Light Confocal Scanning Microscopy Research Articles

Milk fat globule membrane: formation and transformation.

The milk fat globule membrane (MFGM) is formed by complex cell biological processes in the lactating mammary epithelial cell which result in the release of the milk fat globule (MFG) into the secretory alveolus. The MFG is bounded by a continuous unit membrane (UM), separated from the MFG lipid by a thin layer of cytoplasm. This unique apocrine secretion process has been shown in all of the mammary species so far investigated. Once the MFG is released into the alveolus there is a considerable transformation of the UM with its attached cytoplasm. This is the MFGM. The transformation is stable and expressed milk shows the same transformed MFGM structure. Again, this transformation of structure is common to all mammalian species so far investigated. However, the explanation of the transformation very much depends on the method of investigation. Transmission electron microscope (TEM) studies suggest a literal breakdown to a discontinuous UM plus cytoplasm in patches and strands, whereas more recent confocal laser scanning light microscopy (CLSM) studies indicate a separation, in a continuous UM, of two phases, one liquid ordered and the other liquid disordered. This review is designed to show that the TEM and CLSM results show different views of the same structures once certain deficiencies in techniques are factored in.

Visualizing Neurons Under Tension In Vivo with Optogenetic Molecular Force Sensors.

The visualization of mechanical stress distribution in specific molecular networks within a living and physiologically active cell or animal remains a formidable challenge in mechanobiology. The advent of fluorescence-resonance energy transfer (FRET)-based molecular tension sensors overcame a significant hurdle that now enables us to address previously technically limited questions. Here, we describe a method that uses genetically encoded FRET tension sensors to visualize the mechanics of cytoskeletal networks in neurons of living animals with sensitized emission FRET and confocal scanning light microscopy. This method uses noninvasive immobilization of living animals to image neuronal β-spectrin cytoskeleton at the diffraction limit, and leverages multiple imaging controls to verify and underline the quality of the measurements. In combination with a semiautomated machine-vision algorithm to identify and trace individual neurites, our analysis performs simultaneous calculation of FRET efficiencies and visualizes statistical uncertainty on a pixel by pixel basis. Our approach is not limited to genetically encoded spectrin tension sensors, but can also be used for any kind of ratiometric imaging in neuronal cells both in vivo and in vitro.

Exploring soluble and colloidally transported trace elements in stalagmites: The strontium-yttrium connection

While seasonality in speleothem trace element signatures is well-documented, the parameters that control the emergence of laminations vary between elements and tend to be multi-factorial. Here, we examine a series of active and fossil stalagmites from Asturias, Spain, with a particular focus on strontium and yttrium co-variations and fluorescent laminations. Coupled confocal fluorescence scanning light microscopy (layer counting) and time scales derived from accelerated mass spectrometry (F14C) in active stalagmites confirm that fluorescent banding is annual. This banding is coincident with Y peaks and Sr troughs, which are among the most robust trace element markers of seasonality. Strontium concentrations (in particular, the strontium partition coefficient, DSr) are positively correlated with stalagmite growth rate and are likely controlled by solution supersaturation, which is in turn controlled by seasonal variations in cave ventilation. DSr can be estimated after correcting for prior calcite precipitation using coeval Mg/Ca ratios, and is consistent with both empirical and experimental values. Meanwhile, yttrium is a proxy for colloidal organic input, and its concentration in stalagmites is likely controlled by a combination of Y drip water flux, surface retention time (i.e., how long a drip and its associated organic matter are in contact with the stalagmite surface), and dilation within the matrix (hereafter referred to as “dilation”). Persistent Sr-Y anti-correlation can be explained as an interplay between the individual controls on each element, and a breakdown in this relationship may be indicative of past changes in cave ventilation and/or drip hydrology.

