Confocal Laser Scanning Fluorescence Microscopy Research Articles

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.

Evaluation of extracellular polymeric substances matrix volume, surface roughness and bacterial adhesion property of oral biofilm

Background/purposeOral biofilms are highly structured bacterial colonies embedded in a highly hydrated extracellular polymeric substances (EPS) matrix. This study aimed to investigate the characteristics of oral biofilm at different stages of maturation. Materials and methodsOral multispecies biofilms were grown anaerobically from plaque bacteria on collagen coated hydroxyapatite discs in brain heart infusion broth for one and three weeks. The volume of live bacteria and EPS matrix of the biofilms were determined by using corresponding fluorescent probes and confocal laser scanning microscopy. Atomic force microscopy (AFM) was used to quantitatively probe and correlate cell surface adhesion force of biofilms. The surface roughness was quantified in terms of the root mean square average of the height deviations. Adhesion was measured from force–distance data for the retraction of the cell from the surface. ResultsThe volume of live bacteria and EPS of 3-week-old biofilms was higher than 1-week-old biofilms. The surface roughness value in 1-week-old biofilms was significantly higher than that in 3-week-old biofilms. AFM force–distance curve results showed that the adhesion force at the cell–cell interface was significantly more at-tractive than those at bacterial cells surface of both stages biofilms. Adhesion forces between the AFM tip and the surface of bacterial cell were fairly constant, whereas the cell–cell interface experienced greater adhesion forces in the biofilm's development. ConclusionAs oral biofilms become mature, EPS volume and cell–cell adhesion forces increase while the surface roughness decreases.

Fluorescent naphthalimide boronates as theranostics: structural investigations, confocal fluorescence and multiphoton fluorescence lifetime imaging microscopy in living cells.

New design and synthetic strategies were developed to generate functional phenyl boronic acid (BA)-based fluorescent probes incorporating the 1,8-naphthalimide (NI) tag. This fluorescent core was anchored onto the BA unit through small organic linkers consisting of nitrogen groups which can arrest, and internally stabilise the phenyl-boronate units. The newly synthesised fluorophores were characterised spectroscopically by NMR spectroscopy and mass spectrometry and evaluated for their ability to bind to a naturally occurring polysaccharide, β-d-glucan in DMSO and simultaneously as act as in vitro cell imaging reagents. The uptake of these new NI-boronic acid derivatives was studied living cancer cells (HeLa, PC-3) in the presence, and absence, of β-d-glucan. Time-correlated single-photon counting (TCSPC) of DMSO solutions and two-photon fluorescence-lifetime imaging microscopy (FLIM) techniques allowed an insight into the probes' interaction with their environment. Their cellular uptake and distributions were imaged using laser scanning confocal fluorescence microscopy under single- and two-photon excitation regimes (λmax 910 nm). FLIM facilitated the estimation of the impact of the probe's cellular surroundings using the fluorophore lifetime. The extent to which this was mediated by the β-d-glucan was visualised by 2-photon FLIM in living cells. The fluorescence lifetime observed under a range of temperatures varied appreciably, indicating that changes in the environment can be sensed by these probes. In all cases, the cellular membrane penetration of these new probes was remarkable even under variable temperature conditions and localisation was widely concentrated in the cellular cytoplasm, without specific organelle trapping: we conclude that these new probes show promise for cellular imaging in living cancer cells.

Uphill Accumulation of Ionic Species into a Lipid Vesicle by the Concentration Gradient of Counter Ions.

The "uphill (against the concentration gradient)" accumulation of a hydrophobic cation (rhodamine 6G, R6G+) into the inner phase of a giant unilamellar vesicle (GUV) was realized with the concentration gradient of the counter anion (X- = ClO4-, BF4-, or Br-) in the presence of phosphate buffer (P-, pH = 7) in the inner and outer phase of the GUV and detected as the increase of the R6G+ fluorescence intensity in the inner phase using a confocal laser scanning fluorescence microscope. The addition of X- in the outer phase of the GUV caused the accumulation of R6G+ in the inner phase. The degree and kinetics of the accumulation were dependent on the concentration and type of X-; e.g., the inner concentration of R6G+ reached 2.5 times that in the outer phase of GUV after adding 10 mM ClO4-. The accumulation was theoretically simulated by assuming the distribution of ion pairs (R6G+ and X-, R6G+, and P-) between the aqueous phase and the lipid bilayer membrane (ion pair distribution model) and the transmembrane fluxes of R6G+, X- and P-. The theoretical simulation rationalized the accumulation degree and kinetics of the experimental results. The accumulation of the target cation by the concentration gradient of the counter anion demonstrated in this study can be an effective method for the preparation of liposomal drugs.

