Confocal Laser Scanning Fluorescence Microscopy Research Articles

Fiber optic-based integrated system for in vivo multiscale pharmacokinetic monitoring

This paper presents the development of a fiber-optic-based fluorescence detection system for multi-scale monitoring of drug distribution in living animals. The integrated system utilized dual laser sources at the wavelengths of 488 nm and 650 nm and three photomultiplier channels for multi-color fluorescence detection. The emission spectra of fluorescent substances were tracked using the time-resolved fluorescence spectroscopy module to continuously monitor their blood kinetics. The fiber bundle, consisting of 30,000 optic filaments, was designed for wide-field mesoscopic imaging of the drug’s interactions within organs. The inclusion of a gradient refractive index (GRIN) lens within the setup enabled fluorescence confocal laser scanning microscopy to visualize the drug distribution at the cellular level. The system performance was verified by imaging hepatic and renal tissues in mice using cadmium telluride quantum dots (CdTe QDs) and R3. By acquiring multi-level images and real-time data, our integrated system underscores its potential as a potent tool for drug assessment, specifically within the realms of pharmacokinetic and pharmacodynamic investigations.

Biological Efficacy of Ionizing Radiation Sources on 3D Organotypic Tissue Slices Assessed by Fluorescence Microscopy.

Traditional cell-based radiobiological methods are inadequate for assessing the toxicity of ionizing radiation exposure in relation to the microstructure of the extracellular matrix. Organotypic tissue slices preserve the spatial organization observed in vivo, making the tissue easily accessible for visualization and staining. This study aims to explore the use of fluorescence microscopy of physiologically compatible 3D tissue cultures to assess the effects of ionizing radiation. Organotypic tissue slices were obtained by vibratome, and their mechanical properties were studied. Slices were exposed by two ionizing radiation sources; electron beams (80 Gy and 4 Gy), and soft gamma irradiation (80 Gy and 4 Gy). Two tissue culture protocols were used: the standard (37°C), and hypothermic (30°C) conditions. A qualitative analysis of cell viability in organotypic tissue slices was performed using fluorescent dyes and standard laser confocal microscopy. Biological dosimetry is represented by differentially stained 200-µm thick organotypic tissue sections related to living and dead cells and cell metabolic activity. Our results underscore the ability of fluorescence laser scanning confocal microscopy to rapidly assess the radiobiological effects of ionizing radiation in vitro on 3D organotypic tissue slices.

Modulated illumination microscopy: Application perspectives in nuclear nanostructure analysis.

The structure of the cell nucleus of higher organisms has become a major topic of advanced light microscopy. So far, a variety of methods have been applied, including confocal laser scanning fluorescence microscopy, 4Pi, STED and localisation microscopy approaches, as well as different types of patterned illumination microscopy, modulated either laterally (in the object plane) or axially (along the optical axis). Based on our experience, we discuss here some application perspectives of Modulated Illumination Microscopy (MIM) and its combination with single-molecule localisation microscopy (SMLM). For example, spatially modulated illumination microscopy/SMI (illumination modulation along the optical axis) has been used to determine the axial extension (size) of small, optically isolated fluorescent objects between ≤ 200nm and ≥ 40nm diameter with a precision down to the few nm range; it also allows the axial positioning of such structures down to the 1nm scale; combined with laterally structured illumination/SIM, a 3D localisation precision of ≤1nm is expected using fluorescence yields typical for SMLM applications. Together with the nanosizing capability of SMI, this can be used to analyse macromolecular nuclear complexes with a resolution approaching that of cryoelectron microscopy.

