Long-Term Intracellular Tracking of Label-Free Nanoparticles in Live Cells and Tissues with Confocal Microscopy.

The label-free imaging of inorganic nanoparticles (NPs) using confocal laser scanning microscopy (CLSM) provides a powerful and versatile tool for studying interactions between NPs and biological systems. Without the need for exogenous labels or markers, it simply benefits from the differential scattering of visible photons between biomaterials and inorganic NPs. Validation experiments conducted on fixed and living cells in real-time, as well as mouse tissue sections following parenteral administration of NPs. Additionally, by incorporating reporter fluorophores and utilizing both reflectance and fluorescence imaging modalities, the method enables high-resolution multiplex imaging of cellular structures and NPs. Different sizes and concentrations of Au NPs are tested as for Ag, Fe3O4, and CeO2 NPs, all with biological interest. Overall, the comprehensive study of NP imaging by confocal microscopy in reflectance mode provides valuable insights and tools for researchers interested in monitoring the nano-bio interactions.

A role of methionines in the functioning of oxidatively modified fibrinogen.

Posttranslational modifications in fibrinogen resulting from induced oxidation or oxidative stress in the organism can have deleterious influence on optimal functioning of fibrinogen, causing a disturbance in assembly and properties of fibrin. The protective mechanism supporting the ability of fibrinogen to function in ROS-generating environment remains completely unexplored. The effects of very low and moderately low HOCl/-OCl concentrations on fibrinogen oxidative modifications, the fibrin network structure as well as the kinetics of both fibrinogen-to-fibrin conversion and fibrin hydrolysis have been explored in the current study. As opposed to 25 Μm, HOCl/-OCl, 10 µM HOCl/-OCl did not affect the functional activity of fibrinogen. It is shown for the first time that a number of Met residues, AαMet476, AαMet517, AαMet584, BßMet367, γMet264, and γMet94, identified in 10 µM HOCl/-OCl fibrinogen by the HPLC-MS/MS method, operate as ROS scavengers, performing an important antioxidant function. In turn, this indicates that the fibrinogen structure is adapted to the detrimental action of ROS. The results obtained in our study provide evidence for a protective mechanism responsible for maintaining the structure and functioning of fibrinogen molecules in the bloodstream under conditions of mild and moderate oxidative stress.

Microstructural studies of imitation cheese with a shift in continuous phase.

When casein is replaced with starch in imitation cheese, the functionality changes. Three different microscopy methods were applied to understand the microstructural differences in the product depending on which component dominates the microstructure. Confocal Laser Scanning Microscopy (CLSM) for component identification. Scanning Electron Microscopy (SEM) and Cryogenic Scanning Electron Microscopy (Cryo-SEM) for studying surface structures. Differences in the surface structures were detected between SEM and Cryo-SEM. In SEM, starch appeared rough and protein smooth, while in Cryo-SEM no starch roughness of the surface was found. A change in starch modification and effects of protein prehydration was also analysed. Adding octenyl succinic anhydride (OSA) modified starch for emulsifying properties resulted in a microstructure with fragmented protein at a protein level of 7 %, but not at 9 or 12 %. Protein prehydration had limited effect on microstructure. On a macrostructural level, the change to an emulsifying starch increased hardness in imitation cheese with 7 and 9 % protein. Protein prehydration slightly decreased the hardness, but the difference was not significant at all concentrations. This research provides valuable information about the microstructure of imitation cheese at a 50/50 composition, how the microstructure changes with an emulsifying starch and what occurs after a protein prehydration was included in the production.

The Impact of Candida albicans in the Development, Kinetics, Structure, and Cell Viability of Biofilms on Implant Surfaces-An In Vitro Study with a Validated Multispecies Biofilm Model.