Fructus mume Aqueous Extract's Antibacterial Effect on Multi-species Biofilm

Aim: The purpose of this study was to determine the efficacy of several irrigants and an aqueous extract of Fructus mume in eradicating three kinds of biofilm. Materials and Methods: A three-species biofilm of E. faecalis, P. gingivalis and F. nucleatum were cultivated for one week on Thermanox™ plates. Each specimen was immersed for 8 minutes in various irrigants: Fructus mume solution, buffered citric acid, sodium hypochlorite and control (non-irrigated). LIVE/DEAD® BacLightTM staining and confocal light scanning microscopy were used to determine the number of viable bacteria left on the substrate. The same biofilm was then extracted and subjected to scanning electron microscopy (SEM). Results: Images were taken at 12 locations across the biofilm, which were then classified into four areas of concern: Bottom, which would spend the most of the experiment submerged in the solution; It was struck in the centre by the stream of irrigant.; Middle and Upper, where the impression was caused by the irrigant splattering or vaporizing. Results of the amount of viable bacteria residual indicated that Fructus mume showed no significant activity, with an effect like control and even buffered citric acid, and significantly inferior to sodium hypochlorite. Conclusion: Sodium hypochlorite (0.5%) solution was greater to buffered citric acid, Fructus mume and control group as an antimicrobial agent against three-species biofilm.

Antifungal effect of a new photosensitizer derived from BODIPY on Candida albicans biofilms

BackgroundPhotodynamic therapy (PDT) may be an alternative treatment of Candida albicans (C. albicans) infections. The aim of this study was to investigate the antifungal effect of PDT mediated by a new photosensitizer (PS) derived from BODIPY (BDP-4L) on C. albicans biofilms. MethodsC. albicans biofilms were incubated with BDP-4L of different concentrations and then irradiated at the light doses of 1.8, 3.6, 5.4, 7.2 and 9.0 J/cm2. XTT reduction assay was conducted to determine the PS concentration and PDT parameters. Confocal light scanning microscopy (CLSM) and scanning electron microscope (SEM) were used to visualize and quantify the effect of BDP-4L on C. albicans biofilms after PDT. ResultsC. albicans biofilms were inactivated in light dose-dependent and PS concentration-dependent manners using BDP-4L as PS. Without irradiation, no inactivation effect was observed when PS concentrations varied from 5 μM to 80 μM. 40 μM PS with 3.6 J/cm2 irradiation resulted in a significant reduction of 83.8% in biofilm metabolic activities. CLSM assay demonstrated that cell viability was obviously inhibited by 82.6%. SEM images revealed ruptured and rough cell surface, indicating increased cell membrane permeability after PDT. ConclusionsOur results suggested that BDP-4L mediated PDT exhibited a favorable antifungal effect on C. albicans biofilms.

Balbiani body formation and cytoplasmic zonation during early oocyte development in two Clupeiform fishes.

The Balbiani body (Bb) was examined in primary growth phase oocytes for the first time in two clupeoid fish species, the Mediterranean sardine, Sardina pilchardus, and the European anchovy, Engraulis encrasicolus, which belong to different families, Clupeidae and Engraulidae, respectively. Cytoplasmic morphological changes of early secondary growth oocytes were also investigated using confocal laser scanning microscopy, light and transmission electron microscopy. The ultrastructural observations showed that the two species develop a distinct spherical Bb. However, differences in the cytoplasm, mainly in the perinuclear area, were observed. Briefly, in sardine the Bb coexists with a thick perinuclear ring containing mitochondria, nuage, endoplasmic reticulum and small vesicles, while in anchovy this perinuclear ring is thinner, consisting of complexes of nuage and mitochondria. After the disassembly of the Bb, a prominent cytoplasmic zonation develops in the secondary growth oocytes of sardine and anchovy, although with different organelle distribution between the two species. Sardine oocytes exhibit a thick zone of endoplasmic reticulum around the nucleus, whereas in those of anchovy, a thick mitochondria-rich ring surrounding the nucleus was observed. The cytoplasmic characteristics, such as the perinuclear ring in primary oocytes in sardine and the mitochondria-rich ring of early secondary oocytes in anchovy, are also discernible in histological sections by standard procedures and could thus be used as indicators of maturity or imminent spawning period in routine light microscopy observations, providing a valuable tool for applied fisheries biology.

