Confocal Scanning Fluorescence Microscopy Research Articles

Influence of initial root canal treatment on intratubular penetrability and bond strength of nonsurgical retreatment: an in vitro study

To assess the effect of combinations of two different endodontic sealers used in initial and endodontic retreatment on the bond strength of the secondary obturation and the penetrability of the sealers. Forty-eight mandibular premolars were used, receiving standardized endodontic access and biomechanical preparation. Twenty-four teeth received AH Plus sealer (AHP) in primary obturation, and the others received Bio-C® Sealer (BCS) sealer. Retreatment protocol was performed with an R50 instrument. The samples were further subdivided into four groups (n=12) based on the combination of primary/secondary obturation sealers: AHP/AHP; AHP/BCS; BCS/AHP; BCS/BCS. Four samples from each subgroup received the addition of fluorophores to the sealer for penetrability analysis using laser scanning confocal fluorescence microscopy (LSCFM). The root portion on the 8 push-out samples was sectioned into 6 slices of 1.0 mm. Bond strength (BS) was assessed using a universal testing machine until displacement of the filling mass. Failure pattern was evaluated under a stereomicroscope (20x magnification). BS data were analyzed using two-way ANOVA followed by Tukey's test (p<0.05), and the association between the failure pattern and BS value was assessed using the chi-square test (p<0.05). Penetrability was qualitatively evaluated. The highest BS values were observed in the AHP/AHP (4.54±1.5 MPa) and BCS/AHP (5.00±1.0 MPa) groups (p<0.05), with a higher percentage of adhesive failures to the filling material for all groups. LSCFM images indicated greater penetrability of AHP compared to BCS, both in initial treatment and retreatment. AHP sealer exhibited higher BS and greater penetrability compared to BCS sealer.

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.

Fabrication of micro patterns on BaTiO3:Er3+/Yb3+ perovskite films by femtosecond laser micromachining

Femtosecond lasers currently show much promise as potential sources for the fabrication of optical and photonic devices, displaying high performance due to their high resolution, excellent geometric flexibility, and low collateral damage. In particular, devices displaying high performance based on BaTiO3:Er3+/Yb3+ (BTEY) films have been widely investigated, promising for the innovation of perovskite patterning applications. Herein, we fabricated BTEY perovskite films by spin coating at room temperature. XRD pattern and Raman spectroscopy analysis of the BTEY films revealed a tetragonal crystal structure. The study of Williamson-Hall's method calculated the strain and crystallite size for the BTEY film. Different parameters in the exploiting cylindrical focusing of an fs-laser were adopted to modulate the crater cavity of patterning, which gives the opportunities to evaluate their impact on the energy ablation threshold (Eth). It was found that cavity radius is proportional to the energy cross-section, thus providing wide range dependence of damaged spot size versus pulse energy. The Eth was measured using Liu's method, determining a value of Eth that decreased from 33.4 nJ to 22.4 nJ when the number of applied laser pulses per spot increased. It is found that the number of pulses per spot low possesses the best performance, successfully producing highly uniform patterning. Moreover, the evolution with the number of pulses also revealed changes in the surface morphology of the crater cavity via AFM and scanning confocal fluorescence microscopy (SCFM) analysis. These results are crucial for determining the Eth required to damage the material, understating the driving mechanism of formations of cavity profile surfaces, and changes in their optics properties in the edges owing to the balling effects and structural defects.

Single-Molecule Ultrafast Fluorescence-Detected Pump-Probe Microscopy.

We introduce fluorescence-detected pump-probe microscopy by combining a wavelength-tunable ultrafast laser with a confocal scanning fluorescence microscope, enabling access to the femtosecond time scale on the micrometer spatial scale. In addition, we obtain spectral information from Fourier transformation over excitation pulse-pair time delays. We demonstrate this new approach on a model system of a terrylene bisimide (TBI) dye embedded in a PMMA matrix and acquire the linear excitation spectrum as well as time-dependent pump-probe spectra simultaneously. We then push the technique toward single TBI molecules and analyze the statistical distribution of their excitation spectra. Furthermore, we demonstrate the ultrafast transient evolution of several individual molecules, highlighting their different behavior in contrast to the ensemble due to their individual local environment. By correlating the linear and nonlinear spectra, we assess the effect of the molecular environment on the excited-state energy.

Super-resolution for confocal scanning fluorescence microscopy by rotating line-shape point spread function of super-oscillation technique

Super-resolution for confocal scanning fluorescence microscopy by rotating line-shape point spread function of super-oscillation technique

Imaging quality enhancement for high numerical aperture confocal scanning fluorescence microscopy by using two beams

The confocal scanning fluorescence microscopy is a powerful and effective tool which makes a great contribution to explore life sciences. Nevertheless, according to light-diffraction theory, the spatial resolution of this device is predicted to near 200 nm. In order to gain an insight into life science, this spatial resolution of the confocal scanning fluorescence microscopy must be designed to break the limit of theory of light diffraction. In this paper, the improvement of the higher spatial resolution for the confocal scanning fluorescence microscopy is introduced by using two illumination beams for activating the sample. The resolution-improving ability of the proposed method has been demonstrated by the comparison of the imaging results between the confocal scanning fluorescence microscopy with the Gaussian illumination beam and the proposed method.