This study aimed to evaluate the impact of Candida albicans on subgingival biofilm formation on dental implant surfaces. Scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) were used to compare biofilm structure and microbial biomass in the presence and absence of the fungus after periods of 24, 48, and 72 h. Quantitative polymerase chain reaction (qPCR) was used to quantify the number of viable and total micro-organisms for each of the biofilm-forming strains. A general linear model was applied to compare CLSM and qPCR results between the control and test conditions. The biofilm developed with C. albicans at 72 h had a higher bacterial biomass and a significantly higher cell viability (p < 0.05). After both 48 and 72 h of incubation, in the presence of C. albicans, there was a significant increase in counts of Fusobacterium nucleatum and Porphyromonas gingivalis and in the cell viability of Streptococcus oralis, Aggregatibacter actinomycetemcomitans, F. nucleatum, and P. gingivalis. Using a dynamic in vitro multispecies biofilm model, C. albicans exacerbated the development of the biofilm grown on dental implant surfaces, significantly increasing the number and cell viability of periodontal bacteria.

The role of air relative humidity on the wettability of Pseudomonas fluorescens AR11 biofilms.

Biofilms are complex porous materials formed by microorganisms, polysaccharides, proteins, eDNA, inorganic matter, and water. They are ubiquitous in various environmental niches and are known to grow at solid-liquid, solid-air and air-liquid interfaces, often causing problems in several industrial and sanitary fields. Their removal is a challenge in many applications and numerous studies have been conducted to identify promising chemical species as cleaning agents. While these substances target specific components of biofilm structure, the role of water content in biofilm, and how it can influence wettability and detergent absorption have been quite neglected in the literature. Estimating water content in biofilm is a challenging task due to its heterogeneity in morphology and chemical composition. In this study, we controlled water content in Pseudomonas fluorescens AR 11 biofilms grown on submerged glass slides by regulating environmental relative humidity after drying. Interfacial properties of biofilm were investigated by measuring wetting of water and soybean oil. The morphology of biofilm structure was evaluated using Confocal Laser Scanning Microscopy and Scanning Electron Microscopy. The results showed that biofilm water content has a significant and measurable effect on its wettability, leading to the hypothesis that a preliminary control of water content can play a crucial role in biofilm removal process.

Biofilm formation of the food spoiler Brochothrix thermosphacta on different industrial surface materials using a biofilm reactor.

Brochothrix thermosphacta is considered as a major food spoiler bacteria. This study evaluates biofilm formation by B. thermosphacta CD337(2) - a strong biofilm producer strain - on three food industry materials (polycarbonate (PC), polystyrene (PS), and stainless steel (SS)). Biofilms were continuously grown under flow at 25 °C in BHI broth in a modified CDC biofilm reactor. Bacterial cells were enumerated by plate counting, and biofilm spatial organization was deciphered by combining confocal laser scanning microscopy and image analysis. The biofilms had the same growth kinetics on all three materials and reach 8log CFU/cm2 as maximal concentration. Highly structured biofilms were observed on PC and PS, but less structured ones on SS. This difference was confirmed by structural quantification analysis using the image analysis software tool BiofilmQ. Biofilm on SS show less roughness, density, thickness and volume. The biofilm 3D structure seemed to be related to the coupon topography and roughness. The materials used in this study do not affect biofilm growth. However, their roughness and topography affect the biofilm architecture, which could influence biofilm behaviour.

Interactions and effects of a stannous-containing sodium fluoride dentifrice on oral pathogens and the oral microbiome.

Numerous studies have investigated the effects of stannous ions on specific microbes and their efficacy in reducing dental plaque. Nonetheless, our understanding of their impact on the oral microbiome is still a subject of ongoing exploration. Therefore, this study sought to evaluate the effects of a stannous-containing sodium fluoride dentifrice in comparison to a zinc-containing sodium fluoride dentifrice and a control group on intact, healthy oral biofilms. Utilizing the novel 2bRAD-M approach for species-resolved metagenomics, and FISH/CLSM with probes targeting periodontal and caries associated species alongside Sn2+ and Zn2+ ions, we collected and analyzed in situ biofilms from 15 generally healthy individuals with measurable dental plaque and treated the biofilms with dentifrices to elucidate variations in microbial distribution. Although significant shifts in the microbiome upon treatment were not observed, the use of a stannous-containing sodium fluoride dentifrice primarily led to an increase in health-associated commensal species and decrease in pathogenic species. Notably, FISH/CLSM analysis highlighted a marked reduction in representative species associated with periodontitis and caries following treatment with the use of a stannous-containing sodium fluoride dentifrice, as opposed to a zinc-containing sodium fluoride dentifrice and the control group. Additionally, Sn2+ specific intracellular imaging reflected the colocalization of Sn2+ ions with P. gingivalis but not with other species. In contrast, Zn2+ ions exhibited non-specific binding, thus suggesting that Sn2+ could exhibit selective binding toward pathogenic species. Altogether, our results demonstrate that stannous ions could help to maintain a healthy oral microbiome by preferentially targeting certain pathogenic bacteria to reverse dysbiosis and underscores the importance of the continual usage of such products as a preventive measure for oral diseases and the maintenance of health.

