Laser Scanning Microscopy Research Articles

Combining metabolomics and transcriptomics to analyze key response metabolites and molecular mechanisms of Aspergillus fumigatus under cadmium stress

The co-cultivation of fungi with microalgae facilitates microalgae harvesting and enhances heavy metal adsorption. However, the mechanisms of fungal tolerance to cadmium (Cd) have not yet been studied in detail. In this study, functional groups of fungi were analyzed under Cd stress using Fourier transform infrared spectrometer (FTIR), X-ray photoelectron spectroscopy (XPS), scanning electron microscope (SEM), and transmission electron microscope (TEM) to explore their morphology. Confocal laser scanning microscope (CLSM) was used to characterize the changes in the content of extracellular polysaccharides and proteins, and a decrease in the ratio of glutathione (GSH) to oxidized glutathione (GSSG) was monitored. The GSH and GSSG contents in mycelium were 7.4 and 7.9 times higher than that in the control, respectively. After 72 h of Cd treatment, the fungal extracellular polysaccharide and extracellular protein contents increased by 16 and 11.4 mg/g, respectively, compared to the control. This provided several functional groups for the complexation of Cd ions to enhance fungal Cd tolerance. The metabolomic and transcriptomic results revealed a total of 358 differential metabolites after 20, 48, and 72 h in the positive and negative ion modes, and the number of differential metabolites specific to each group was 104, 14, and 89, respectively. There were 927, 1167, and 1287 up-regulated genes, and 1301, 1480, and 1683 down-regulated genes at 20, 48, and 72 h, respectively. Energy metabolism, amino acid metabolism, and the ABC transport system are the key metabolic pathways for tolerance enhancement and heavy metal detoxification in fungi. The expression of S-cysteinosuccinic acid was significantly up-regulated after Cd stress and associated with enhanced fungal tolerance and resistance to Cd.

Process development and validation of next generation 3D calibration standards for application in optical microscopy

Abstract A scalable wafer-based fabrication process for a new generation of 3D standards enabling the 3D calibration of optical microscopes is presented and validated. The 3D standards are based on step pyramids with several layers in the µm range and a system of cylindrical knops distributed across the layers as marks for coordinate based calibration. This enables calibration for the three coordinate axes and the orthogonality error between them in a single measurement step. The requirements necessary for such a calibration, as optical non-transparency, reproducible flatness of the pyramid step heights and the lowest possible deviations of the lateral marks coordinates, are met by optimizing the manufacturing process: The deviation of the height steps distributed over the wafer is ±3.6 nm and is primarily caused by the layer deposition processes. The lateral manufacturing accuracy was determined using calibrated scanning electron microscope (SEM) and show a mean deviation of 20 or 60 nm, depending on the lateral size of the structures. The electron beam lithography process and the level of inaccuracy of the SEM standard have an influence on the lateral scaling accuracy. Based on the tactilely generated height values and the coordinates of the mark determined by a calibrated SEM, an example calibration of a confocal laser scanning microscope was successfully performed and showed good conformity to conventional calibration techniques.

A trans-acting sRNA SaaS targeting hilD, cheA and csgA to inhibit biofilm formation of S. Enteritidis

IntroductionSalmonella Enteritidis has brought great harm to public health, animal production and food safety worldwide. The biofilm formed by Salmonella Enteritidis plays a critical role in microbial cross-contamination. Small non-coding RNAs (sRNAs) have been demonstrated to be responsible for regulating the formation of biofilm. The sRNA SaaS has been identified previously, that promotes pathogenicity by regulating invasion and virulence factors. However, whether the SaaS is implicated in regulating biofilm formation in abiotic surfaces remains unclear. ObjectivesThis study aimed to clarify the effect of SaaS in Salmonella Enteritidis and explore the modulatory mechanism on the biofilm formation. MethodsMotility characteristics and total biomass of biofilm of test strains were investigated by the phenotypes in three soft agar plates and crystal violet staining in polystyrene microplates. Studies of microscopic structure and extracellular polymeric substances (EPS) of biofilm on solid surfaces were carried out using confocal laser scanning microscope (CLSM) and Raman spectra. Transcriptomics and proteomics were applied to analyze the changes of gene expression and EPS component. The RNA-protein pull-down and promoter–reporter β-galactosidase activity assays were employed to analyze RNA binding proteins and identify target mRNAs, respectively. ResultsSaaS inhibits biofilm formation by repressing the adhesion potential and the secretion of EPS components. Integration of transcriptomics and proteomics analysis revealed that SaaS strengthened the expression of the flagellar synthesis system and downregulated the expression of curli amyloid fibers. Furthermore, RNA-protein pull-down interactome datasets indicated that SaaS binds to Hfq (an RNA molecular chaperone protein, known as a host factor for phage Qbeta RNA replication) uniquely among 193 candidate proteins, and promoter–reporter β-galactosidase activity assay confirmed target mRNAs including hilD, cheA, and csgA. ConclusionSaaS inhibits the properties of bacterial mobility, perturbs the secretion of EPS, and contributes to the inhibition of biofilm formation by interacting with target mRNA (hilD, cheA, and csgA) through the Hfq-mediated pathway.