Interfacial engineering method to regulate the performances of bilayer emulsions co-stabilized by casein/butyrylated dextrin nanoparticles and chitosan

In present study, bilayer emulsions with different interfacial structures stabilized by casein/butyrylated dextrin nanoparticles (CDNP), chitosan (CS) and chitosan nanoparticles (CSNP) were prepared to overcome the limitations of conventional emulsions. The effects of chitosan morphology and incorporation sequences on the bilayer emulsions were examined. Bilayer emulsions prepared with CDNP as the inner layer and CS/CSNP as the outer layer were observed to have smaller droplet sizes (1.39 ± 86.74 um and 1.45 ± 7.87 um). Bilayer emulsions prepared with CDNP as the inner layer and CS as the outer layer exhibited the lowest creaming index (2.38 %) after 14 days of storage, indicating excellent stability. Furthermore, bilayer emulsion prepared with CDNP as the inner layer and CS as the outer layer also exhibited a uniform water distribution, excellent protein oxidative stability, and uniformly distributed droplets by the measurement of Low-field NMR, intrinsic tryptophan fluorescence and laser confocal laser scanning microscopy. These results indicated that the study provided a theoretical basis for the development and design of bilayer emulsions with different interfacial structures. This study also provides a new material for the preparation of delivery systems that protect biologically active compounds. Bilayer emulsions are promising for applications in traditional and manufactured food products.

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

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 apossible cause for this observation. Intraoral biofilm was collected from splints equipped with buccally and palatally exposed test specimens, which were worn by 12volunteers for atotal of 48 h. The test specimens consisted of standard bracket material cylinders on top of ahydroxyapatite 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. 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. 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.

Antibiofilm Properties and Demineralization Suppression in Early Enamel Lesions Using Dental Coating Materials.

This study aimed to investigate the effects of dental coating materials on Streptococcus mutans biofilm formation. The test materials were PRG Barrier Coat (PRG), BioCoat Ca (BioC), and FluorDental Jelly (FluorJ). Bovine enamel specimens were demineralized to mimic early enamel lesions. The biofilm was developed on a specimen treated with one of the materials by using a modified Robbins device flow-cell system. Scanning electron and fluorescence confocal laser scanning microscopy, viable and total cell counts, and gene expression assessments of the antibiofilm were performed. Ion incorporation was analyzed using a wavelength-dispersive X-ray spectroscopy electron probe microanalyzer. All materials allowed biofilm formation but reduced its volume. FluorJ was the only material that inhibited biofilm accumulation and had a bactericidal effect, revealing 0.66 log CFU in viable cells and 1.23 log copy reduction in total cells compared with the untreated group after 24 h of incubation. The ions released from PRG varied depending on the element. BioC contributed to enamel remineralization by supplying calcium ions while blocking the acid produced from the biofilm. In summary, the dental coating materials physically prevented acid attacks from the biofilm while providing ions to the enamel to improve its mechanical properties.

Biodegradable Nanocomposite of ZnS(Mn) Quantum Dots Immobilized Graphene Oxide for Bioimaging Applications.

Developing a biocompatible and biodegradable graphene-based fluorescent nanoprobe with the ability to visualize live cells could be interesting for intracellular imaging and monitoring the efficiency of chemotherapy. Herein, we report a biodegradable and biocompatible hybrid fluorescent graphene oxide (GO)-ZnS(Mn) composite synthesized via in situ growth of ZnS(Mn) quantum dots (QDs) on the surface of GO in the aqueous medium. The prepared 'GO-ZnS(Mn)' composite was characterized by X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), thermogravimetric analysis (TGA) and high-resolution transmission electron microscopy (HR-TEM) along with selected area electron diffraction (SAED). Further, the fluorescence properties of the GO-ZnS(Mn) composite were studied using fluorescence emission spectroscopy. The composite material exhibited a strong and broad visible light fluorescence from 500 to 600 nm by excitation with 365 nm (UV) light. The cytotoxic experiments of folic acid (FA) conjugated GO-ZnS(Mn) using MTT [(3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide)] assay revealed that the composite had excellent biocompatibility even at higher concentrations up to 200 µg/mL in HeLa cell lines. Next, the bioimaging experiments carried out using confocal fluorescence laser scanning microscopy (CLSM) revealed that GO-ZnS(Mn) composite was taken up by the HeLa cells effectively within 12 h of incubation via receptor (folate) mediated endocytosis with strong fluorescence throughout the cell surface. Finally, the biodegradability of GO-ZnS(Mn) composite was studied by treating it with human myeloperoxidase enzyme (hMPO) isolated from the primary immune cells, neutrophils, which is important to understand the in vivo fate of GO-Zns(Mn). The HR-TEM and Raman analyses confirmed the biodegradation of GO-ZnS(Mn) within 15 h of hMPO treatment. Thus, the biodegradable GO-ZnS (Mn) composite could be helpful for chemotherapy and bioimaging applications.