Examination of Subbasal Nerve Plexus and Central Corneal Stromal Microstructure in Subjects With Congenital Aniridia, Using in Vivo Confocal Laser Scanning Microscopy.

PURPOSE: During life up to 70% of aniridia subjects develop aniridia-associated keratopathy (AAK). AAK is characterized by limbal stem cell insufficiency, impaired corneal epithelial cell differentiation and abnormal cell adhesion, which leads to centripetal spreading vascularization, conjunctivalization, and thickening of the cornea. Our aim was to examine the subbasal nerve plexus and central corneal stromal microstructure in subjects with congenital aniridia, using in vivo confocal laser scanning microscopy CLSM. METHODS: 31 eyes of 18 patients (55.6% males, mean age: 25.22 ± 16.35 years) with congenital aniridia and 46 eyes of 29 healthy subjects (41.4% males, mean age 30 ± 14.82 years) were examined using the Rostock Cornea Module of Heidelberg Retina Tomograph-III. At the subbasal nerve plexus, corneal nerve fiber density (CNFD), corneal nerve fiber length (CNFL), corneal total branch density (CTBD), and corneal nerve fiber width (CNFW) were analyzed using ACCMetrics software. Keratocyte density in the anterior, middle and posterior stroma was assessed manually. RESULTS: The CNFD (2.02 ± 4.08 vs 13.99 ± 6.34/mm2), CNFL (5.78 ± 2.68 vs 10.56 ± 2.82 mm/mm2) and CTBD (15.08 ± 15.62 vs 27.44 ± 15.05/mm2) were significantly lower in congenital aniridia subjects than in controls (p < 0.001 for all). CNFW was significantly higher in aniridia subjects than in controls (0.03 ± 0.004 vs 0.02 ± 0.003 mm/mm2) (p = 0.003). Keratocyte density was significantly lower in all stromal layers of aniridia subjects than in controls (p < 0.001 for all). Stromal alterations included confluent keratocytes, keratocytes with long extensions and hyperreflective dots between keratocytes in aniridia. CONCLUSIONS: Decrease in CNFD, CNFL, and CTBD, as well as increase in CNFW well refer to the congenital aniridia-associated neuropathy. The decreased keratocyte density and the stromal alterations may be related to an increased cell death in congenital aniridia, nevertheless, stromal changes in different stages of AAK have to be further analyzed in detail.

A Comparative Study of New Fluorescent Anthraquinone and Benzanthrone α-Aminophosphonates: Synthesis, Spectroscopy, Toxicology, X-ray Crystallography, and Microscopy of Opisthorchis felineus.

In this research, we explore the synthesis of and characterize α-aminophosphonates derived from anthraquinone and benzanthrone, focusing on their fluorescence properties and potential applications in confocal laser scanning microscopy (CLSM). The synthesized compounds exhibit notable solvatochromic behavior, emitting fluorescence from green to red across various solvents. Spectroscopic analysis, including 1H-, 13C-, and 31P-NMR, FTIR, and mass spectrometry, confirms the chemical structures. The compounds' toxicity is evaluated using etiolated wheat sprouts, revealing varying degrees of impact on growth and oxidative damage. Furthermore, the study introduces these α-aminophosphonates for CLSM imaging of the parasitic flatworm Opisthorchis felineus, demonstrating their potential in visualizing biological specimens. Additionally, an X-ray crystallographic study of an anthraquinone α-aminophosphonate provides valuable structural insights.

Biofilm volume and acidification within initial biofilms formed in situ on buccally and palatally exposed bracket material.