Bacterial neuraminidase inhibitory chalcones from flowers of Coreopsis lanceolata, their kinetic characterization and antibiofilm effect

BackgroundBacteria within biofilms are thousand times more resistant to antibiotics. Neuraminidase is a crucial enzyme for bacterial adhesion and biofilm formation, it hydrolyzes glycosidic residue of glycoproteins, glycolipids, and oligosaccharides. Coreopsis lanceolata L. flowers may have a significant potential of bacterial neuraminidase (BNA) inhibition because of high natural abundance of chalcones. PurposeThe investigation of bacterial biofilm inhibitors has emerged as a novel therapeutic strategy against antibiotic resistance. Therefore, individual chalcones were isolated from C. lanceolata and their capacity to inhibit BNA and formation of Escherichia coli biofilm were evaluated. MethodsDifferent chromatographic techniques were used to isolate the compounds (1-12). Enzyme inhibition and detailed kinetic behavior of compounds was determined by estimation of kinetic parameters (Michaelis-Menten constants (Km), maximum velocity (Vmax), dissociation constant for binding with the free enzyme (KI) and enzyme-substate complex (KIS)). Binding affinities (KSV) and binding modes of inhibitors were elucidated by fluorescence quenching and molecular docking, respectively. The natural abundance of chalcones was established through UPLC-Q-TOF/MS. The most potent inhibitor (1) was tested for its ability to inhibit the formation of E. coli biofilm, which was examined by crystal violet assay, scanning electron microscope (SEM) and confocal laser scanning microscope (CLSM). ResultsA series of eight chalcones (1-8) and four chalcone glucosides (9-12), inhibited BNA in a dose-dependent manner with IC50 of 8.3 ∼ 77.0 µM. The most potent chalcones were butein (1, IC50 = 8.3 µM) and its glucoside 9 (IC50 = 13.8 µM). The aglycones (1-8) showed non-competitive inhibition, while chalcone glucosides (9-12) displayed a mixed type I (KI < KIS). Inhibitory behaviors were doubly confirmed by KSV and matched with tendency of IC50. The functional group responsible for BNA inhibition were disclosed as 4ʹ-hydroxyl group on B-ring by structure activity relationship (SAR) and molecular docking experiments. Butein (1) suppressed E. coli biofilm formation by > 50 % at 100 µM according to crystal violet assay, which was confirmed by SEM and CLSM imaging. ConclusionThe results showed that chalcones (1-8) and chalcone glucosides (9-12), metabolites isolated from the flowers of C. lanceolata, had BNA inhibitory and antibiofilm formation effect on E. coli.

Encapsulation of zingerone by self-assembling peptides derived from fish viscera: Characterization, interaction and effects on colon epithelial cells

The purpose of the present work was to encapsulate zingerone (a bioactive compound from ginger) by self-assembling peptides derived from fish viscera. The encapsulation conditions were investigated and the structure of fish peptides-zingerone complex was characterized. The interaction between zingerone and fish peptides was investigated using fluorescence spectroscopy. Further research was performed on the in vitro release of zingerone and fish peptide-zingerone as well as their antiproliferative effects on colon epithelial Caco-2 cells. The results demonstrated that zingerone can be successfully encapsulated by self-assembling peptides derived from fish viscera with high encapsulation efficiency and loading capacity. Furthermore, transmission electron microscope and confocal laser scanning microscope observations revealed the successful encapsulation of zingerone by fish viscera peptides. In addition, in vitro release and antiproliferative activity against Caco-2 cells can be significantly increased by encapsulating zingerone via peptide self-assembly. The current study advances knowledge of encapsulation of bioactive compounds through peptide self-assembly.

