Flexible Sheet-Type Sensor for Noninvasive Measurement of Cellular Oxygen Metabolism on a Culture Dish

A novel flexible sensor was developed for the noninvasive oxygen metabolism measurement of cultivated cells and tissues. This device is composed of a transparent double-layered polymer sheet of ethylene-vinyl alcohol (EVOH) and poly(dimethylsiloxane) (PDMS) having an array of microhole structures of 90 μm diameter and 50 μm depth on its surface. All the microhole structures were equipped with a 1-μm-thick optical chemical sensing layer of platinum porphyrin-fluoropolymer on their bottom. The three-dimensional microstructures of the sensor were fabricated by a newly developed simple and low-cost production method named self-aligned hot embossing. The device was designed to be attached slightly above the cells cultivated on a dish to form a temporarily closed microspace over the target cells during measurement. Since the change in oxygen concentration is relatively fast in the microcompartmentalized culture medium, a rapid evaluation of the oxygen consumption rate is possible by measuring the phosphorescence lifetime of the platinum porphyrin-fluoropolymer. The combined use of the device and an automated optical measurement system enabled the high-throughput sensing of cellular oxygen consumption (100 points/min). We monitored the oxygen metabolism of the human breast cancer cell line MCF7 on a Petri dish and evaluated the oxygen consumption rate to be 0.72 ± 0.12 fmol/min/cell. Furthermore, to demonstrate the utility of the developed sensing system, we demonstrated the mapping of the oxygen consumption rate of rat brain slices and succeeded in visualizing a clear difference among the layer structures of the hippocampus, i.e., the cornu ammonis (CA1 and CA3) and dentate gyrus (DG).     

Stochastic Assembly of Bacteria in Microwell Arrays Reveals the Importance of Confinement in Community Development

The structure and function of microbial communities is deeply influenced by the physical and chemical architecture of the local microenvironment and the abundance of its community members. The complexity of this natural parameter space has made characterization of the key drivers of community development difficult. In order to facilitate these characterizations, we have developed a microwell platform designed to screen microbial growth and interactions across a wide variety of physical and initial conditions. Assembly of microbial communities into microwells was achieved using a novel biofabrication method that exploits well feature sizes for control of innoculum levels. Wells with incrementally smaller size features created populations with increasingly larger variations in inoculum levels. This allowed for reproducible growth measurement in large (20 μm diameter) wells, and screening for favorable growth conditions in small (5, 10 μm diameter) wells. We demonstrate the utility of this approach for screening and discovery using 5 μm wells to assemble P. aeruginosa colonies across a broad distribution of innoculum levels, and identify those conditions that promote the highest probability of survivial and growth under spatial confinement. Multi-member community assembly was also characterized to demonstrate the broad potential of this platform for studying the role of member abundance on microbial competition, mutualism and community succession.     

Disturbance-free rapid solution exchange for magnetic tweezers single-molecule studies

Single-molecule manipulation technologies have been extensively applied to studies of the structures and interactions of DNA and proteins. An important aspect of such studies is to obtain the dynamics of interactions; however the initial binding is often difficult to obtain due to large mechanical perturbation during solution introduction. Here, we report a simple disturbance-free rapid solution exchange method for magnetic tweezers single-molecule manipulation experiments, which is achieved by tethering the molecules inside microwells (typical dimensions–diameter (D): 40–50 μm, height (H): 100 μm; H:D∼2:1). Our simulations and experiments show that the flow speed can be reduced by several orders of magnitude near the bottom of the microwells from that in the flow chamber, effectively eliminating the flow disturbance to molecules tethered in the microwells. We demonstrate a wide scope of applications of this method by measuring the force dependent DNA structural transitions in response to solution condition change, and polymerization dynamics of RecA on ssDNA/SSB-coated ssDNA/dsDNA of various tether lengths under constant forces, as well as the dynamics of vinculin binding to α-catenin at a constant force (< 5 pN) applied to the α-catenin protein.     