PURPOSE: Acidification by bacterial biofilms at the bracket/tooth interface is one of the most common problems in fixed orthodontic treatments, which can lead to white spot lesions (WSL) and caries. As lingual brackets were shown to exhibit reduced WSL formation clinically, the aim of this in situ study was to compare initial intraoral biofilm formation and acidification on bracket-like specimens placed buccally and palatally in the upper jaw as a possible cause for this observation. METHODS: Intraoral biofilm was collected from splints equipped with buccally and palatally exposed test specimens, which were worn by 12 volunteers for a total of 48 h. The test specimens consisted of standard bracket material cylinders on top of a hydroxyapatite disc to represent the bracket/tooth interface. They were analyzed for three-dimensional biofilm volume and live/dead distribution by fluorescence staining and confocal laser scanning microscopy as well as for acidification by fluorescence-based pH ratiometry. RESULTS: Similar general biofilm morphology with regard to volume and viability could be detected for buccally and palatally exposed specimens. For pH values, biofilms from both positions showed increased acidification at the bottom layer. Interestingly, the pH value at the top layers of the biofilms was slightly lower on palatally than on buccally exposed specimens, which may likely be due to anatomic conditions. CONCLUSION: Based on the results of this study, initial intraoral biofilm formation and acidification is almost similar on the bracket material/biomimetic tooth interface when placed buccally or palatally in the upper jaw. As lingual brackets were shown to exhibit reduced WSL formation clinically, future studies should investigate further factors like bracket geometry.

In vitro efficacy of Er:YAG laser-activated irrigation versus passive ultrasonic irrigation and sonic-powered irrigation for treating multispecies biofilms in artificial grooves and dentinal tubules: an SEM and CLSM study.

BACKGROUND: Multispecies biofilms located in the anatomical intricacies of the root canal system remain the greatest challenge in root canal disinfection. The efficacy of Er:YAG laser-activated irrigation techniques for treating multispecies biofilms in these hard-to-reach areas has not been proved. The objective of this laboratory study was to evaluate the effectiveness of two Er:YAG laser-activated irrigation techniques, namely, photon-induced photoacoustic streaming (PIPS) and shock wave-enhanced emission photoacoustic streaming (SWEEPS), in treating multispecies biofilms within apical artificial grooves and dentinal tubules, in comparison with conventional needle irrigation (CNI), passive ultrasonic irrigation (PUI), and sonic-powered irrigation (EDDY). Two types of multispecies root canal biofilm models were established in combination with two assessment methods using scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) with the aim to obtain more meaningful results. METHODS: Ninety extracted human single-rooted premolars were chosen for two multispecies biofilm models. Each tooth was longitudinally split into two halves. In the first model, a deep narrow groove was created in the apical segment of the canal wall. After cultivating a mixed bacterial biofilm for 4 weeks, the split halves were reassembled and subjected to five irrigation techniques: CNI, PUI, EDD, PIPS, and SWEEPS. The residual biofilms inside and outside the groove in Model 1 were analyzed using SEM. For Model 2, the specimens were split longitudinally once more to evaluate the percentage of killed bacteria in the dentinal tubules across different canal sections (apical, middle, and coronal thirds) using CLSM. One-way analysis of variance and post hoc multiple comparisons were used to assess the antibiofilm efficacy of the 5 irrigation techniques. RESULTS: Robust biofilm growth was observed in all negative controls after 4 weeks. In Model 1, within each group, significantly fewer bacteria remained outside the groove than inside the groove (P < 0.05). SWEEPS, PIPS and EDDY had significantly greater biofilm removal efficacy than CNI and PUI, both from the outside and inside the groove (P < 0.05). Although SWEEPS was more effective than both PIPS and EDDY at removing biofilms inside the groove (P < 0.05), there were no significant differences among these methods outside the groove (P > 0.05). In Model 2, SWEEPS and EDDY exhibited superior bacterial killing efficacy within the dentinal tubules, followed by PIPS, PUI, and CNI (P < 0.05). CONCLUSION: Er:YAG laser-activated irrigation techniques, along with EDDY, demonstrated significant antibiofilm efficacy in apical artificial grooves and dentinal tubules, areas that are typically challenging to access.

Comparative evaluation of dentinal tubule penetration and push-out bond strength of new injectable hydraulic calcium disilicate based root canal sealer: A single blinded in vitro study.