Characterization of modified starch-based complexes-stabilized linolenic acid emulsions and their enhanced oxidative stability in vitro gastrointestinal digestion

A new approach of fabricating α-linolenic acid emulsions with enhanced oxidative stability in vitro digestion was established, using covalent octenyl succinic anhydride starch (OSAS)-soy protein (SP)-epigallocatechin-3-gallate (EGCG) complexes as emulsifiers. The physicochemical characteristics and surface morphology of emulsions were mainly characterized by rheological measurements, laser scanning microscope (CLSM) and cryo-scanning electron microscopy (Cryo-SEM). Results indicated that emulsions had dense interfacial layers and strong network structures. As a result, the stability and antioxidant ability of emulsions were improved significantly. In addition, the oxidative stability of emulsions in vitro gastrointestinal digestion was explored. Results showed that emulsions could maintain better oxidative stability owing to antioxidant activity of covalent OSAS-SP-EGCG complexes under gastrointestinal conditions. In particular, lipid hydroperoxide and malondialdehyde contents of emulsions prepared by 1:4 complexes were lower than 0.35 mmol/L and 20.5 nmol/mL, respectively, approximately half those of emulsions stabilized by OSAS (0.65 mmol/L and 39.5 nmol/mL). It was indicated that covalent OSAS-SP-EGCG complexes could effectively inhibit α-linolenic acid oxidation in emulsions during vitro gastrointestinal digestion. This work will provide a theoretical basis for the development of α-linolenic acid emulsions, which will help to broaden application of α-linolenic acid in food industry.

Bacterial sealing ability of calcium silicate-based sealer for endodontic surgery: an in-vitro study

BackgroundApical surgery with standard retrograde maneuvers may be challenging in certain cases. Simplifying apical surgery to reduce operating time and streamline retrograde manipulation is an emerging need in clinical endodontics.Aim of the studyThe aim of the study was to compare the bacterial sealing ability of a calcium silicate-based sealer with the single cone technique combined with root end resection only, and calcium silicate-based sealer as a retrograde filling versus MTA retrofilling, and to analyze bacterial viability using confocal laser scanning microscope (CLSM).Materials and methodsIn this in vitro experimental study, 50 extracted human maxillary incisor teeth were instrumented and randomly divided into five groups: three experimental groups, a positive control group, and a negative control group (n = 10/group). In the experimental groups, the roots were obturated using the single cone technique (SCT) and a calcium silicate-based sealer. In group 1, the roots were resected 3 mm from the apex with no further retrograde preparation or filling. In groups 2 and 3, the roots were resected, retroprepared, and retrofilled with either a calcium silicate-based sealer or MTA, respectively. Group 4 (positive control) was filled with a single gutta-percha cone without any sealer. In group 5 (negative control), the canals were left empty, and the roots were sealed with wax and nail varnish. A bacterial leakage model using Enterococcus faecalis was employed to assess the sealing ability over a 30-day period, checking for turbidity and analyzing colony forming units (CFUs) per milliliter. Five specimens from each group were examined using CLSM for bacterial viability. Data for the bacterial sealing ability were statistically analyzed using chi-squared and Kruskal-Wallis tests.ResultsThe three experimental groups did not show significant differences in terms of bacterial leakage, or bacterial counts (CFUs) (P > 0.05). However, significant differences were observed when comparing the experimental groups to the positive control group. Notably, the calcium silicate-based sealer, when used as a retrofilling, yielded the best sealing ability. CLSM imaging revealed viable bacterial penetration in all the positive control group specimens while for the experimental groups, dead bacteria was the prominent feature seen.ConclusionWithin the limitations of this study, it could be concluded that the bacterial sealing ability of calcium silicate-based sealer with the single cone technique combined with root end resection only and calcium silicate-based sealer as a retrograde filling were comparable with MTA retrofilling during endodontic surgical procedures.