Cytological observation and transcriptome analysis reveal dynamic changes of Rhizoctonia solani colonization on leaf sheath and different genes recruited between the resistant and susceptible genotypes in rice.

Sheath blight, caused by Rhizoctonia solani, is a big threat to the global rice production. To characterize the early development of R. solani on rice leaf and leaf sheath, two genotypes, GD66 (a resistant genotype) and Lemont (a susceptible genotype), were observed using four cytological techniques: the whole-mount eosin B-staining confocal laser scanning microscopy (WE-CLSM), stereoscopy, fluorescence microscopy, and plastic semi-thin sectioning after in vitro inoculation. WE-CLSM observation showed that, at 12h post-inoculation (hpi), the amount of hyphae increased dramatically on leaf and sheath surface, the infection cushions occurred and maintained at a huge number from about 18 to 36 hpi, and then the infection cushions disappeared gradually from about 42 to 72 hpi. Interestingly, R. solani could not only colonize on the abaxial surfaces of leaf sheath but also invade the paraxial side of the leaf sheath, which shows a different behavior from that of leaf. RNA sequencing detected 6,234 differentially expressed genes (DEGs) for Lemont and 7,784 DEGs for GD66 at 24 hpi, and 2,523 DEGs for Lemont and 2,719 DEGs for GD66 at 48 hpi, suggesting that GD66 is recruiting more genes in fighting against the pathogen. Among DEGs, resistant genes, such as OsRLCK5, Xa21, and Pid2, displayed higher expression in the resistant genotype than the susceptible genotype at both 24 and 48 hpi, which were validated by quantitative reverse transcription-PCR. Our results indicated that the resistance phenotype of GD66 was the consequence of recruiting a series of resistance genes involved in different regulatory pathways. WE-CLSM is a powerful technique for uncovering the mechanism of R. solani invading rice and for detecting rice sheath blight-resistant germplasm.

Fabrication of hydroxyapatite porous body with connective pores using the self-networking property of rice starch powder by heat treatment

• Porous hydroxyapatite ceramics was fabricated. • Gelatinization-retrogradation phenomena of starch was used as pore-networking agent. • Randomly-developed open pores were introduced into the hydroxyapatite matrix. • Pore structure was characterized by confocal scanning laser fluorescence microscopy. Hydroxyapatite porous ceramics were fabricated utilizing the property called gelatinization-retrogradation in which starch particles used as a pore-forming agent form a self-network structure by heat treatment. The pore structures in the hydroxyapatite samples were observed by combining the immersion transmission method and a confocal scanning laser fluorescence microscope. The three-dimensional image created by stacking the cross-sectional observation images showed that randomly-developed open pores were introduced into the matrix.

Local pH Modulation during Electro-Enzymatic O2 Reduction: Characterization of the Influence of Ionic Strength by In Situ Fluorescence Microscopy.

Understanding how environmental factors affect the bioelectrode efficiency and stability is of uttermost importance to develop high-performance bioelectrochemical devices. By coupling fluorescence confocal microscopy in situ to electrochemistry, this work focuses on the influence of the ionic strength on electro-enzymatic catalysis. In this context, the 4 e-/4 H+ reduction of O2 into water by the bilirubin oxidase from Myrothecium verrucaria (MvBOD) is considered as a model. The effects of salt concentration on the enzyme activity and stability were probed by enzymatic assays performed in homogeneous catalysis conditions and monitored by UV-vis absorption spectroscopy. They were also investigated in heterogeneous catalysis conditions by electrochemical measurements with MvBOD immobilized at a graphite microelectrode. We demonstrate that the catalytic activity and stability of the enzyme both in solution and in the immobilized state at the bioelectrode were conserved with an electrolyte concentration of up to 0.5 M, both in a buffered and a non-buffered electrolyte. Relying on this, we used fluorescence confocal laser scanning microscopy coupled in situ to electrochemistry to explore the local pH of the electrolyte at the vicinity of the electrode surface at various ionic strengths and for several overpotentials. 3D proton depletion profiles generated by the interfacial electro-enzymatic reaction were recorded in the presence of a pH-sensitive fluorophore. These concentration profiles were shown to contract with increasing ionic strength, thus highlighting the need for a minimal electrolyte concentration to ensure availability of charged substrates at the electrode surface during electro-enzymatic experiments.