Microbial biofilm community structure and composition on the lithic substrates of Herculaneum Suburban Baths

In this work, we want to investigate the impact of different substrates and different environmental condition on the biofilm communities growing on plaster, marble, and mortar substrates inside the Herculaneum Suburban Baths. To do so, we measured environmental conditions and sampled biofilm communities along the walls of the baths and used culture-dependent and -independent molecular techniques (DGGE) to identify the species at each sampling sites. We used the species pool to infer structure and richness of communities within each site in each substrate, and confocal light scanning microscopy to assess the three-dimensional structure of the sampled biofilms. To gather further insights, we built a meta-community network and used its local realizations to analyze co-occurrence patterns of species. We found that light is a limiting factor in the baths environment, that moving along sites equals moving along an irradiation gradient, and that such gradient shapes the community structure, de facto separating a dark community, rich in Bacteria, Fungi and cyanobacteria, from two dim communities, rich in Chlorophyta. Almost all sites are dominated by photoautotrophs, with Fungi and Bacteria relegated to the role of rare species., and structural properties of biofilms are not consistent within the same substrate. We conclude that the Herculaneum suburban baths are an environment-shaped community, where one dark community (plaster) and one dim community (mortar) provides species to a “midway” community (marble).

Effect of Brefeldin A on the Development of <i>Sargassum cymosum</i> Zygotes (Phaeophyceae, Fucales): Structural and Ultrasctructural Analysis

To evaluate the effects of brefeldin A (BFA) on Golgi bodies and indirectly on the polarized development of S. cymosum zygotes, zygotes were cultured at concentrations of 4 μM, 8 μM, 16 μM and 32 μM of BFA, prepared in sterilized seawater. After 12 hours, the samples were fixed and processed for transmission electron microscopy (TEM), confocal laser scanning microscopy, light microscopy and cell counting. For recovery analysis, after treatment, the samples were again placed in sterile seawater for over 12 hours. In the control, after 12 hours, the embryos were adhered and with advanced stage of development, presenting up to 6 cell divisions. In the treated samples, the embryos did not adhere and only in the lower concentrations were they visualized with cellular divisions, is that in the highest concentration, the majority of the zygotes were dead. After recovery, embryos showed development only in the lowest concentrations. By confocal microscopy, the treated samples had an irregular distribution of chloroplasts and physodes. Also through TEM, it was possible to observe the loss of organization of Golgi bodies, in turn leading to the formation of vesicles and fusion of physodes. We can conclude that Golgi bodies are responsible for the production and secretion of molecules related to the adhesion and formation of cell wall membrane, also aiding in the polarization and orientation of physodes. The fused physodes occupied much of the cytoplasm, preventing other cytoplasmic processes, thus directly affecting the development of this alga.

Combination of Dark-Field and Confocal Microscopy for the Optical Detection of Silver and Titanium Nanoparticles in Mammalian Cells.

We describe here two optical microscopy techniques-dark-field confocal light scanning microscopy (DF-CLSM) and dark-field wide-field confocal microscopy (DF-WFCM), that can be used to study interaction between nanoparticles and cells in 3D space. Dark field microscopy can detect very small structures below the diffraction limit of conventional light microscopes, while a confocal setup provides vertical sectioning capabilities to render specimens in 3D. The use of DF-WFCM instead of DF-CLSM allows faster sample processing but yields lower resolution. We used a retinal pigment epithelial cell line ARPE-19 to illustrate different optical and lighting conditions necessary for optimal imaging of metal and metal oxide nanoparticles (TiO2 and Ag). Our experimental setup primarily involved an E-800 Nikon and Nikon Ni upright microscopes and a Nikon Ti2 microscope connected to a xenon light source along with special dark-field objectives. For confocal studies we used either Leica and Nikon inverted confocal microscopes. For microscopic analyses, ARPE-19 cells were fixed in situ in cultured chamber slides or collected from T-25 flasks and then fixed in suspension. At the lowest concentrations of TiO2 or Ag tested (0.1-0.3μg/mL), we were able to detect as few as 5-10 nanoparticles per cell due to intense light scattering by the particles. The degree of brightness detected indicated that the uptake of nanoparticles within ARPE-19 cells could be monitored using dark-field microscopy. Here we describe how to use wide-field microscopy to follow nanoparticle uptake by cells and how to assess some aspects of cellular health in in vitro cell cultures exposed to nanoparticles.