Spatially resolved in situ analyses of the adsorption behavior of different admixtures on binders

AbstractAdmixtures play a key role in improving the performance of the cement paste matrix. A more efficient matrix can allow a reduction the binder content while maintaining the same properties. There is currently a lack of knowledge about the spatial distribution of admixtures in situ on binder surfaces which would facilitate the use of admixtures in general in inorganic binder systems. Imaging techniques such as confocal laser scanning microscopy (CLSM) and fluorescence microscopy can be used to study the spatiotemporal adsorption of superplasticizers and plasticizers on binder particles. However, in order to make the admixtures visible using these methods, they must first be coupled to a fluorescent molecule (staining). Different types of superplasticizers and plasticizers, including polycarboxylate ethers (PCE) and lignosulfonate (LS) were stained and tested. It was shown that not only adsorption but also desorption can be observed and that desorption cannot play a relevant role in a binder system. The absence of desorption could be used to observe a real system in permanent preparations, so that the adsorption on individual cement grains could be followed in a time‐dependent manner. The newly developed method provides unprecedented insight into the adsorption of additives.

Physiological Concentrations of Calcium Interact with Alginate and Extracellular DNA in the Matrices of Pseudomonas aeruginosa Biofilms to Impede Phagocytosis by Neutrophils.

Biofilms are communities of interacting microbes embedded in a matrix of polymer, protein, and other materials. Biofilms develop distinct mechanical characteristics that depend on their predominant matrix components. These matrix components may be produced by microbes themselves or, for infections in vivo, incorporated from the host environment. Pseudomonas aeruginosa (P. aeruginosa) is a human pathogen that forms robust biofilms that extensively tolerate antibiotics and effectively evade clearance by the immune system. Two of the important bacterial-produced polymers in the matrices of P. aeruginosa biofilms are alginate and extracellular DNA (eDNA), both of which are anionic and therefore have the potential to interact electrostatically with cations. Many physiological sites of infection contain significant concentrations of the calcium ion (Ca2+). In this study, we investigate the structural and mechanical impacts of Ca2+ supplementation in alginate-dominated biofilms grown in vitro, and we evaluate the impact of targeted enzyme treatments on clearance by immune cells. We use multiple-particle tracking microrheology to evaluate the changes in biofilm viscoelasticity caused by treatment with alginate lyase or DNase I. For biofilms grown without Ca2+, we correlate a decrease in relative elasticity with increased phagocytic success. However, we find that growth with Ca2+ supplementation disrupts this correlation except in the case where both enzymes are applied. This suggests that the calcium cation may be impacting the microstructure of the biofilm in nontrivial ways. Indeed, confocal laser scanning fluorescence microscopy and scanning electron microscopy reveal unique Ca2+-dependent eDNA and alginate microstructures. Our results suggest that the presence of Ca2+ drives the formation of structurally and compositionally discrete microdomains within the biofilm through electrostatic interactions with the anionic matrix components eDNA and alginate. Further, we observe that these structures serve a protective function as the dissolution of both components is required to render biofilm bacteria vulnerable to phagocytosis by neutrophils.

On-line heating rolling behavior of Mg9999 sheets under large single pass reduction

Pure magnesium foil has a great potential for rechargeable magnesium battery anode application, the formation of edge crack and break during Mg sheet rolling highly decreases the yield and thus increase the cost. In this paper, pure magnesium sheets of different thicknesses (0.3 mm, 0.4 mm, 0.5 mm and 0.6 mm) were prepared from 1 mm pure magnesium sheets by on-line heating rolling in a single pass. The edge cracking and breaking behavior of pure magnesium sheets were characterized by scanning electron microscope (SEM), X-ray diffractometer (XRD), electron backscatter diffractometer (EBSD), energy dispersive spectroscopy (EDS) and laser scanning confocal fluorescence microscope (LSCM). The results show that the number of edge cracks of pure magnesium sheets increases with the increase of rolling reduction. As the rolling reduction increases to 70%, due to the severe edge crack of 0.3 mm thick sheet and the formation of grooves on the magnesium sheet surface, the breaking of pure magnesium sheets occurs. The mechanism of edge cracking and breaking of ultra-thin pure magnesium rolled sheet is discussed.