Comparative Analysis of Interfacial Adaptation and Depth Penetration of Recent HiFlow versus Regular Bioceramic Sealers in Conjunction with BC Gutta-Percha Points Using Two Different Obturation Techniques-A Preliminary Report of an Ex Vivo Study.

This study aimed to assess the adaptability and penetration depth capacity of recent bioceramic systems, including regular EndoSequence (BC) versus HiFlow (BCH) sealers in the presence of BC points. A total of 54 single-rooted teeth were instrumented and obturated with either the cold or warm compaction technique (n = 9), using either BC, BCH, or AH Plus (AHP) combined with BC points. The adaptation, film thickness, and gaps/voids were evaluated by scanning electron microscopy. The sealer/dentin interface was evaluated by Raman spectroscopy, and depth penetration was evaluated by a confocal laser scanning microscope. According to the normality test, the data were statistically analyzed by ANOVA or Kruskal-Wallis and Mann-Whitney U tests at p < 0.05. BCH sealer showed the significantly thinnest film with the greatest flow (p > 0.001), with further improvement when subjected to the warm compaction technique. Moreover, it exhibited close adaptation with deep penetration into radicular dentin, forming a tag-like structure. The Raman spectra also indicated close contact with the dentin surface. The use of BC sealer with BC points exhibited homogenous, single-unit obturation, either with a cold or warm technique. Furthermore, the use of the warm compaction technique with BCH sealer achieved a gap-free interface associated with tag-like structures, which exhibit the monoblock phenomenon.

Synthesis of microporous titanium surface with aligned pores through changes of currents during electrochemical process

This paper reports the synthesis of microporous Ti surfaces with aligned pores by applying a different current during electrochemical process for application in cell-surface interaction study and biomedical implants. As the microporous Ti surfaces were synthesized by current from (0.5–4)A, the microstructure of the Ti surfaces was shifted from a relative smooth to microporous one that was observed to have an aligned pore surface. Optical images showed that the micropore sizes in ∼(85.4–224.4)µm. The roughness of the microporous Ti was significantly higher than that of the Ti by a factor of approximately 5 to 7. Confocal laser scanning microscope showed excellent cell attachment and preferential growth on the microporous Ti with aligned channels after 72 h of culturing which could be important for cell-surface interactions study and biomedical implants.

Effect of Bleaching on Surface Roughness and Color Parameters of Coffee-Stained Nanohybrid Dental Composites with Different Viscosities

Abstract Objective The aim of this study was to test the effect of different bleaching protocols on surface roughness, color stability, and translucency parameter of coffee-stained nanohybrid dental composites with different viscosities. Materials and Methods Five nanohybrid dental composites with different viscosities (n = 250)—Neo Spectra LV, Neo Spectra HV, Neo Spectra Flow (Dentsply, Konstanz, Germany), Grandio, and Grandio Flow (Voco, Cuxhaven, Germany)—were used to test surface roughness, color stability, and translucency parameter after bleaching of coffee-stained specimens using either in-office bleaching, home bleaching, or a combination of both. The viscosity of the five types of dental composites was tested using a viscometer. A scanning laser microscope (SLM) was used to examine the surface topography of representative samples from each dental composite after the combined effect of both bleaching agents. Effect of composite type, bleaching protocol, and their interaction was assessed by two-way analysis of variance. For multiple comparisons, Tukey's post hoc test was used with Bonferroni correction. Results Surface roughness of all coffee-stained dental composites increased after bleaching; however, those of Grandio and Grandio Flow increased significantly (p &lt; 0.0001). This was confirmed by SLM images that showed rough surface with protruding fillers after in-office and home bleaching protocol. Results revealed that bleaching, regardless of the protocol, decreased the delta E values of all dental composites; however, it was still higher than the accepted threshold value of 3.7. Neither of the bleaching protocols enhanced translucency parameter of all dental composites. Neo Spectra Flow and Voco Flow had the highest translucency parameter after the three bleaching protocols. Conclusion Bleaching does not improve color stability and translucency of coffee-stained nanohybrid dental composites regardless of the viscosity. Surface roughness of coffee-stained nanohybrid dental composites increases after application of bleaching agents.