The distinct effects of aspirin on platelet aggregation induced by infectious bacteria

Bacteria induce platelet aggregation triggered by several mechanisms. The goal of this work was to characterize platelet aggregates induced by different bacterial strains and to quantify the effect of aspirin treatment using aggregation tests, as well as a novel approach based on confocal analysis. Blood samples were obtained from either healthy donors (n = 27) or patients treated with long-term aspirin (n = 15). The bacterial species included were Staphylococcus aureus, Enterococcus faecalis, and Streptococcus sanguinis. The different aggregate’s ultrastructures depending on the bacterial strain were analyzed using Scanning electron microscopy. Quantification of the size of the platelet aggregates, their mean number as well as the bacterial impregnation within the aggregates was performed using confocal laser scanning light microscopy. Light Transmission Aggregometry was also performed. Our results reported distinct characteristics of platelet aggregates depending on the bacterial strain. Using confocal analysis, we have shown that aspirin significantly reduced platelet aggregation induced by S. aureus (p = .003) and E. faecalis (p = .006) with no effect in the case of S. sanguinis (p = .529). The results of the aggregometry were concordant with those of the confocal technique in the case of S. aureus and S. sanguinis. Interestingly, aggregation induced by E. faecalis was detected only with confocal analysis. In conclusion, our confocal scanning microscopy allowed a detailed study of the platelet aggregation induced by bacteria. We showed that aspirin acts on bacterial-induced platelet aggregation depending on the species. These results are in favor of the use of aspirin considering the species and the bacterial strain involved.

Carbon dot-protoporphyrin IX conjugates for improved drug delivery and bioimaging.

Photodynamic therapy (PDT) uses photosensitisers such as protoporphyrin IX (PpIX) to target tumours via the release of toxic singlet oxygen when irradiated. The effectivity of the treatment is limited by the innate properties of the photosensitizers; they typically exhibit inefficient accumulation in target tissue and high dark toxicity. Carbon dots (CDs) are biocompatible fluorescent nanoparticles which can improve PpIX cellular uptake and solubility. In this work, we present conjugates synthesised by host-guest encapsulation (PpIX@CD) and amide cross-linking (PpIX-CD). Characterization demonstrated conjugates have a loading efficiency of 34–48% and similar singlet oxygen production to PpIX. PpIX-containing CDs showed a 2.2 to 3.7-fold decrease in dark toxicity. PpIX-CD and PpIX@CD showed equivalent light-induced toxicity to PpIX in concentrations >1 μg/ml, leading to a 3.2 to 4.1-fold increase in photo-toxicity index (PI). The less soluble fraction of cross-linked conjugates (PpIX-CD)p did not show significant difference from PpIX. Confocal light scanning microscopy demonstrated rapid intracellular uptake and accumulation of conjugates. Our results demonstrate the variations between cross-linking strategies in CD-based conjugates, highlighting their potential as carriers in drug delivery and bioimaging applications.