Influence of Pectinase Adsorption on Scouring Efficiency of Mercerized Cotton Yarns

The low scouring efficiency of mercerized cotton yarns scoured with alkaline and acid pectinases, reported in previously published articles, raised the hypothesis of the lack of pectinase adsorption onto mercerized cotton. In an attempt to identify whether low pectinase adsorption occurs, the adsorption of alkaline and acid pectinases on raw and mercerized cotton yarns is measured using the Lowry protein assay, while laser-scanning confocal fluorescence microscopy is used to visually show the presence/absence of the enzymes on the surface of the cotton yarns. In addition, the surface zeta potential of the raw and mercerized cotton yarns is measured to identify the influence of the surface charge on the adsorption ability of the used enzymes. Data suggest that although used substrates have adsorbed more alkaline than acid pectinase, the adsorption of both enzymes is more intensive onto the mercerized cotton relative to raw cotton. The similar surface zeta potential of the raw and mercerized yarns indicates that higher enzyme adsorption on mercerized cotton is not influenced by the surface charge. Laser-scanning confocal fluorescence microscopy micrographs depict an evident increase of fluorescing signal with scouring time to raw and mercerized cotton yarn. The more intensive signals after 60 min of enzymatic scouring with both enzymes suggest enzyme presence on the yarn surface after a long treatment time. The obtained results confirm the enzyme retention on the yarn surface, thus excluding the hypothesis that the absence of enzyme adsorption is a factor for the low scouring efficiency of mercerized cotton yarns.

Consuming intracellular glucose and regulating the levels of O2/H2O2 via the closed cascade catalysis system of Cu-CeO2 nanozyme and glucose oxidase

Imbalances in the intracellular environment caused by high levels of glucose, H2O2, and hypoxia can greatly impact cancer development and treatment. However, there is limited research on regulating the levels of these species simultaneously in tumor cells. Here, a pH-responsive nanozyme-enzyme hybrid system was developed to regulate intracellular glucose, H2O2 and O2. The system, named DMSN@Cu-CeO2@GOx, consists of Cu-CeO2 nanoparticles and glucose oxidase (GOx) immobilized in dendritic mesoporous silica (DMSN) spheres. GOx efficiently consumes glucose in tumor cells, causing a drop in pH and producing a significant amount of H2O2. Cu-CeO2 then catalyzes the conversion of H2O2 to O2 due to its high catalase-like (CAT) activity in weakly acidic conditions. The process was monitored by fluorescence probes, and the mechanism was investigated through fluorescence spectroscopy and confocal laser scanning microscopy. The cascade catalytic system with excellent biocompatibility continuously consumes glucose and elevates the level of O2 in cells. This hybrid nanomaterial offers a means to regulate the glucose/H2O2/O2 levels in cells and may provide insights into starvation therapy by modulating reactive species within cells.

DS24 Prospective study comparing ex vivo confocal laser scanning microscopy and frozen section histopathology of 23 basal cell carcinomas treated with Mohs micrographic surgery

