteria survived HPP and actively repaired their membrane pores.

The deep sedimentary biosphere, extending 100s of meters below the seafloor harbors unexpected diversity of Bacteria, Archaea, and microbial eukaryotes. Far less is known about microbial eukaryotes in subsurface habitats, albeit several studies have indicated that fungi dominate microbial eukaryotic communities and fungal molecular signatures (of both yeasts and filamentous forms) have been detected in samples as deep as 1740 mbsf. Here, we compare and contrast fungal ribosomal RNA gene signatures and whole community metatranscriptomes present in sediment core samples from 6 and 95 mbsf from Peru Margin site 1229A and from samples from 12 and 345 mbsf from Canterbury Basin site U1352. The metatranscriptome analyses reveal higher relative expression of amino acid and peptide transporters in the less nutrient rich Canterbury Basin sediments compared to the nutrient rich Peru Margin, and higher expression of motility genes in the Peru Margin samples. Higher expression of genes associated with metals transporters and antibiotic resistance and production was detected in Canterbury Basin sediments. A poly-A focused metatranscriptome produced for the Canterbury Basin sample from 345 mbsf provides further evidence for active fungal communities in the subsurface in the form of fungal-associated transcripts for metabolic and cellular processes, cell and membrane functions, and catalytic activities. Fungal communities at comparable depths at the two geographically separated locations appear dominated by distinct taxa. Differences in taxonomic composition and expression of genes associated with particular metabolic activities may be a function of sediment organic content as well as oceanic province. Microscopic analysis of Canterbury Basin sediment samples from 4 and 403 mbsf produced visualizations of septate fungal filaments, branching fungi, conidiogenesis, and spores. These images provide another important line of evidence supporting the occurrence and activity of fungi in the deep subseafloor biosphere.

To understand the response of the Lyme disease spirochete Borrelia burgdorferi exposed to stress conditions and assess the viability of this spirochete, we used a correlative cryo-fluorescence and cryo-scanning microscopy approach. This approach enables simple exposition of bacteria to various experimental conditions that can be stopped at certain time intervals by cryo-immobilization, examination of cell viability without necessity to maintain suitable culture conditions during viability assays, and visualization of structures in their native state at high magnification. We focused on rare and transient events e.g., the formation of round bodies and the presence of membranous blebs in spirochetes exposed to culture medium, host sera either without or with the bacteriolytic effect and water. We described all crucial steps of the workflow, particularly the influence of freeze-etching and accelerating voltage on the visualization of topography. With the help of newly designed cryo-transport device, we achieved greater reproducibility.

Artificial snow production is a crucial part of modern skiing resorts in Austria and globally, and will develop even more so with changing precipitation patterns and a warming climate trend. Producing artificial snow requires major investments in energy, water, infrastructure and manpower for skiing resorts. In addition to appropriate meteorological conditions, the efficiency of artificial snow production depends on heterogeneous ice-nucleation, which can occur at temperatures as high as -2°C when induced by specific bacterial ice nucleating particles (INPs). We aimed to investigate the presence, source and ice nucleating properties of these particles in the water cycle of an alpine ski resort in Obergurgl, Tyrol, Austria. We sampled artificial snow, river water, water pumped from a storage pond and compared it to samples collected from fresh natural snow and aged piste snow from the area. Particles from each sampled system were characterized in order to determine their transport mechanisms at a ski resort. We applied a physical droplet freezing assay [DRoplet Ice Nuclei Counter Zurich (DRINCZ)] to heated and unheated samples to characterize the biological and non-biological component of IN-activity. Bacterial abundance and community structure of the samples was obtained using quantitative PCR and Illumina Mi-Seq Amplicon Sequencing, and their chemical properties were determined by liquid ion-chromatography, energy dispersive X-ray spectroscopy (EDX) and scanning electron microscopy (SEM). The results show the flow of biological and inorganic material from the river to the slopes, an uptake of new microorganisms through the air and the piping, and possible proliferation or introduction of ice nucleation active biological particles in aged piste snow. Natural snow, as the first stage in this system, had the lowest amount of ice nucleation active particles and the least amount of biological and mineral particles in general, yet shares some microbial characteristics with fresh artificial snow.

Based on serial sectioning, focused ion beam scanning electron microscopy (FIB/SEM), and electron tomography, we depict in detail the highly unusual anatomy of the marine hyperthermophilic crenarchaeon, Ignicoccus hospitalis. Our data support a complex and dynamic endomembrane system consisting of cytoplasmic protrusions, and with secretory function. Moreover, we reveal that the cytoplasm of the putative archaeal ectoparasite Nanoarchaeum equitans can get in direct contact with this endomembrane system, complementing and explaining recent proteomic, transcriptomic and metabolomic data on this inter-archaeal relationship. In addition, we identified a matrix of filamentous structures and/or tethers in the voluminous inter-membrane compartment (IMC) of I. hospitalis, which might be responsible for membrane dynamics. Overall, this unusual cellular compartmentalization, ultrastructure and dynamics in an archaeon that belongs to the recently proposed TACK superphylum prompts speculation that the eukaryotic endomembrane system might originate from Archaea.