Deficient SUMO Attachment to Flp Recombinase Leads to Homologous Recombination-dependent Hyperamplification of the Yeast 2 μm Circle Plasmid

Many Saccharomyces cerevisiae mutants defective in the SUMO pathway accumulate elevated levels of the native 2 μm circle plasmid (2 μm). Here we show that accumulation of 2 μm in the SUMO pathway mutants siz1Δ siz2Δ, slx5Δ, and slx8Δ is associated with formation of an aberrant high-molecular-weight (HMW) form of 2 μm. Characterization of this species from siz1Δ siz2Δ showed that it contains tandem copies of the 2 μm sequence as well as single-stranded DNA. Accumulation of this species requires both the 2 μm–encoded Flp recombinase and the cellular homologous recombination repair (HRR) pathway. Importantly, reduced SUMO attachment to Flp is sufficient to induce formation of this species. Our data suggest a model in which Flp that cannot be sumoylated causes DNA damage, whose repair via HRR produces an intermediate that generates tandem copies of the 2 μm sequence. This intermediate may be a rolling circle formed via break-induced replication (BIR), because mutants defective in BIR contain reduced levels of the HMW form. This work also illustrates the importance of using cir° strains when studying mutants that affect the yeast SUMO pathway, to avoid confusing direct functions of the SUMO pathway with secondary effects of 2 μm amplification.     

Spatial Physiological Heterogeneity in Pseudomonas aeruginosa Biofilm Is Determined by Oxygen Availability

The role of oxygen availability in determining the local physiological activity of Pseudomonas aeruginosa growing in biofilms was investigated. Biofilms grown in an ambient-air environment expressed approximately 1/15th the alkaline phosphatase specific activity of planktonic bacteria subjected to the same phosphate limitation treatment. Biofilms grown in a gaseous environment of pure oxygen exhibited 1.9 times the amount of alkaline phosphatase specific activity of air-grown biofilms, whereas biofilms grown in an environment in which the air was replaced with pure nitrogen prior to the inducing treatment did not develop alkaline phosphatase activity. Frozen cross sections of biofilms stained for alkaline phosphatase activity with a fluorogenic stain demonstrated that alkaline phosphatase activity was concentrated in distinct bands adjacent to the gaseous interfaces. These bands were approximately 30 μm thick with biofilms grown in air, 2 μm thick with biofilms grown in pure nitrogen, and 46 μm thick with biofilms grown in pure oxygen. Overall biofilm thickness ranged from approximately 117 to approximately 151 μm. Measurements with an oxygen microelectrode indicated that oxygen was depleted locally within the biofilm and that the oxygen-replete zone was of a dimension similar to that of the biologically active zone, as indicated by alkaline phosphatase induction. These experiments revealed marked spatial physiological heterogeneity within P. aeruginosa biofilms in which active protein synthesis was restricted by oxygen availability to the upper 30 μm of the biofilm. Such physiological heterogeneity has implications for microbial ecology and for understanding the reduced susceptibilities of biofilms to antimicrobial agents.     

Method To Immobilize the Aphid-Pathogenic Fungus Erynia neoaphidis in an Alginate Matrix for Biocontrol

Erynia neoaphidis is an important fungal pathogen of aphid pests worldwide. There have been few reported attempts to formulate this natural agent for use in biocontrol. In the current study, factors involved in the immobilization of E. neoaphidis hyphae in an alginate matrix were investigated. Hyphae of two isolates cultured in liquid medium were 220 to 620 μm in length and 7 to 19 μm in diameter with a 74 to 83% cytoplasmic content. The optimal concentration of low-viscosity sodium alginate for production of conidia from entrapped hyphae was 1.5% (wt/vol), and 0.1 and 0.25 M calcium chloride were equally suitable for use as the gelling solution. Alginate beads were rinsed with 10% sucrose after gelling. However, beads should not be left for longer than 40 min in 0.1 M calcium chloride or 10% sucrose to prevent a 10% loss in conidial production. A 40% (vol/vol) concentration of fungal biomass produced significantly more conidia than either 20% or the standard concentration of 10%. This effect persisted even after beads were dried overnight in a laminar flow hood and stored at 4°C for 4 days. Conidia from freshly produced alginate beads caused 27 to 32% infection in Pea aphids as determined by standardized laboratory bioassays. This finding was not significantly different from infections in aphids inoculated with fresh mycelial mats or plugs from Petri dish cultures. In conclusion, algination appears to be a promising technique for utilizing E. neoaphidis in the biocontrol of aphid pests.     