PH-induced complex coacervation of Gel and Agar: Phase behavior and structural properties

In this study, we investigated the behavioral patterns of gelatin (Gel) and Agar in terms of composites and phase separation. As the pH (4.5–7.0) increased, the electrostatic interaction between the gel and the agar weakened, and a decrease in the degree of agglomeration of the microstructure was observed in the confocal laser scanning microscope (CLSM) plots. Specifically, When the ratio of Gel to Agar was 2:1 and the pH was 4.5 (pHopt), a large amount of complex coalescence appeared. The Absorption and fluorescence spectra demonstrated the impact of Agar interaction on the tertiary conformation of the Gel, while circular dichroism (CD) detected changes in its secondary structure. Furthermore, Fourier Transform Infrared Resonance spectroscopy (FTIR) demonstrated that the complexes were produced through electrostatic interactions between the -NH3+ groups of Gel and –COO- groups of Agar. The Gel-Agar coacervation process induced structural modifications, which enhanced the Gel’s thermal stability. The findings of this study are expected to be an essential contribution to the potential application of Gel-Agar coacervates for encapsulating functional ingredients.

Using salted egg white in steamed bread: Impact on functional and structural characteristics

Steamed bread has long been an important part of Chinese cuisine. This study investigated the effects of salted egg white (SEW) (5, 10, 15, and 20% w/w) on the quality of steamed breads. Findings revealed that SEW notably enhanced the bread's volume and texture, with a 20% inclusion significantly boosting water retention and rheological properties, albeit reducing bread's lightness. In addition, the H-bond absorption band intensity in the Fourier transform infrared spectroscopy (FTIR) analysis showed increased peak intensities with higher SEW levels, indicative of protein structure alterations. X-ray diffraction confirmed the presence of an amylose–lipid complex. Scanning electron microscope (SEM) and Confocal laser scanning microscope (CLSM) imaging depicted a smooth, consistent protein network with SEW addition. Consumer sensory evaluation responded favourably to the SEW15 steamed bread, suggesting its potential for food industry application. Overall, the study considers SEW an effective ingredient for improving steamed bread quality.

Multi-scale tissue fluorescence mapping with fiber optic ultraviolet excitation and generative modeling

Cellular imaging of thick samples requires physical sectioning or laser scanning microscopy, which can be restrictive, involved, and generally incompatible with high-throughput requirements. We developed fiber optic microscopy with ultraviolet (UV) surface excitation (FUSE), a portable and quantitative fluorescence imaging platform for thick tissue that enabled quick sub-cellular imaging without thin sections. We substantially advanced prior UV excitation approaches with illumination engineering and computational methods. Optical fibers delivered &lt;300nm light with directional control, enabling unprecedented 50× widefield imaging on thick tissue with sub-nuclear clarity, and 3D topography of surface microstructure. Probabilistic modeling of high-magnification images using our normalizing flow architecture FUSE-Flow (made freely available as open-source software) enhanced low-magnification imaging with measurable localized uncertainty via variational inference. Comprehensive validation comprised multi-scale fluorescence histology compared with standard H&amp;E histology, and quantitative analyses of senescence, antibiotic toxicity, and nuclear DNA content in tissue models via efficient sampling of thick slices from entire murine organs up to 0.4×8×12mm and 1.3 million cells per surface. This technology addresses long-standing laboratory gaps in high-throughput studies for rapid cellular insights.

Effect of Repolishing on Color Stability, Translucency, and Surface Roughness of Aged Monochromatic Dental Composites

Abstract Objective This study aimed to test repolishing effect on color stability, translucency, and surface roughness of aged monochromatic dental composite in artificial saliva, tea, mouthwash, and coffee after 1 month, simulating 2.5 years of clinical use. Materials and Methods Omnichroma, a monochromatic dental composite, and Z250 XT a nanohybrid multishade dental composite were used in this study. Specimens (n = 80) from each dental composite were prepared to determine color stability, translucency parameter, and surface roughness after repolishing of aged specimens in artificial saliva, tea, mouthwash, and coffee for 1 month. Scanning electron microscope and laser scanning microscope were used to study surface topography after repolishing. Data was analyzed using analysis of variance, Scheffe test, and independent t-test. Results A significant difference between both dental composites after repolishing in terms of color stability and translucency parameter as that of Omnichroma was higher but sill color changes of Omnichroma were perceptible. There was no difference between both composites in regard to surface roughness after repolishing; however, values of both composites were above the accepted value of 0.2 µm and laser scanning microscope images confirmed these findings. Conclusions Repolishing did not enhance color and surface roughness of aged monochromatic dental composites.