Objective: To evaluate and compare dentinal tubule penetration and push-out bond strength of BIO-C ION+, AH Plus and NanoSeal-S using Confocal Laser Scanning Microscopy (CLSM) and Universal Testing Machine (UTM). Materials & method: Sixty human mandibular premolars were prepared using ProTaper Gold till F3. Samples were then divided into 3 groups: Group I (n = 20) BIO-C ION + sealer, Group II (n = 20) AH Plus and Group III (n = 20) NanoSeal-S sealer. Groups were then sub-divided into two sub groups: In Subgroup A (n = 10) samples were obturated using single-cone with 0.1 % Rhodamine B dye and in Subgroup B (n = 10) samples were obturated using single cone. The samples were then transversely sectioned into coronal, middle and apical segments, samples in subgroup A & B were then submitted to CLSM analysis and UTM respectively. Results: The Bond Strength data showed following means (MPa): Group I Subgroup B: (BIO-C ION+) coronal (1.64), middle (1.25), apical (0.93); Group II Subgroup B: (AH Plus) coronal (2.20), middle (1.85) apical (1.38) and Group III Subgroup B: (NanoSeal-S) coronal (1.26), middle (0.94), apical (0.58). The dentinal tubule penetration data showed following means: (µm) Group I Subgroup A (BIO-C ION+) coronal (1184.69), middle (997.03), apical (637.26); Group II Subgroup-A AH Plus (864.14) and NanoSeal-S (495.64). Statistical analysis (two-way ANOVA, Tukey's Post Hoc Test) showed significant difference among sealers (p < 0.001) and root canal thirds (p < 0.001). Conclusion: The results of the study concluded that BIO-C ION + sealer showed maximum dentinal tubule penetration and AH Plus demonstrated maximum push-out bond strength.

Novel cytoskeletal traits in the intestinal parasites (Squirmida, Platyproteum vivax) of Pacific peanut worms (Sipuncula, Phascolosoma agassizii).

The cytoskeletal organization of a squirmid, namely Platyproteum vivax, was investigated with confocal laser scanning microscopy (CLSM) to refine inferences about convergent evolution among intestinal parasites of marine invertebrates. Platyproteum inhabits Pacific peanut worms (Phascolosoma agassizii) and has traits that are similar to other lineages of myzozoan parasites, namely gregarine apicomplexans within Selenidium, such as conspicuous feeding stages, called "trophozoites," capable of dynamic undulations. SEM and CLSM of P. vivax revealed an inconspicuous flagellar apparatus and a uniform array of longitudinal microtubules organized in bundles (LMBs). Extreme flattening of the trophozoites and a consistently oblique morphology of the anterior end provided a reliable way to distinguish dorsal and ventral surfaces. CLSM revealed a novel system of microtubules oriented in the flattened dorsoventral plane. Most of these dorsoventral microtubule bundles (DVMBs) had a punctate distribution and were evenly spaced along a curved line spanning the longitudinal axis of the trophozoites. This configuration of microtubules is inferred to function in maintaining the flattened shape of the trophozoites and facilitate dynamic undulations. The novel traits in Platyproteum are consistent with phylogenomic data showing that this lineage is only distantly related to Selenidium and other marine gregarine apicomplexans with dynamic intestinal trophozoites.

Effects of freezing and drying programs on IgY aggregation and activity during microwave freeze-drying: Protective effects and interactions of trehalose and mannitol.

The acquisition of high quality lyophilized IgY products, characterized by an aesthetically pleasing visage, heightened stability, and a marked preservation of activity, constitutes an indispensable pursuit in augmenting the safety and pragmatic utility of IgY. Within this context, an exploration was undertaken to investigate an innovative modality encompassing microwave freeze-drying (MFD) as a preparatory methodology of IgY. Morphological assessments revealed that both cryogenic freezing and subsequent MFD procedures resulted in aggregation of IgY, with the deleterious influence posed by the MFD phase transcending that of the freezing phase. The composite protective agent comprised of trehalose and mannitol engendered a safeguarding effect on the structural integrity of IgY, thereby attenuating reducing aggregation between IgY during the freeze-drying process. Enzyme-linked immunosorbent assay (ELISA) outcomes demonstrated a discernible correlation between IgY aggregation and a notable reduction in its binding affinity towards the pertinent antigen. Comparative analysis vis-à-vis the control sample delineated that when the trehalose-to-mannitol ratio was upheld at 1:3, a two-fold outcome was achieved: a mitigation of the collapse susceptibility within the final product as well as a deterrence of IgY agglomeration, concomitant with an elevated preservation rate of active antibodies (78.57 %).