Live Cell Imaging of Butterfly Pupal and Larval Wings In Vivo

Butterfly wing color patterns are determined during the late larval and early pupal stages. Characterization of wing epithelial cells at these stages is thus critical to understand how wing structures, including color patterns, are determined. Previously, we successfully recorded real-time in vivo images of developing butterfly wings over time at the tissue level. In this study, we employed similar in vivo fluorescent imaging techniques to visualize developing wing epithelial cells in the late larval and early pupal stages 1 hour post-pupation. Both larval and pupal epithelial cells were rich in mitochondria and intracellular networks of endoplasmic reticulum, suggesting high metabolic activities, likely in preparation for cellular division, polyploidization, and differentiation. Larval epithelial cells in the wing imaginal disk were relatively large horizontally and tightly packed, whereas pupal epithelial cells were smaller and relatively loosely packed. Furthermore, larval cells were flat, whereas pupal cells were vertically elongated as deep as 130 μm. In pupal cells, many endosome-like or autophagosome-like structures were present in the cellular periphery down to approximately 10 μm in depth, and extensive epidermal feet or filopodia-like processes were observed a few micrometers deep from the cellular surface. Cells were clustered or bundled from approximately 50 μm in depth to deeper levels. From 60 μm to 80 μm in depth, horizontal connections between these clusters were observed. The prospective eyespot and marginal focus areas were resistant to fluorescent dyes, likely because of their non-flat cone-like structures with a relatively thick cuticle. These in vivo images provide important information with which to understand processes of epithelial cell differentiation and color pattern determination in butterfly wings.     

Scan-Free Absorbance Spectral Imaging A(x,y, λ) of Single Live Algal Cells for Quantifying Absorbance of Cell Suspensions

Label-free, non-invasive, rapid absorbance spectral imaging A(x,y,λ) microscopy of single live cells at 1.2 μm × 1.2 μm resolution with an NA = 0.85 objective was developed and applied to unicellular green algae Chlamydomonas reinhardtii. By introducing the fiber assembly to rearrange a two-dimensional image to the one-dimensional array to fit the slit of an imaging spectrograph equipped with a CCD detector, scan-free acquisition of three-dimensional information of A(x,y,λ) was realized. The space-resolved absorbance spectra of the eyespot, an orange organelle about 1 μm, were extracted from the green-color background in a chlorophyll-rich single live cell absorbance image. Characteristic absorbance change in the cell suspension after hydrogen photoproduction in C. reinhardtii was investigated to find a single 715-nm absorption peak was locally distributed within single cells. The formula to calculate the absorbance of cell suspensions from that of single cells was presented to obtain a quantitative, parameter-free agreement with the experiment. It is quantitatively shown that the average number of chlorophylls per cell is significantly underestimated when it is evaluated from the absorbance of the cell suspensions due to the package effect.     

Cryptocotyle lata (Trematoda: Heterophyidae) Adult from a Korean Raccoon Dog,Nyctereutes procyonoides koreensis

Total 513 heterophyid flukes were collected from a carcass of wild Korean raccoon dog, Nyctereutes procyonoides koreensis, in Korea. With morphological and molecular characteristics, the flukes were identified to Cryptocotyle lata. The adult C. lata were minute, transparent, pentagonal, 522 μm long by 425 μm wide. Ceca extended into post-testicular region. Ventrogenital sac elliptical, 79 μm by 87 μm with genital pore and ventral sucker. Two testes semielliptical and slightly lobed, located in the posterior region, right testis 173 μm by 155 μm, left testis 130 μm by 134 μm. In a phylogenetic tree, the fluke specimen of this study was grouped with C. lata divergent from Cryptocotyle lingua. We report here N. procyonoides koreensis first as a natural definitive host of C. lata.     