Study of proanthocyanidin promotes osteogenic differentiation of human periodontal ligament stem cells through the transcription factor EB-induced autophagy-lysosome pathway

Objective: To investigate the mechanism of proanthocyanidin (PA) in regulating the osteogenic differentiation of human periodontal ligament stem cells (PDLSCs), and to explore the effects of PA on the expression and nuclear translocation of transcription factor EB (TFEB) and on the autophagy-lysosome pathway. Methods: PDLSCs were divided into control group and PA group, which were subjected to RNA sequencing analysis (RNA Seq) to detect differentially expressed genes. The osteogenic differentiation ability and autophagy level were observed by real-time fluorescence quantitative PCR (RT-qPCR) analysis, alkaline phosphatase (ALP) staining and transmission electron microscope (TEM), respectively. Scratch assay and Transwell assay were used to detect the migration ability of PDLSCs. Lysotracker and immunofluorescence staining were used to detect the biogenesis of lysosomes. The total protein expression of transcription factor EB (TFEB) as well as that in cytoplasm and nucleus were detected by Western blotting. Confocal laser scanning microscope (CLSM) was used to observe the nuclear translocation of TFEB. The PDLSCs were treated with small interfering RNA (siRNA) technology to knock down the expression levels of TFEB gene with or without PA treatment. Western blotting was used to analyze the expressions of autophagy-related proteins Beclin1 and microtubule-associated protein 1 light chain 3 (LC3B), as well as osteogenic-related proteins runt-related transcription factor 2 (RUNX2), ALP, and osteocalcin in PDLSCs. Results: Compared with the control group, the osteogenic-related and autophagy-related genes showed differential expression in PDLSCs after PA treatment (P<0.05). The mRNA expression levels of osteogenic-related genes RUNX2 (2.32±0.15) and collagen type Ⅰ alpha 1 (COL1α1) (1.80±0.18), as well as the autophagy related genes LC3B (1.87±0.08) and Beclin1 (1.63±0.08) were significantly increased in the PA group, compared with the control group (1.01±0.16, 1.00±0.10, 1.00±0.07, 1.00±0.06, respectively, all P<0.01). Compared with the control group, the PA group had higher ALP activity, and more autophagosomes and autophagolysosomes observed by TEM. PA promoted the migration of PDLSCs (P<0.05) and the increased number of lysosomes and the expression of lysosomal associated membrane protein 1 (LAMP1). In the PA group, the relative expression level of total TFEB protein (1.49±0.07) and the nuclear/cytoplasmic expression of TFEB protein (1.52±0.12) were significantly higher than the control group (1.00±0.11, 1.00±0.13, respectively) (t=6.43, P<0.01; t=5.07, P<0.01). The relative nuclear/cytoplasmic fluorescence intensity of TFEB in the PA group (0.79±0.09) was increased compared with the control group (0.11±0.08) (t=8.32, P<0.01). Knocking down TFEB significantly reduced the expression of TFEB (1.00±0.15 vs 0.64±0.04), LAMP1 (1.00±0.10 vs 0.69±0.09), Beclin1 (1.00±0.05 vs 0.60±0.05), and LC3B Ⅱ/Ⅰ (1.00±0.06 vs 0.73±0.07) in PDLSCs (P<0.05, P<0.05, P<0.01, P<0.01). When TFEB gene was knocked down, the expression levels of Beclin1 (1.05±0.11), LC3B Ⅱ/Ⅰ (1.02±0.09), RUNX2 (1.04±0.10), ALP (1.04±0.16), and osteocalcin (1.03±0.15) proteins were significantly decreased in the PA group compared with the pre-knockdown period (1.28±0.03, 1.44±0.11, 1.38±0.11, 1.62±0.11, 1.65±0.17, respectively) (P<0.05, P<0.01, P<0.05, P<0.01, and P<0.01, respectively). Conclusions: PA promotes the osteogenic differentiation of PDLSCs through inducing the expression and nuclear translocation of TFEB and activating the autophagy-lysosome pathway.