Abstract Ex vivo confocal laser scanning microscopy (CLSM) is an emerging technique that is used for the rapid analysis of freshly excised tissue during Mohs micrographic surgery (MMS). Previous studies on fluorescence CLSM have shown 88% and 99% sensitivity and specificity, respectively, of diagnosing basal cell carcinomas (BCCs) vs. conventional histology. The fourth-generation Vivascope 2500 allows fresh tissue to be scanned after minimal preparation with acetic acid and acridine orange, creating digitally coloured mosaic images, mimicking conventional histological haematoxylin and eosin slides. The scanning speed of < 3 min creates the potential to improve patient experiences, allowing multiple stages and repair in a single session, reducing overall operating times and improving efficiency. After a 2018 pilot study on a limited case series, we have begun the process of testing the real-world use of this technology. We present an initial prospective case series to assess BCC excision of stage 1 Mohs specimens using the Vivascope 2500 vs. conventional frozen section histology. Caldicot approval was sought. Over a 3-month period, 23 cases of stage 1 facial BCCs on one Mohs list were processed. Samples were obtained as standard bevelled Mohs excisions, and Mohs maps were created for all cases. There were three false-negative BCCs, of which two were infiltrative BCCs with small foci missed on the initial review on CLSM images and one superficial BCC not visible due to lack of epidermal capture. Of the four false-positive cases, the foci appearing to be BCC included inflammation in one, follicular structures in two and conjunctival glands misinterpreted as BCC in one eyelid case. Time taken to process specimens and read the digital images by CLSM was compared with the time taken to obtain and read frozen section slides. The shortest time recorded for CLSM and frozen sections was 6 min and 31 min, respectively. The longest time recorded for CLSM and frozen sections was 20 min and 57 min, respectively. In summary, CLSM shows promise in MMS for selected BCCs. Improved techniques to flatten the epidermis for better visualization and further experience with image reading and interpretation have the potential to minimize errors.

Optimized silk fibroin nanoparticle functionalization with anti-CEA nanobody enhancing active targeting of colorectal cancer cells

This work aimed to establish a simple and feasible method to obtain silk fibroin nanoparticles (SFNPs) with uniform particles size, and then modify the SFNPs with nanobody (Nb) 11C12 targeting the proximal membrane end of carcinoembryonic antigen on the surface of colorectal cancer (CRC) cells. The regenerated silk fibroin (SF) was isolated using ultrafiltration tubes with a 50 kDa molecular weight cut-off, and the retention fraction (named as SF > 50 kDa) was further self-assembled into SFNPs by ethanol induction. Scanning electron microscope (SEM) and high-resolution transmission electron microscop showed that the SFNPs with uniform particles size were formed. Due to electrostatic adsorption and pH responsiveness, SFNPs have been proved to effectively load and release the anticancer drug doxorubicin hydrochloride (DOX) (DOX@SFNPs). Further, targeting molecule Nb 11C12 was used to modify these nanoparticles, constituting the targeted outer layer of the drug delivery system (DOX@SFNPs-11C12), achieving precise localization to cancer cells. The release amount of DOX observed from in vitro drug release profiles increased as follows: pH 7.4 < pH 6.8 < pH 5.4, demonstrating that the DOX release could be accelerated in a weakly acidic environment. In vitro cytotoxicity experiments displayed that SFNPs-11C12 nanoparticles exhibited good safety and biocompatibility. Drug-loaded nanoparticles, DOX@SFNPs-11C12, led to higher LoVo cells apoptosis compared to DOX@SFNPs. Fluorescence spectrophotometer characterization and confocal laser scanning microscopy further showed that the internalization of DOX was highest in the DOX@SFNPs-11C12, certifying that the introduced targeting molecule enhanced the uptake of drug delivery system by LoVo cells. This study provides a simple and operational approach to developing an optimized SFNPs drug delivery system modified by targeting Nb, which can be a good candidate for CRC therapy.

Physicochemical Properties and Biocompatibility of Various Bioceramic Root Canal Sealers: In Vitro Study

IntroductionThe aim of this study was to compare the physicochemical properties and biocompatibility of various calcium silicate–based bioceramic sealers (CSBSs). MethodsFour recently developed CSBSs, including AH Plus Bioceramic Sealer (AHB), EndoSequence BC Sealer (ESB), TotalFill BC Sealer (TTB), and Bio-C Sealer (BIC), were compared with the epoxy resin–based sealer AH Plus (AHP). Their physical properties, including flow, setting time, radiopacity, dimensional stability, and pH, were evaluated according to the International Organization for Standardization (ISO) 6876. Their cytotoxicity in human periodontal ligament fibroblast (hPDLF) was assessed through the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and compared. Additionally, cell attachment to the sealer surface was analyzed using green fluorescent protein and confocal laser scanning microscopy to evaluate cell viability. Data were analyzed using a one-way analysis of variance to determine the difference between groups for categorical variables, followed by Tukey's post hoc test at the significance level of 95%. ResultsThe flow, setting time, and radiopacity of all tested CSBSs satisfied the ISO 6876/2012 standards. Further, these CSBSs showed shrinkage after immersion in distilled water for 30 days and complied with the ISO 6876/2001 requirements. The pH values of AHB, ESB, TTB, and BIC were greater than 11, whereas AHP had a pH of 6.69 after 4 weeks. CSBS showed excellent biocompatibility compared with that of AHP (P < .05). Confocal laser scanning microscopy showed that alive hPDLFs were attached well to all the tested CSBSs but not to AHP. ConclusionsCSBSs have similar physical characteristics within the ISO standards and higher biocompatibility than epoxy resin–based sealers.