In vivo CD8+ T Cell Dynamics in the Liver of Plasmodium yoelii Immunized and Infected Mice

Plasmodium falciparum malaria remains one of the most serious health problems globally and a protective malaria vaccine is desperately needed. Vaccination with attenuated parasites elicits multiple cellular effector mechanisms that lead to Plasmodium liver stage elimination. While granule-mediated cytotoxicity requires contact between CD8+ effector T cells and infected hepatocytes, cytokine secretion should allow parasite killing over longer distances. To better understand the mechanism of parasite elimination in vivo, we monitored the dynamics of CD8+ T cells in the livers of naïve, immunized and sporozoite-infected mice by intravital microscopy. We found that immunization of BALB/c mice with attenuated P. yoelii 17XNL sporozoites significantly increases the velocity of CD8+ T cells patrolling the hepatic microvasculature from 2.69±0.34 μm/min in naïve mice to 5.74±0.66 μm/min, 9.26±0.92 μm/min, and 7.11±0.73 μm/min in mice immunized with irradiated, early genetically attenuated (Pyuis4-deficient), and late genetically attenuated (Pyfabb/f-deficient) parasites, respectively. Sporozoite infection of immunized mice revealed a 97% and 63% reduction in liver stage density and volume, respectively, compared to naïve controls. To examine cellular mechanisms of immunity in situ, naïve mice were passively immunized with hepatic or splenic CD8+ T cells. Unexpectedly, adoptive transfer rendered the motile CD8+ T cells from immunized mice immotile in the liver of P. yoelii infected mice. Similarly, when mice were simultaneously inoculated with viable sporozoites and CD8+ T cells, velocities 18 h later were also significantly reduced to 0.68±0.10 μm/min, 1.53±0.22 μm/min, and 1.06±0.26 μm/min for CD8+ T cells from mice immunized with irradiated wild type sporozoites, Pyfabb/f-deficient parasites, and P. yoelii CS_280–288 peptide, respectively. Because immobilized CD8+ T cells are unable to make contact with infected hepatocytes, soluble mediators could potentially play a key role in parasite elimination under these experimental conditions.     

Video-Rate Confocal Microscopy for Single-Molecule Imaging in Live Cells and Superresolution Fluorescence Imaging

There is no confocal microscope optimized for single-molecule imaging in live cells and superresolution fluorescence imaging. By combining the swiftness of the line-scanning method and the high sensitivity of wide-field detection, we have developed a, to our knowledge, novel confocal fluorescence microscope with a good optical-sectioning capability (1.0 μm), fast frame rates (<33 fps), and superior fluorescence detection efficiency. Full compatibility of the microscope with conventional cell-imaging techniques allowed us to do single-molecule imaging with a great ease at arbitrary depths of live cells. With the new microscope, we monitored diffusion motion of fluorescently labeled cAMP receptors of Dictyostelium discoideum at both the basal and apical surfaces and obtained superresolution fluorescence images of microtubules of COS-7 cells at depths in the range 0–85 μm from the surface of a coverglass.     

Oxygen Concentration Inside a Functioning Photosynthetic Cell

The excess oxygen concentration in the photosynthetic membranes of functioning oxygenic photosynthetic cells was estimated using classical diffusion theory combined with experimental data on oxygen production rates of cyanobacterial cells. The excess oxygen concentration within the plesiomorphic cyanobacterium Gloeobactor violaceus is only 0.025 μM, or four orders of magnitude lower than the oxygen concentration in air-saturated water. Such a low concentration suggests that the first oxygenic photosynthetic bacteria in solitary form could have evolved ∼2.8 billion years ago without special mechanisms to protect them against reactive oxygen species. These mechanisms instead could have been developed during the following ∼500 million years while the oxygen level in the Earth’s atmosphere was slowly rising. Excess oxygen concentrations within individual cells of the apomorphic cyanobacteria Synechocystis and Synechococcus are 0.064 and 0.25 μM, respectively. These numbers suggest that intramembrane and intracellular proteins in isolated oxygenic photosynthetic cells are not subjected to excessively high oxygen levels. The situation is different for closely packed colonies of photosynthetic cells. Calculations show that the excess concentration within colonies that are ∼40 μm or larger in diameter can be comparable to the oxygen concentration in air-saturated water, suggesting that species forming colonies require protection against reactive oxygen species even in the absence of oxygen in the surrounding atmosphere.     