Droplet evaporation: Colloidal interactions vs. evaporation kinetics

The drying of a colloidal droplet has attracted considerable attention because of its inherent complexity and significance in applications such as coating, printing, and layering processes. There has been great interest in controlling the well-known ‘coffee-ring’ effect, in which the contact line of an evaporating drop is pinned to the substrate so that dispersed particles are deposited onto the three-phase contact line. Large micro-scale convection-driven flows typically determine the structure of the deposited film. Interparticle forces which operate over nanometer-separations, at least in aqueous systems, are relatively unimportant. Our aim is to explore if much longer-range colloidal forces, comparable to hydrodynamic flows, could be used to control particle drying. We generate controllable micrometer-range electrostatic interactions in a suspension by using charge control additives; an anionic surfactant and oil-soluble electrolyte in nonpolar solvents. The drying of droplets containing monodisperse, fluorescent poly-(methyl methacrylate) (PMMA) particles was studied by confocal light scanning microscopy (CLSM). The existence and extent of a ‘coffee-ring’ deposit was characterized. We find that the drying patterns are controlled by a competition between the evaporation rate of the solvent and the strength and range of the electrostatic interactions.

Antifungal effect of photodynamic therapy mediated by curcumin on Candida albicans biofilms in vitro.

Canida albicans can cause opportunistic infections ranging from superficial mucous membrane lesions to life-threatening disease. The aim of this study is to investigate the antifungal effect of photodynamic therapy (PDT) mediated by curcumin (CUR) on C. albicans biofilms in vitro. One standard strain ATCC 90028 and two clinical isolates from HIV (CCA1) and oral lichen planus (CCA2) patients' oral cavities were used in this study. Biofilms were photosensitized with 60 μM CUR and irradiated by light emitting diode (LED) under the wavelength of 455 nm and energy densities of 2.64, 5.28, 7.92, 10.56, 13.2 J/cm2. Then the antifungal effects of CUR-PDT were evaluated by XTT reduction assay and confocal light scanning microscopy (CLSM) observations. The effects of CUR-PDT on the expression levels of hypha-specific and biofilm-related genes including EFG1, UME6, HGC1 and ECE1 were assessed by quantitative Real-time PCR (qRT-PCR) method. The inhibition rates after CUR-PDT in three biofilms(ATCC 90028, CCA1, CCA2)were 90.87%, 66.44% and 86.74% respectively (p < 0.05). Relative gene expression levels of EFG1, UME6, HGC1 and ECE1 were all downregulated after CUR-PDT, with fold-decrease of 6.865, 3.382, 2.167 and 6.887 in ATCC 90028, 2.466, 2.146, 1.627 and 3.102 in CCA1, and 5.406, 2.347, 2.073and 3.711 in CCA2 (p < 0.05). Curcumin-mediated PDT could effectively inactivateCandida albicans biofilms in vitro. Expression of genes involved in biofilms formation were downregulated after CUR-PDT.

Transdentinal cytotoxicity and macrophage phenotype of a novel quaternary ammonium silane cavity disinfectant

ObjectiveTo evaluate the transdentinal cytotoxicity and macrophage phenotype response to a novel quaternary ammonium silane (QAS) cavity disinfectant. MethodsNIH 3T3 mouse fibroblasts were cultured in Dulbecco’s Modified Eagle's Medium and incubated for 3 days. The cells (3×104) were seeded on the pulpal side of dentine discs and the occlusal side of the discs were treated with different cavity disinfectants: Group 1: de-ionized water (control); Group 2: 2% chlorhexidine (CHX); Group 3: 2% QAS; Group 4: 5% QAS, and Group 5: 10% QAS. Cell morphology of NIH 3T3 cells was examined using scanning electron microscopy (SEM) and cell viability was assessed using Trypan blue assay. The eluates were collected and applied on cells seeded in 24-well plates. The total protein production, alkaline phosphatase activity and deposition of mineralized nodules were evaluated after 7 and 14 days. Immunofluorescence staining was performed on the samples with primary antibodies of CD68+, CD80+, and CD163+ assessing the macrophage M1/M2 phenotypes. The macrophages were imaged using a confocal scanning light microscope with an excitation wavelength of 488nm. ResultsNo significant difference in cell viability (p<0.0001), total protein production (p<0.01) and mineralized nodule production (p<0.05) was found between 2% QAS and the control, which was significantly higher than 2% CHX, 5% and 10% QAS after 14 days. Alkaline phosphatase production of 2% QAS was significantly lower than the control (p<0.001), but higher than 2% CHX at 14 days. The M1/M2 macrophage ratio was also significantly lower in the 2% and 10% QAS groups (p<0.05) compared to the control and 2% CHX groups. SignificanceThe 2% QAS cavity disinfectant does not have cytotoxic effects on 3T3 NIH mouse fibroblast cells and the predominance of the anti-inflammatory phenotype after its application may stimulate healing and tissue repair.