Key mechanism of enzymatic dehairing technology for leather-making: permeation behaviors of protease into animal hide and the mechanism of charge regulation

Enzymes have been widely used as alternatives to conventional chemicals in cleaner leather processes due to their advantages of meeting increasing environmental demands. Especially, enzymatic unhairing based on protease has been applied to leather-making for a long time, however, it still has the key problem of slow permeation in the animal hide, resulting in slow unhairing rate, poor hair removal effect, excessive proteolysis of hide collagen, and decreased leather quality. Aiming at the key problem of bio-unhairing technology, fluorescent labeling technique and confocal laser scanning microscopy were used to investigate the protease permeation behaviors into the animal hide based on well-prepared labeled proteases, as well as the quantitative analysis of the protease amount in different hide layers. The results show that the protease mainly permeates the bovine hide through transfollicular routes from the hair side, and although the intercellular-interfibrillar pathway also exists, it does not play an important role. Additionally, the protease permeation behaviors into the hide are greatly impacted by the charge states of the hide and protease proteins, depending on the isoelectric points (pI) of the proteins and solution pH values. When the solution pH is not between the pI values of the two proteins, the similarly charged protease can quickly and deeply penetrate the hide because of the electrostatic repulsion. The established mechanism provides a theoretical basis for developing an efficient enzymatic unhairing technology for leather-making, and this can also be applied to other processes involving the enzyme permeation into the hide or leather.Graphical

Cross-Linking of Oxidized Hydroxypropyl Cellulose in Paper: Influence of Molecular Weight and Polymer Distribution on Paper Wet Strength Development.

With the overarching aim for the development of sustainable, nontoxic wet strength agents for paper, a novel polymer gel system based on oxidized hydroxypropyl cellulose (keto-HPC) cross-linked with polyamines was investigated in detail to gain a deeper insight into the wet strength mechanism. When applied to paper, this wet strength system significantly increases the relative wet strength by using only low amounts of polymer, and it is therefore comparable with established wet strength agents based on fossil resources, such as polyamidoamine epichlorohydrin resins. With the help of ultrasonic treatment, keto-HPC was degraded with respect to its molecular weight and further cross-linked in paper using polymeric amine-reactive counterparts. The resulting polymer-cross-linked paper mechanical properties were analyzed with respect to the dry and wet tensile strength, respectively. In addition, we analyzed the polymer distribution using fluorescence confocal laser scanning microscopy (CLSM). If high-molecular-weight samples are being used for cross-linking, we do find accumulation of the polymer mainly on the surface of the fibers and at fiber crossing points, accompanied with enhancing strong effects on paper's wet tensile strength. In contrast, if low-molecular-weight (i.e., degraded) keto-HPC is being applied, the macromolecules are capable of entering the inner porous structure of the paper fibers, and almost no accumulation at the fiber crossing points is observed, which also results in a lowered wet paper tensile strength, respectively. This insight into wet strength mechanisms of the keto-HPC/polyamine system can thus lead to new opportunities for the development of alternative biobased wet strength agents where molecular weight dependence of the wet tensile properties allows for a fine tuning of mechanical properties in the wet state.