Mechanosensitivity of a Rapid Bioluminescence Reporter System Assessed by Atomic Force Microscopy

Cells are sophisticated integrators of mechanical stimuli that lead to physiological, biochemical, and genetic responses. The bioluminescence of dinoflagellates, alveolate protists that use light emission for predator defense, serves as a rapid noninvasive whole-cell reporter of mechanosensitivity. In this study, we used atomic force microscopy (AFM) to explore the relationship between cell mechanical properties and mechanosensitivity in live cells of the dinoflagellate Pyrocystis lunula. Cell stiffness was 0.56 MPa, consistent with cells possessing a cell wall. Cell response depended on both the magnitude and velocity of the applied force. At the maximum stimulation velocity of 390 μm s⁻¹, the threshold response occurred at a force of 7.2 μN, resulting in a contact time of 6.1 ms and indentation of 2.1 μm. Cells did not respond to a low stimulation velocity of 20 μm s⁻¹, indicating a velocity dependent response that, based on stress relaxation experiments, was explained by the cell viscoelastic properties. This study demonstrates the use of AFM to study mechanosensitivity in a cell system that responds at fast timescales, and provides insights into how viscoelastic properties affect mechanosensitivity. It also provides a comparison with previous studies using hydrodynamic stimulation, showing the discrepancy in cell response between direct compressive forces using AFM and those within flow fields based on average flow properties.     

Margollus bokanicus n. sp. from Iran,the Fourth Species of a Rare Nematode Genus (Dorylaimida,Tylencholaimellidae)

Margollus bokanicus n. sp., collected from natural habitats in Khorasaneh district, Bokan, West Azarbaijan province, Iran, is described. Morphological and morphometric data are provided as well as drawings and light microscopy illustrations. The new species is characterized by a medium size body length (0.60 to 0.73 mm), labial and postlabial sclerotizations, lip region 7-μm wide, offset by constriction and long neck (167 to 207 μm), long pharyngeal basal bulb (27 to 36 μm) or 16% to 17% of total neck length, female genital system monodelphic–opisthodelphic, anterior branch reduced to a uterine sac (26–29 μm) or 1.1 to 1.3 times the body diameter, long posterior uterus (25–28 μm) or 1.1 to 1.3 times the body diameter, V = 40 to 47, cylindroid female tail (17 to 24 μm, c = 31 to 38, c’ = 1.1 to 1.4), and males unknown. This taxon is easily distinguishable from other Margollus species by its smaller general size and more posterior vulva. A compendium of Margollus species is also presented.     

Mycobacterium gilvum Illustrates Size-Correlated Relationships between Mycobacteria and Acanthamoeba polyphaga

Mycobacteria are isolated from soil and water environments, where free-living amoebae live. Free-living amoebae are bactericidal, yet some rapidly growing mycobacteria are amoeba-resistant organisms that survive in the amoebal trophozoites and cysts. Such a capacity has not been studied for the environmental rapidly growing organism Mycobacterium gilvum. We investigated the ability of M. gilvum to survive in the trophozoites of Acanthamoeba polyphaga strain Linc-AP1 by using optical and electron microscopy and culture-based microbial enumerations in the presence of negative controls. We observed that 29% of A. polyphaga cells were infected by M. gilvum mycobacteria by 6 h postinfection. Surviving M. gilvum mycobacteria did not multiply and did not kill the amoebal trophozoites during a 5-day coculture. Extensive electron microscopy observations indicated that M. gilvum measured 1.4 ± 0.5 μm and failed to find M. gilvum organisms in the amoebal cysts. Further experimental study of two other rapidly growing mycobacteria, Mycobacterium rhodesiae and Mycobacterium thermoresistibile, indicated that both measured <2 μm and exhibited the same amoeba-mycobacterium relationships as M. gilvum. In general, we observed that mycobacteria measuring <2 μm do not significantly grow within and do not kill amoebal trophozoites, in contrast to mycobacteria measuring >2 μm (P < 0.05). The mechanisms underlying such an observation remain to be determined.     