Bio-mining drugs from the sea: High antibiofilm properties of haemocyanin purified from the haemolymph of flower crab Portunus pelagicus (L.) (Decapoda: Portunidae)

Marine organisms are an outstanding source of molecules with wide biological activities. In this research, we investigated the antimicrobial protein haemocyanin isolated from the haemolymph of flower crab (Portunus pelagicus) using gel filtration chromatography. Purified P. pelagicus haemocyanin (Pp-Hc) was subjected to SDS-PAGE analysis and the fraction containing Pp-Hc ran as a distinct band, its molecular weight was 78 kDa. The Pp-Hc was characterized through HPLC, XRD, CD and FTIR analyses. The antibacterial potential of Pp-Hc was evaluated against five Gram-positive and ten Gram-negative bacteria through the agar well diffusion method, which clearly revealed the high bactericidal activity of Pp-Hc. Furthermore, Pp-Hc was studied for its antibiofilm activity against five biofilm-forming Gram-negative bacteria, i.e., Vibrio alginolyticus, Vibrio harveyi, Vibrio parahaemolyticus, Pseudomonas aeruginosa, and Proteus vulgaris. Confocal laser scanning microscopy (CLSM) and light microscopy evidenced significant antibiofilm properties of Pp-Hc at 100 μg/ml concentration. Based on exopolysaccharide (EPS) quantification and cell surface hydrophobicity (CSH) index, the antibiofilm action of Pp-Hc was linked to its ability to reduce EPS production and increase cell surface hydrophobicity. The pathogen recognition mechanism of purified Pp-Hc showed a wide spectrum of binding activities, leading to high toxicity against both Gram-positive and Gram-negative bacterial species. Furthermore, the purified Pp-Hc showed high antifungal activity against the pathogen Candida albicans. Haemocyanin is not only involved in transporting oxygen, it has a relevant function in the flower crab immune system. This work adds useful knowledge for aquaculture concerns dealing with microbial pathogen control. Overall, our results outlined the high potential of purified Pp-Hc as a starting material to develop new and effective antimicrobial drugs for aquaculture purposes.

Stability, microstructure and rheological behavior of konjac glucomannan-zein mixed systems

This study aims to investigate the stability, microstructure and rheological properties of konjac glucomannan (KGM)/zein mixed systems in different mixing ratios. A phase diagram was established by centrifugation and visual observation. KGM/zein could form a stable homogeneous dispersion with appropriate mixing formula, and the particle size in mixed systems increased with increasing zein content. During storage, zein particles increased in size but were homogeneously distributed in the continuous phase of KGM without flocculation as observed by confocal light scanning microscopy. The rate of particle size change slowed down with increasing concentration of KGM. Transmission electron microscopy and atomic force microscopy images showed that zein particles were distributed in the KGM molecular network. The mixed systems showed shear-thinning behavior, and the temperature dependence of the viscosity was well-fitted by the Arrhenius equation. Based on dynamic viscoelasticity analysis, the mixed systems showed typical behaviors for entangled polymer solutions. The shift of cross-over frequency of storage (G′) and loss (G″) moduli to higher frequencies with increasing concentration of zein implied the shortening of the lifetime of the temporary entangled junction in the mixed systems.