Comparison of cell viability assessment and visualization of Aspergillus niger biofilm with two fluorescent probe staining methods

Filamentous fungi are ubiquitous and frequent components of biofilms. A means to visualize them and quantify their viability is essential for understanding their development and disruption. However, quantifying filamentous fungal biofilms poses challenges because, unlike yeasts and bacteria, they are not composed of discrete cells of similar size. This research focused on filamentous fungal biofilms that are representative of those in the built environment. The objective of this study was to develop a rapid method to examine biofilm structure and quantify live (metabolically active/ membrane undamaged) and dead (inactive/ membrane damaged) cells in Aspergillus niger biofilms utilizing a fluorescent probe staining method and confocal laser scanning microscopy (CLSM). For this, we compared two commercially available probe staining kits that have been developed for bacterial and yeast systems. One method utilized the classic cell stain FUN 1 that exhibits orange-red fluorescent intravacuolar structures in metabolically active cells, while dead cells are fluoresced green. The second method utilized a combination of SYTO9 and propidium iodide (PI), and stains cells based on their membrane morphology. SYTO9 is a green fluorescent stain with the capacity to penetrate the living cell walls, and PI is a red fluorescent stain that can only penetrate dead or dying cells with damaged cell membranes. Following staining, the biofilms were imaged using CLSM and biofilm volumes and thickness were quantified using COMSTAT, a computer program that measures biofilm accumulation from digital image stacks. The results were compared to independent measurements of live-dead cell density, as well as a classic cell viability assay-XTT. The data showed that the combination of SYTO9 and PI is optimal for staining filamentous fungal biofilms.

Histologic Cell Shape Descriptors for the Retinal Pigment Epithelium in Age-Related Macular Degeneration: A Comparison to Unaffected Eyes.

PurposePhenotype alterations of the retinal pigment epithelium (RPE) are a main characteristic of age-related macular degeneration (AMD). Individual RPE cell shape descriptors may help to delineate healthy from AMD-affected cells in early disease stages.MethodsTwenty-two human RPE flatmounts (7 eyes with AMD [early, 3; geographic atrophy, 1; neovascular, 3); 15 unaffected eyes [8 aged ≤51 years; 7 aged >80 years)] were imaged at the fovea, perifovea, and near periphery (predefined sample locations) using a laser-scanning confocal fluorescence microscope. RPE cell boundaries were manually marked with computer assistance. For each cell, 11 shape descriptors were calculated and correlated with donor age, cell autofluorescence (AF) intensity, and retinal location. Statistical analysis was performed using an ensemble classifier based on logistic regression.ResultsIn AMD, RPE was altered at all locations (most pronounced at the fovea), with area, solidity, and form factor being the most discriminatory descriptors. In the unaffected macula, aging had no significant effect on cell shape factors; however, with increasing distance to the fovea, area, solidity, and convexity increased while form factor decreased. Reduced AF in AMD was significantly associated with decreased roundness and solidity.ConclusionsAMD results in an altered RPE with enlarged and deformed cells that could precede clinically visible lesions and thus serve as early biomarkers for AMD onset. Our data may also help guide the interpretation of RPE morphology in in vivo studies utilizing high-resolution single-cell imaging.Translational RelevanceOur histologic RPE cell shape data have the ability to identify robust biomarkers for the early detection of AMD-affected cells, which also could serve as a basis for automated segmentation of RPE sheets.

Adsorption of Candida albicans on Ti-6Al-4V surface and its corrosion effects in artificial saliva

In this study, the corrosion behavior and mechanism of Ti-6Al-4V in artificial saliva with Candida albicans were investigated using electrochemical and surface analysis techniques. Fluorescence microscopy (FM) and confocal laser scanning microscopy (CLSM) showed that C. albicans could easily adsorb on the surface of Ti-6Al-4V alloy to form non-dense biofilm. The non-compact biofilm provided necessary conditions for pitting corrosion on Ti-6Al-4V alloys by scanning electron microscopy (SEM) observation. The potentiodynamic polarization (PDP) curves and electrochemical impedance spectroscopy (EIS) revealed that C. albicans significantly reduced the corrosion resistance of Ti-6Al-4V alloys. The cyclic voltammetry (CV) and differential pulse voltammetry (DPV) results indicated that C. albicans biofilm promoted electron transfer from the anodic sites to cathodic depolarizer during the corrosion process, showing that the role of oral fungi must be considered when evaluating the performance of oral materials. This study may provide a new clue for evaluating the corrosion resistance of dental implant materials in the oral environment.