Spectroscopic analysis of the sum-frequency response of the carbon-hydrogen stretching modes in collagen type I.

We studied the origin of the vibrational signatures in the sum-frequency generation (SFG) spectrum of fibrillar collagen type I in the carbon-hydrogen stretching regime. For this purpose, we developed an all-reflective, laser-scanning SFG microscope with minimum chromatic aberrations and excellent retention of the polarization state of the incident beams. We performed detailed SFG measurements of aligned collagen fibers obtained from rat tail tendon, enabling the characterization of the magnitude and polarization-orientation dependence of individual tensor elements Xijk2 of collagen's nonlinear susceptibility. Using the three-dimensional atomic positions derived from published crystallographic data of collagen type I, we simulated its Xijk2 elements for the methylene stretching vibration and compared the predicted response with the experimental results. Our analysis revealed that the carbon-hydrogen stretching range of the SFG spectrum is dominated by symmetric stretching modes of methylene bridge groups on the pyrrolidine rings of the proline and hydroxyproline residues, giving rise to a dominant peak near 2942cm-1 and a shoulder at 2917cm-1. Weak asymmetric stretches of the methylene bridge group of glycine are observed in the region near 2870cm-1, whereas asymmetric CH2-stretching modes on the pyrrolidine rings are found in the 2980 to 3030cm-1 range. These findings help predict the protein's nonlinear optical properties from its crystal structure, thus establishing a connection between the protein structure and SFG spectroscopic measurements.

The effect of different irrigation activation methods on the penetration of the irrigation solution into the dentinal tubules in teeth with calcium hydroxide applied and oval-shaped canals: Confocal laser scanning microscope analysis.

This study aimed to evaluate the efficacy of different irrigation techniques including standard needle irrigation (SNI), passive ultrasonic irrigation (PUI), EndoActivator (EA), XP-Endo Finisher (XPF), photon-induced photoacoustic streaming (PIPS), and shock wave enhanced emission photo-acoustic streaming (SWEEPS) systems on the penetration of irrigation solutions into dentinal tubules in teeth with calcium hydroxide (CH) applied and oval-shaped canals by confocal laser scanning microscope (CLSM). Ninety mandibular incisor teeth with oval-shaped canals were included in this study. After the preparation of teeth using Resiproc Blue R25 (VDW, Munich, Germany) canals were filled with a paste based on CH. The teeth were stored in 100% humidity at a temperature of 37°C for 14 days. According to the irrigation activation systems, the teeth were divided into six groups (n = 15); SNI, PUI, EA, XPF, PIPS, and SWEEPS. In each group, 3 irrigation/activation cycles of 20 s of irrigation and 20 s of activation were performed with the relevant activation method. The root canals were filled with fluorescein sodium (Sigma, Alldrich Co., St. Louis, MO, USA) and activated with the relevant activation method for 30 s. Specimens were sectioned horizontally to 1 ± 0.1 mm at 2, 5, and 8 mm from the apex and then examined under the CLSM. Maximum penetration depth, maximum penetration area and penetration percentage were measured by using Image J software. Data were analyzed using a two-way analysis of variance (ANOVA) and posthoc Tukey tests (p = .05). No difference was found between the activation systems in terms of maximum penetration depth and maximum penetration area of irrigation solutions in the apical section (p > .05). The penetration percentage of irrigation solutions was higher in PUI and PIPS compared with EA and XPF at the apical section (p < .05). No difference was found between SNI, PUI, PIPS, and SWEEPS in terms of the penetration percentage of irrigation solutions in all sections (p > .05). The penetration percentage of irrigation solutions was higher in the middle and coronal sections compared to the apical section in EA and XPF (p < .05). Tubule penetration of irrigation solutions in SNI was similar between sections. The Tubule penetration area of irrigation solutions in PUI, EA, XPF, PIPS, and SWEEPS was lower in the apical section than in the middle and coronal sections. RESEARCH HIGHLIGHTS: The tubule penetration depth and area of the irrigation solutions were similar between the activation systems tested in the apical sections. The Tubule penetration area of irrigation solutions in middle and coronal sections was higher in PUI and PIPS than in SNI.

Comparative evaluation of different surface pretreatment methods on the depth of penetration of adhesive resin in sandwich technique: A confocal laser scanning microscopy study.