Visualization of etching cycles efficacy at the resin infiltration into artificial enamel caries: in-vitro study on bovine teeth.

This study evaluated the effect of repeated etching cycles on resin infiltrant penetration. Enamel samples measuring 4 × 4 × 3 mm3 were obtained from the facial aspect of 50 extracted bovine teeth. Samples were immersed in a demineralization solution for 21 days to create artificial lesions and divided into five equal groups (n = 10). A 15% hydrochloric acid gel was administered to each group. The acid etching application time differed between groups: Group 1; 2 min, Group 2; 2 × 2 min, Group 3; 3 × 2 min, Group 4; 4 × 2 min, and Group 5; 5 × 2 min. Resin infiltration was visualized using a confocal laser scanning microscopy. The lesion, penetration and erosion depth (µm) were calculated, and data were statistically analyzed. The highest penetration depth (75.59 ± 9.42 µm) was seen in Group 5, followed by Groups 4, 3, 2 and 1. However, there were no statistically significant differences in the penetration depths between Groups 4 and 5 and between Groups 2, 3 and 4 (p > 0.05). In conclusion, a repeated etching cycle enhanced resin infiltrant penetration.

A tool for live-cell confocal imaging of temperature-dependent organelle dynamics.

Intracellular organelles alter their morphology in response to ambient conditions such as temperature to optimize physiological activities in cells. Observing organelle dynamics at various temperatures deepens our understanding of cellular responses to the environment. Confocal laser microscopy is a powerful tool for live-cell imaging of fluorescently labeled organelles. However, the large contact area between the specimen and the ambient air on the microscope stage makes it difficult to maintain accurate cellular temperatures. Here, we present a method for precisely controlling cellular temperatures using a custom-made adaptor that can be installed on a commercially available temperature-controlled microscope stage. Using this adaptor, we observed temperature-dependent organelle dynamics in living plant cells; morphological changes in chloroplasts and peroxisomes were temperature dependent. This newly developed adaptor can easily be placed on a temperature-controlled stage to capture intracellular responses to temperature at unprecedentedly high resolution.

Comparison of Easydo Activator, ultrasonic and needle irrigation techniques on sealer penetration and smear layer removal in vitro.

The effects of Easydo Activator (EA), a new sonic irrigation system, on sealer penetration at the root apex were compared to needle irrigation (NI) and passive ultrasonic irrigation (PUI) in this study. Forty-two single-rooted teeth were prepared and randomly divided into three groups (n = 14): group 1: NI; group 2: PUI; and group 3: EA. A solution of 3% sodium hypochlorite (NaOCl) was used for irrigation. Nine teeth in each group were filled with AH Plus sealer mixed with CY5 fluorescent dye and a single gutta-percha cone. The sealer penetration area, maximum penetration depth and percentage of sealer penetration at 5 mm and 1 mm from the apex were analyzed by confocal laser scanning microscopy (CLSM). The remaining 5 teeth in each group were subjected to test smear layer scores by scanning electron microscopy (SEM). The CLSM evaluation showed that increases in the area, depth and percentage of sealer penetration were detected at 1 and 5 mm from the root apex in the PUI group compared with the NI group, and greater increases were observed in the EA group (P < 0.05). The SEM experiment showed that the lowest scores for the smear layer and debris removal were achieved by the EA group when compared with the PUI and NI groups (P < 0.05). In conclusion, EA was superior to PUI and NI regarding sealer penetration at the root apex during endodontic treatment, and it could provide a new technical idea for clinical root canal therapy.

Epimorphic development in tropical shallow-water Nymphonidae (Arthropoda: Pycnogonida) revealed by fluorescence imaging.