Adding tetrahydrofuran to dilute acid pretreatment provides new insights into substrate changes that greatly enhance biomass deconstruction by Clostridium thermocellum and fungal enzymes

BackgroundConsolidated bioprocessing (CBP) by anaerobes, such as Clostridium thermocellum, which combine enzyme production, hydrolysis, and fermentation are promising alternatives to historical economic challenges of using fungal enzymes for biological conversion of lignocellulosic biomass. However, limited research has integrated CBP with real pretreated biomass, and understanding how pretreatment impacts subsequent deconstruction by CBP vs. fungal enzymes can provide valuable insights into CBP and suggest other novel biomass deconstruction strategies. This study focused on determining the effect of pretreatment by dilute sulfuric acid alone (DA) and with tetrahydrofuran (THF) addition via co-solvent-enhanced lignocellulosic fractionation (CELF) on deconstruction of corn stover and Populus with much different recalcitrance by C. thermocellum vs. fungal enzymes and changes in pretreated biomass related to these differences.ResultsCoupling CELF fractionation of corn stover and Populus with subsequent CBP by the anaerobe C. thermocellum completely solubilized polysaccharides left in the pretreated solids within only 48 h without adding enzymes. These results were better than those from the conventional DA followed by either CBP or fungal enzymes or CELF followed by fungal enzyme hydrolysis, especially at viable enzyme loadings. Enzyme adsorption on CELF-pretreated corn stover and CELF-pretreated Populus solids were virtually equal, while DA improved the enzyme accessibility for corn stover more than Populus. Confocal scanning light microscopy (CSLM), transmission electron microscopy (TEM), and NMR characterization of solids from both pretreatments revealed differences in cell wall structure and lignin composition, location, coalescence, and migration-enhanced digestibility of CELF-pretreated solids.ConclusionsAdding THF to DA pretreatment (CELF) greatly enhanced deconstruction of corn stover and Populus by fungal enzymes and C. thermocellum CBP, and the CELF–CBP tandem was agnostic to feedstock recalcitrance. Composition measurements, material balances, cellulase adsorption, and CSLM and TEM imaging revealed adding THF enhanced the enzyme accessibility, cell wall fractures, and cellular dislocation and cell wall delamination. Overall, enhanced deconstruction of CELF solids by enzymes and particularly by C. thermocellum could be related to lignin removal and alteration, thereby pointing to these factors being key contributors to biomass recalcitrance as a barrier to low-cost biological conversion to sustainable fuels.

Ontogenetic development and functioning of the anterior end of Cossura pygodactylata Jones, 1956 (Annelida: Cossuridae)

Juvenile and adult Cossura pygodactylata Jones 1956 from the White Sea were studied using confocal laser scanning microscopy, light microscopy, scanning and transmission electron microscopy. Transformations of the anterior musculature and digestive tract during ontogenesis were investigated. The early juveniles were shown to be lecithotrophic; their pharyngeal cavities were not connected to the intestines, which contained yolk granules. The juveniles bore prototrochs, which are used for movement, although juveniles had parapodial musculature similar to that of the adults. The juveniles presumably inhabit the upper semi-liquid layer of the silt. The muscles of the prostomium and circumbuccal complex change dramatically during ontogenesis. The ultrastructure of the buccal tentacles is redescribed. The tentacles consist of outer ciliated epithelial cells and an inner cylinder formed by epithelio-muscle cells. The blood sinus is situated between the central cylinder and the epithelium. Both juveniles and adults have developed circulatory systems. The whole dorsal vessel forms the heart with walls that consist of cells with circular cross-striated muscular fibres. The inner lumen is occluded by the heart body which is formed by a single row of cells that are tightly pressed together and connected by adherens junctions along their anterior and posterior surfaces. They contain granules and vesicles and bear numerous processes on the outer surface. The heart body most likely has a secretory haemopoetic function. A hypothetical mechanism of protraction and retraction of the buccal tentacles is suggested, and the participation of muscle contraction and relaxation in these movements is described. It is proposed that the protraction of the tentacles is provided by cell rigidity and increases in the blood volume in the tentacles blood sinuses. The development of the circulatory system is likely related to the need to keep the tentacles exposed during feeding while the anterior part of the body cavity is filled with muscle cell processes and there is no coelomic liquid flow. The proposed mechanism of feeding inside the sediment contrasts with that of surface feeding suggested by Tzetlin (Mem Mus Natl Hist Nat 162:137–143, 1994).