Extraction of bioactive polysaccharide from Ulva prolifera biomass waste toward potential biomedical application

Ulva prolifera macroalgae blooming caused by water eutrophication seriously affects the marine ecological environment. Exploring an efficient approach to turning algae biomass waste into high-value-added products is significant. The present work aimed to demonstrate the feasibility of the bioactive polysaccharide extraction from Ulva prolifera and to evaluate its potential biomedical application. A short autoclave process was proposed and optimized using the response surface methodology to extract Ulva polysaccharides (UP) with high molar mass. Our results indicated that UP with high molar mass (9.17 × 105 g/mol) and competitive radical scavenging activity (up to 53.4 %) could be effectively extracted with the assistance of Na2CO3 (1.3 %, wt.) at a solid-liquid ratio of 1/10 in 26 min. The obtained UP mainly composes of galactose (9.4 %), glucose (73.1 %), xylose (9.6 %), and mannose (4.7 %). The biocompatibility of the UP and its potential application as a bioactive ingredient in 3D cell culture has been evaluated and confirmed by confocal laser scanning microscopy and fluorescence microscope imaging inspection. This work demonstrated the feasibility of extracting bioactive sulfated polysaccharides with potential applications in biomedicine from biomass waste. Meanwhile, this work also provided an alternative solution to deal with the environmental challenges incurred by algae blooming worldwide.

Growth phase-dependent production of the adhesive nanofiber protein AtaA in Acinetobacter sp. Tol 5.

AtaA, the sticky, long, and peritrichate nanofiber protein from Acinetobacter sp. Tol 5, mediates autoagglutination and is highly adhesive to various material surfaces, resulting in a biofilm. Although the production of the adhesive nanofiber protein is likely to require a large amount of energy and material sources, the relationship between AtaA fiber production and cell growth remains unknown. Here, we report the growth phase-dependent AtaA fiber production in Tol 5. We examined the ataA gene expression in different growth phases using a reporter gene assay with an originally developed reporter plasmid and using reverse transcription-quantitative polymerase chain reaction. Bacterial cells with surface-displayed AtaA at different growth phases were immunostained and analyzed using fluorescence flow cytometry and confocal laser scanning microscopy. The results indicate that Tol 5 modulated the amount of surface-displayed AtaA at the transcriptional level. AtaA production was low in the early growth phase but remarkably increased in the late growth phase, covering the whole bacterial cell with AtaA fibers in the stationary phase. Tol 5 displayed AtaA fibers poorly in the early growth phase and showed less autoagglutination and adhesiveness than those in the stationary phase. Although Tol 5 grew as fast as its ataA-deficient mutant in the early growth phase, the optical density of Tol 5 culture was slightly lower than that of the ataA-deficient mutant in the late growth phase. Based on these experimental results, we propose the growth-phase-dependent production of AtaA fiber for efficient and fast cell growth.

Artificial confocal microscopy for deep label-free imaging.

Widefield microscopy of optically thick specimens typically features reduced contrast due to "spatial crosstalk", in which the signal at each point in the field of view is the result of a superposition from neighbouring points that are simultaneously illuminated. In 1955, Marvin Minsky proposed confocal microscopy as a solution to this problem. Today, laser scanning confocal fluorescence microscopy is broadly used due to its high depth resolution and sensitivity, but comes at the price of photobleaching, chemical, and photo-toxicity. Here, we present artificial confocal microscopy (ACM) to achieve confocal-level depth sectioning, sensitivity, and chemical specificity, on unlabeled specimens, nondestructively. We equipped a commercial laser scanning confocal instrument with a quantitative phase imaging module, which provides optical path-length maps of the specimen in the same field of view as the fluorescence channel. Using pairs of phase and fluorescence images, we trained a convolution neural network to translate the former into the latter. The training to infer a new tag is very practical as the input and ground truth data are intrinsically registered, and the data acquisition is automated. The ACM images present significantly stronger depth sectioning than the input (phase) images, enabling us to recover confocal-like tomographic volumes of microspheres, hippocampal neurons in culture, and 3D liver cancer spheroids. By training on nucleus-specific tags, ACM allows for segmenting individual nuclei within dense spheroids for both cell counting and volume measurements. In summary, ACM can provide quantitative, dynamic data, nondestructively from thick samples, while chemical specificity is recovered computationally.