New Anti-Inflammatory Metabolites by Microbial Transformation of Medrysone

Microbial transformation of the anti-inflammatory steroid medrysone (1) was carried out for the first time with the filamentous fungi Cunninghamella blakesleeana (ATCC 8688a), Neurospora crassa (ATCC 18419), and Rhizopus stolonifer (TSY 0471). The objective was to evaluate the anti-inflammatory potential of the substrate (1) and its metabolites. This yielded seven new metabolites, 14α-hydroxy-6α-methylpregn-4-ene-3,11,20-trione (2), 6β-hydroxy-6α-methylpregn-4-ene-3,11,20-trione (3), 15β-hydroxy-6α-methylpregn-4-ene-3,11,20-trione (4), 6β,17α-dihydroxy-6α-methylpregn-4-ene-3,11,20-trione (5), 6β,20S-dihydroxy-6α-methylpregn-4-ene-3,11-dione (6), 11β,16β-dihydroxy-6α-methylpregn-4-ene-3,11-dione (7), and 15β,20R-dihydroxy-6α-methylpregn-4-ene-3,11-dione (8). Single-crystal X-ray diffraction technique unambiguously established the structures of the metabolites 2, 4, 6, and 8. Fungal transformation of 1 yielded oxidation at the C-6β, -11β, -14α, -15β, -16β positions. Various cellular anti-inflammatory assays, including inhibition of phagocyte oxidative burst, T-cell proliferation, and cytokine were performed. Among all the tested compounds, metabolite 6 (IC₅₀ = 30.3 μg/mL) moderately inhibited the reactive oxygen species (ROS) produced from zymosan-induced human whole blood cells. Compounds 1, 4, 5, 7, and 8 strongly inhibited the proliferation of T-cells with IC₅₀ values between <0.2–10.4 μg/mL. Compound 7 was found to be the most potent inhibitor (IC₅₀ < 0.2 μg/mL), whereas compounds 2, 3, and 6 showed moderate levels of inhibition (IC₅₀ = 14.6–20.0 μg/mL). Compounds 1, and 7 also inhibited the production of pro-inflammatory cytokine TNF-α. All these compounds were found to be non-toxic to 3T3 cells (mouse fibroblast), and also showed no activity when tested against HeLa (human epithelial carcinoma), or against PC3 (prostate cancer) cancer cell lines.     

Use of an Oxygen-Insensitive Microscale Biosensor for Methane To Measure Methane Concentration Profiles in a Rice Paddy

An oxygen-insensitive microscale biosensor for methane was constructed by furnishing a previously described biosensor with an oxygen guard. The guard consisted of a glass capillary containing heterotrophic bacteria, which consumed oxygen diffusing through the tip membrane, thus preventing it from diffusing into the methane-sensing unit. Oxygen microprofiles were measured through the oxygen guard capillary, demonstrating the principle and limitations of the method. When the tip of the guard capillary was exposed to 100% oxygen at 21°C, heterotrophic oxygen consumption prevented oxygen from diffusing further than 170 μm into the capillary, whereas atmospheric levels of oxygen were consumed within 50 μm. The capacity of the oxygen guard for scavenging oxygen decreased with decreasing temperature, and atmospheric levels of oxygen caused oxygen penetration to 200 μm at 5°C. The sensors could be manufactured with tip diameters as small as 25 μm, and response times were about 1 min at room temperature. Pore water profiles of methane concentrations in a rice paddy soil were measured, and a strong correlation between the depths of oxygen penetration and methane appearance was observed as a function of the light regimen; this finding confirmed the role of microbenthic photosynthesis in limiting methane emissions from surfaces of waterlogged sediments and soils.     

Successional Development of Sulfate-Reducing Bacterial Populations and Their Activities in a Wastewater Biofilm Growing under Microaerophilic Conditions