Probing the future of correlative microscopy

Instrumental developments in microscopy, such as confocal scanning light microscopy and super resolution light microscopy, have firmly established imaging as a key technology in modern life science research. However, this would not have been possible without the availability of the right probes—the concurrent emergence of green fluorescent protein (GFP) completely transformed the field. We can surely assume that GFP and its derivatives have now penetrated most biomedical research labs that use imaging as a tool. The importance of fluorescent probes in light microscopy can further be measured by the award of the Nobel Prize for Chemistry in 2008 to Tsien, Shimomura and Chalfie. The role of probes is as pertinent in the emerging field of correlative microscopy, though arguably far more complex. In correlative microscopy, two or more imaging modalities are applied to a single sample, with the combined images yielding more information than when each modality is used independently. To reflect this, we often use the phrase ‘1+1=3’. The most established correlative microscopy technique is correlative light electron microscopy (CLEM). Usually confocal LM and transmission EM (TEM) are correlated, though recently there have been major developments in both light and electron microscopy leading to other combinations. Correlative light and scanning EM in particular is a growing area due to the availability of new 3D automated microscopes like the serial block face SEM, the focused ion beam SEM, and array tomography [5]. However, whereas fluorescent molecules (either as an expression construct or through antibody or ligand coupling) are the most commonly used to detect a protein of interest in LM, this fluorescence is not directly visible in the electron microscope. Here, an electron-dense moiety such as a gold particle is generally required. Thus, the most direct way to produce a marker/probe (these terms are used interchangeably in the field and in this issue) is to couple both a fluorescent molecule and an electron-dense particle to the protein of interest. Unfortunately though, it is not that simple, and in most instances, the direct coupling of, e.g. a gold particle next to a fluorescent molecule decreases or completely masks the fluorescence emission (see, e.g. [2]). Hence, there have been major efforts to create the ‘optimal’ CLEM probe, one that is visible directly in the LM and EM and can be easily tagged to a protein of interest. Most likely there will never be a single optimal CLEM probe that can be used for all applications, and one has to carefully analyse which probe is best suited for each application. This special issue deals with these latter two cases: the development of new probes and the application of a probe to a specific biological question. The issue starts with a paper by the group of John Robinson discussing possibly the oldest CLEM probe—fluoronanogold. Developed at the beginning of the 1990s, it combines a fluorescent moiety with a very small nanogold particle. As the quenching of fluorescence is strongly influenced by the size (and distance) of the gold particle to the fluorophore [2], this probe does not seem to suffer serious quenching. A disadvantage is that the 1.4-nm gold is too small to be directly visible in the EM against the background contrast of cells or tissues. Hence, silver enhancement has been a necessity. Originally used on Tokuyasu sections, the application of fluoronanogold in pre-embedment * Paul Verkade bixpv@bristol.ac.uk

In vitro digestion and physicochemical properties of wheat starch/flour modified by heat-moisture treatment

Physicochemical and digestive properties between wheat starch (WS) and wheat flour (WF) which had been modified by using heat-moisture treatment (HMT) were investigated. Reduced peak viscosity and increased pasting temperature were found in both WS and WF after HMT. Samples treated with a higher moisture treatment (25% and 35%) exhibited biphasic endotherms. HMT significantly changed crystal structure of WS and WF, and patterns transferred from A to A + V with moisture content increase indicating a formation of starch–lipid complex. Besides, HMT caused the clumping of starch granules and the aggregation of denatured protein, observed using confocal laser scanning microscopy (CLSM) and light microscopy. The higher moisture contents produced HMT samples with higher resistant starch content. In addition, HMT wheat flour showed a higher resistant and slowly digestible starch content when compared to the other counterpart of WS.