Nanomechanical subsurface characterisation of cellulosic fibres

AbstractThe mechanical properties of single fibres are highly important in the paper production process to produce and adjust properties for the favoured fields of application. The description of mechanical properties is usually characterised via linearized assumptions and is not resolved locally or spatially in three dimensions. In tensile tests or nanoindentation experiments on cellulosic fibres, only mechanical parameter for the whole fibre, such as elastic modulus or hardness, is usually obtained. To obtain a more detailed mechanical picture of the fibre, it is crucial to determine mechanical properties in depth. To this end, we discuss an atomic force microscopy-based approach to examine stepwise the local stiffness as a function of indentation depth via static force-distance curves. To our knowledge, we are the first authors to apply this method cellulosic fibres. The method was applied to linter fibres (extracted from a finished paper sheet) as well as to natural raw cotton fibres to better understand the influence of the pulp treatment process in paper production on the mechanical properties. Both types of fibres were characterised in dry and wet conditions with respect to alterations in their mechanical properties. The used stepwise analysis method of the force-distance curves allowed subsurface imaging of the fibres. It could be revealed how the walls in the fibre structure protects the fibre against mechanical loading. Via a combined 3D display of the mapped topography and the fitted elastic moduli in z-direction, a spatially resolved mechanical map of the fibre interior near the surface could be established. Additionally, we labelled the fibres with different carbohydrate binding modules tagged with fluorescent proteins to compare the AFM results with fluorescence confocal laser scanning microscopy imaging. Nanomechanical subsurface imaging in combination with fluorescent protein labelling is thus a tool to better understand the mechanical behaviour of cellulosic fibres, which have a complex, hierarchical structure.Graphical abstract

Microscopic structure, viscoelastic behaviour and 3D printing potential of milk protein concentrate‐hydrocolloid complex coacervates

SummaryTo prepare a milk protein‐enriched ink for extrusion‐based 3D food printing, this study investigated the effects of a wide range of hydrocolloids on the microstructures, viscoelastic characteristics and 3D printing performance of milk protein concentrate (MPC). The distributions of hydrocolloids and milk protein in mixed coacervates were characterised by fluorescent covalent labelling and confocal laser scanning microscope (CLSM), and the microstructure of the coacervates was observed by scanning electron microscopy (SEM). In addition, the rheological properties of prepared protein coacervates, including steady shear test, dynamic oscillatory test, thixotropy and creep recovery were investigated. Meanwhile, Burger’s model was fitted to the creep behaviour to further study their viscoelastic properties. The results showed that κ‐carrageenan, pectin, guar gum and sodium alginate significantly increased the zero‐shear viscosity, thixotropy and solid‐like behaviour while xanthan showed an opposite phenomenon. Results showed that the presence of hydrocolloids improved the 3D printability of MPC by forming a complex network between protein particles and hydrocolloids, and guar gum, pectin and κ‐carrageenan better help maintain the deposited 3D structures of MPC ink than xanthan.

Degree-of-spatial-coherence laser scanning confocal fluorescence microscope

The modulation of degree of spatial coherence (DOSC) plays an optical gating which highly correlates with the coherence volume (CV) in the proposed DOSC laser scanning confocal fluorescence microscope (DOSC-LSCFM). The axial resolution at sub-diffraction-limit performance was demonstrated and discussed.

Raman Research on Bleomycin-Induced DNA Strand Breaks and Repair Processes in Living Cells.

Even several thousands of DNA lesions are induced in one cell within one day. DNA damage may lead to mutations, formation of chromosomal aberrations, or cellular death. A particularly cytotoxic type of DNA damage is single- and double-strand breaks (SSBs and DSBs, respectively). In this work, we followed DNA conformational transitions induced by the disruption of DNA backbone. Conformational changes of chromatin in living cells were induced by a bleomycin (BLM), an anticancer drug, which generates SSBs and DSBs. Raman micro-spectroscopy enabled to observe chemical changes at the level of single cell and to collect hyperspectral images of molecular structure and composition with sub-micrometer resolution. We applied multivariate data analysis methods to extract key information from registered data, particularly to probe DNA conformational changes. Applied methodology enabled to track conformational transition from B-DNA to A-DNA upon cellular response to BLM treatment. Additionally, increased expression of proteins within the cell nucleus resulting from the activation of repair processes was demonstrated. The ongoing DNA repair process under the BLM action was also confirmed with confocal laser scanning fluorescent microscopy.