The aim of the study was to evaluate and compare the depth of penetration of adhesive resin in resin-modified glass ionomer cement (RMGIC) after different surface pretreatment methods using a confocal laser scanning microscope (CLSM). Class I cavity preparation was done in 45 freshly extracted premolars and divided into three groups, according to the surface pretreatment of RMGIC which was placed in a thickness of 1 mm in the cavity. In the control group, no pretreatment was done, and other two groups were pretreated with acid etching and air abrasion (AA). The fifth-generation dentin bonding agent mixed with rhodamine B dye 0.1% was applied on RMGIC in all the samples and cured for 15 s. Samples were restored using composite resin and sectioned longitudinally. The depth of penetration of adhesive resin was evaluated using CLSM. Depth of penetration of adhesive resin was highest with AA (153.70 ± 10.23), followed by acid etching (122.71 ± 12.25) and control group (77.12 ± 6.37). Based on the findings of this research, AA enhances the depth of penetration of adhesive resin in the RMGIC surface. Thus, AA before placement of composite resin on the RMGIC can be effective in a clinical scenario.

Influence of root canal moisture on the penetration of TotalFill bioceramic sealer into the dentinal tubules: A confocal laser scanning microscopy study

Abstract Introduction: Bioceramic (BC) sealers exhibit multiple desirable biologic and physical properties. The aim of this study was to evaluate the effect of root canal moisture on the depth of TotalFill BC sealer penetration into dentinal tubules at different levels using a confocal laser scanning microscope (CLSM). Materials and Methods: The manuscript of this laboratory study has been written according to preferred reporting items for laboratory studies in endodontology 2021 guidelines. Forty single-rooted extracted human premolars were decoronated using a low-speed diamond disc, followed by root canal preparation using nickel–titanium files under copious irrigation. The samples were randomly allocated for obturation into four groups n = 10 based on the sealer and root canal moisture status. Group A: TotalFill BC sealer in wet canal, Group B: TotalFill BC sealer in dry canal, Group C: AH Plus sealer in wet canal, and Group D: AH Plus sealer in dry canal. The roots were sectioned at three different distances from the apex. The root segments were examined using CLSM to measure the extent of sealer penetration into the dentinal tubules. The data underwent analysis through a three-way mixed ANOVA test, followed by post hoc assessments (P &lt; 0.05). Results: In wet canals, the tubular penetration of TotalFill BC sealer was notably greater than that of AH plus, with a statistically significant difference. However, AH Plus sealer had a statistically significant tubular penetration in the dry canals compared to the wet. Greater sealer penetration was observed at 5 mm from the apex, followed by 3 mm, and the least penetration depth was found at 1 mm from the apex. Conclusions: The presence of root canal moisture and distance from the apex had a significant impact on the penetration of TotalFill BC sealer into the dentinal tubules.

Natural product nobiletin-loaded Pickering emulsion stabilized by bovine serum albumin/carboxymethyl inulin complexes: preparation and digestive characteristics.

To expand the application of nobiletin (NOB) in semi-solid functional foods, bovine serum albumin (BSA)/carboxymethyl inulin (CMI) complexes-stabilized Pickering emulsion (BCPE) (φoil = 60%, v/v) was fabricated, and the swallowing index and bioavailability of the NOB-loaded Pickering emulsion was evaluated. Confocal laser scanning microscope (CLSM) and cryo-scanning electron microscopy (cryo-SEM) images revealed that BSA/CMI complexes attached to the oil-water interface. NOB-loaded BCPE exhibited a viscoelastic and shear-thinning behavior. Fork drip test results suggested that the textural value of unloaded and NOB-loaded emulsions was International Dysphagia Diet Standardisation Initiative Level 4, which could be swallowed directly without chewing. The in vitro lipolysis model suggested that NOB had a faster digestive profile and a higher bioaccessibility in the BCPE than in the oil suspension. The in vivo rat model revealed that the oral bioavailability of NOB was increased by 2.07 folds in BCPE compared to its bioavailability in unformulated oil. Moreover, BCPE led to a higher plasma concentration of the major demethylated metabolite of NOB (4'-demethylnobiletin) than the unformulated oil. Accordingly, BCPE enhanced the oral bioavailability of NOB by improving bioaccessibility, absorption, and biotransformation.