BACKGROUND: Extant lineages of sea spiders (Pycnogonida) exhibit different types of development. Most commonly, pycnogonids hatch as a minute, feeding protonymphon larva with subsequent anamorphic development. However, especially in cold water habitats at higher latitudes and in the deep sea, some taxa have large, lecithotrophic larvae, or even undergo extended embryonic development with significantly advanced postlarval hatching stages. Similar biogeographic trends are observed in other marine invertebrates, often referred to as "Thorson's rule". RESULTS: To expand our knowledge on the developmental diversity in the most speciose pycnogonid genus Nymphon, we studied the developmental stages of the two tropical representatives N. floridanum and N. micronesicum., We compared classical scanning electron microscopy with fluorescence-based approaches to determine which imaging strategy is better suited for the ethanol-fixed material available. Both species show epimorphic development and hatch as an advanced, lecithotrophic postlarval instar possessing the anlagen of all body segments. Leg pairs 1-3 show a considerable degree of differentiation at hatching, but their proximal regions remain coiled and hidden under the cuticle of the hatching instar. The adult palp and oviger are not anteceded by three-articled larval limbs, but differentiate directly from non-articulated limb buds during postembryonic development. CONCLUSIONS: Fluorescence imaging yielded more reliable morphological data than classical scanning electron microscopy, being the method of choice for maximal information gain from rare and fragile sea spider samples fixed in high-percentage ethanol. The discovery of epimorphic development with lecithotrophic postlarval instars in two small Nymphon species from tropical shallow-water habitats challenges the notion that this developmental pathway represents an exclusive cold-water adaptation in Nymphonidae. Instead, close phylogenetic affinities to the likewise more direct-developing Callipallenidae hint at a common evolutionary origin of this trait in the clade Nymphonoidea (Callipallenidae + Nymphonidae). The lack of functional palpal and ovigeral larval limbs in callipallenids and postlarval hatchers among nymphonids may be a derived character of Nymphonoidea. To further test this hypothesis, a stable and well-resolved phylogenetic backbone for Nymphonoidea is key.

Does Systemic Hematological Therapy Influence the Course of Paraproteinemic Keratopathy?

The purpose of this article is to evaluate the course of paraproteinemic keratopathy (PPK) in patients undergoing systemic therapy for the underlying hematological disease. Baseline and follow-up examinations included hematological work-up, best-corrected visual acuity, slit-lamp biomicroscopy, and in vivo confocal laser scanning microscopy (IVCM). We included 22 patients with bilateral PPK (aged 68 ± 10.4 years, 11 males). Ten patients with multiple myeloma (MM) underwent on-label systemic therapy. During follow-up, we observed a regression of corneal opacities in three patients under slit-lamp examination and under IVCM, while PPK remained unchanged in seven patients. In three patients with monoclonal gammopathy of ocular significance (MGOS), systemic therapy was initiated off-label to reduce the serum paraprotein load before penetrating keratoplasty (PKP). These patients showed no signs of PPK recurrence for up to 24 months after PKP. In one patient without systemic therapy, a recurrence in corneal grafts occurred within 12 months of PKP. In eight patients without systemic therapy, PPK remained stable. In conclusion, systemic therapy for MM patients reduced corneal opacity in 30% of treated patients. Furthermore, systemic therapy performed before PKP in patients without conventional systemic therapy indication (MGOS) likely postpones PPK recurrence in the corneal graft.

Microfluidic reactor designed for time-lapsed imaging of pretreatment and enzymatic hydrolysis of lignocellulosic biomass.

The effect of tissue-specific biochemical heterogeneities of lignocellulosic biomass on biomass deconstruction is best understood through confocal laser scanning microscopy (CLSM) combined with immunohistochemistry. However, this process can be challenging, given the fragility of plant materials, and is generally not able to observe changes in the same section of biomass during both pretreatment and enzymatic hydrolysis. To overcome this challenge, a custom polydimethylsiloxane (PDMS) microfluidic imaging reactor was constructed using standard photolithographic techniques. As proof of concept, CLSM was performed on 60 µm-thick corn stem sections during pretreatment and enzymatic hydrolysis using the imaging reactor. Based on the fluorescence images, the less lignified parenchyma cell walls were more susceptible to pretreatment than the lignin-rich vascular bundles. During enzymatic hydrolysis, the highly lignified protoxylem cell wall was the most resistant, remaining unhydrolyzed even after 48 h. Therefore, imaging thin whole biomass sections was useful to obtain tissue-specific changes during biomass deconstruction.