A combination of fluorescence in situ hybridization, microprofiles, denaturing gradient gel electrophoresis of PCR-amplified 16S ribosomal DNA fragments, and 16S rRNA gene cloning analysis was applied to investigate successional development of sulfate-reducing bacteria (SRB) community structure and in situ sulfide production activity within a biofilm growing under microaerophilic conditions (dissolved oxygen concentration in the bulk liquid was in the range of 0 to 100 μM) and in the presence of nitrate. Microelectrode measurements showed that oxygen penetrated 200 μm from the surface during all stages of biofilm development. The first sulfide production of 0.32 μmol of H₂S m⁻² s⁻¹ was detected below ca. 500 μm in the 3rd week and then gradually increased to 0.70 μmol H₂S m⁻² s⁻¹ in the 8th week. The most active sulfide production zone moved upward to the oxic-anoxic interface and intensified with time. This result coincided with an increase in SRB populations in the surface layer of the biofilm. The numbers of the probe SRB385- and 660-hybridized SRB populations significantly increased to 7.9 × 10⁹ cells cm⁻³ and 3.6 × 10⁹ cells cm⁻³, respectively, in the surface 400 μm during an 8-week cultivation, while those populations were relatively unchanged in the deeper part of the biofilm, probably due to substrate transport limitation. Based on 16S rRNA gene cloning analysis data, clone sequences that related to Desulfomicrobium hypogeium (99% sequence similarity) and Desulfobulbus elongatus (95% sequence similarity) were most frequently found. Different molecular analyses confirmed that Desulfobulbus, Desulfovibrio, and Desulfomicrobium were found to be the numerically important members of SRB in this wastewater biofilm.     

Brine Assemblages of Ultrasmall Microbial Cells within the Ice Cover of Lake Vida,Antarctica

The anoxic and freezing brine that permeates Lake Vida''s perennial ice below 16 m contains an abundance of very small (≤0.2-μm) particles mixed with a less abundant population of microbial cells ranging from >0.2 to 1.5 μm in length. Fluorescent DNA staining, electron microscopy (EM) observations, elemental analysis, and extraction of high-molecular-weight genomic DNA indicated that a significant portion of these ultrasmall particles are cells. A continuous electron-dense layer surrounding a less electron-dense region was observed by EM, indicating the presence of a biological membrane surrounding a cytoplasm. The ultrasmall cells are 0.192 ± 0.065 μm, with morphology characteristic of coccoid and diplococcic bacterial cells, often surrounded by iron-rich capsular structures. EM observations also detected the presence of smaller unidentified nanoparticles of 0.020 to 0.140 μm among the brine cells. A 16S rRNA gene clone library from the brine 0.1- to 0.2-μm-size fraction revealed a relatively low-diversity assemblage of Bacteria sequences distinct from the previously reported >0.2-μm-cell-size Lake Vida brine assemblage. The brine 0.1- to 0.2-μm-size fraction was dominated by the Proteobacteria-affiliated genera Herbaspirillum, Pseudoalteromonas, and Marinobacter. Cultivation efforts of the 0.1- to 0.2-μm-size fraction led to the isolation of Actinobacteria-affiliated genera Microbacterium and Kocuria. Based on phylogenetic relatedness and microscopic observations, we hypothesize that the ultrasmall cells in Lake Vida brine are ultramicrocells that are likely in a reduced size state as a result of environmental stress or life cycle-related conditions.     

Live-Cell Superresolution Imaging by Pulsed STED Two-Photon Excitation Microscopy

Two-photon laser scanning microscopy (2PLSM) allows fluorescence imaging in thick biological samples where absorption and scattering typically degrade resolution and signal collection of one-photon imaging approaches. The spatial resolution of conventional 2PLSM is limited by diffraction, and the near-infrared wavelengths used for excitation in 2PLSM preclude the accurate imaging of many small subcellular compartments of neurons. Stimulated emission depletion (STED) microscopy is a superresolution imaging modality that overcomes the resolution limit imposed by diffraction and allows fluorescence imaging of nanoscale features. Here, we describe the design and operation of a superresolution two-photon microscope using pulsed excitation and STED lasers. We examine the depth dependence of STED imaging in acute tissue slices and find enhancement of 2P resolution ranging from approximately fivefold at 20 μm to approximately twofold at 90-μm deep. The depth dependence of resolution is found to be consistent with the depth dependence of depletion efficiency, suggesting resolution is limited by STED laser propagation through turbid tissue. Finally, we achieve live imaging of dendritic spines with 60-nm resolution and demonstrate that our technique allows accurate quantification of neuronal morphology up to 30-μm deep in living brain tissue.