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.

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.

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.

A Functional Relationship Between UNC45A and MYO5B Connects Two Rare Diseases With Shared Enteropathy.

Background & AimsUNC45A is a myosin (co-)chaperone, and mutations in the UNC45A gene were recently identified in osteo-oto-hepato-enteric (O2HE) syndrome patients presenting with congenital diarrhea and intrahepatic cholestasis. Congenital diarrhea and intrahepatic cholestasis are also the prime symptoms in patients with microvillus inclusion disease (MVID) and mutations in MYO5B, encoding the recycling endosome–associated myosin Vb. The aim of this study was to determine whether UNC45A and myosin Vb are functionally linked.MethodsCRISPR-Cas9 gene editing and site-directed mutagenesis were performed with intestinal epithelial and hepatocellular cell lines, followed by Western blotting, quantitative polymerase chain reaction, and scanning electron and/or confocal fluorescence microscopy to determine the relationship between (mutants of) UNC45A and myosin Vb.ResultsUNC45A depletion in intestinal and hepatic cells reduced myosin Vb protein expression, and in intestinal epithelial cells, it affected 2 myosin Vb-dependent processes that underlie MVID pathogenesis: rat sarcoma-associated binding protein (RAB)11A-positve recycling endosome positioning and microvilli development. Reintroduction of UNC45A in UNC45A-depleted cells restored myosin Vb expression, and reintroduction of UNC45A or myosin Vb, but not the O2HE patient UNC45A-c.1268T>A variant, restored recycling endosome positioning and microvilli development. The O2HE patient-associated p.V423D substitution, encoded by the UNC45A-c.1268T>A variant, impaired UNC45A protein stability but as such not the ability of UNC45A to promote myosin Vb expression and microvilli development.ConclusionsA functional relationship exists between UNC45A and myosin Vb, thereby connecting 2 rare congenital diseases with overlapping enteropathy at the molecular level. Protein instability rather than functional impairment underlies the pathogenicity of the O2HE syndrome–associated UNC45A-p.V423D mutation.

Glutathione-responsive molecular nanoparticles from a dianionic bolaamphiphile and their use as carriers for targeted delivery

The formation in aqueous media of molecular nanoparticles from a bolaamphiphile (SucIleCsa) incorporating a disulfide moiety is described. The particles can be loaded efficiently with the lipophilic mitochondrial marker DiOC6(3), quenching its fluorescence, which is recovered upon reductive particle disassembly. DiOC6(3) transport into human colorectal adenocarcinoma cells (HT-29) is demonstrated using flow cytometry and confocal scanning fluorescence microscopy. A significant increase in intracellular fluorescence is observed when the cells are stimulated to produce glutathione (GSH). These new molecular nanoparticles can be considered a theranostic tool that simultaneously achieves targeted delivery of lipophilic substances and signals high levels of GSH.

Development of high-speed, integrated high-resolution optical coherence microscopy and dual-channel fluorescence microscopy for the simultaneous co-registration of reflectance and fluorescence signals

Optical coherence microscopy is an imaging technique capable of generating three-dimensional images of structures in tissue, but with limited functional imaging ability. Fluorescence microscopy offers functional information of cellular activities. The integration of both imaging modalities can enable the evaluation of the impact of structural information on cells activities. To develop an integrated system combining high-resolution optical coherence microscopy (HR-OCM) and dual-channel scanning confocal fluorescence microscopy (DC-SCFM) to enable the co-registration of structural and functional information. Different strategies to enable the simultaneous recording of information, as well as to overcome the focal plane mismatch between both imaging modalities were developed. The system's performances were evaluated in imaging fluorescence microspheres embedded in multi-layer tape and silicone phantom. HR-OCM achieved an axial resolution of 2.4-µm in tissue over an imaging depth of 1-mm. A speed of 250 kHz and a lateral resolution of 2-µm over a field-of-view of 1.1 mm ×1.1 mm were demonstrated with the combined system. DC-SCFM allowed to discriminate between green and red microspheres while HR-OCM enabled their depth localization. The combined system is synergistic in generating structural and functional information of samples, and has the potential to enable new investigations of biological processes in animal models.

Catalase-loaded polymersomes as a promising safe ingredient to active photoprotection

The topical application of enzymes for UV-induced damage repair, such as catalase, represents an interesting strategy for "active" photoprotection. However, catalase, a large and hydrophilic molecule, needs to penetrate the skin to present activity and, in this sense, nanocarriers can provide an aid for barrier crossing. Among the nanocarriers, polymersomes (Ps) obtained by the self-aggregation of amphiphilic block copolymers represent an alternative for topical enzyme delivery. Here, catalase-loaded Ps of Pluronic® L121, an amphiphilic triblock copolymer, were prepared by direct dissolution and in vitro evaluated regarding safety and efficacy. The Ps presented a suitable photo/cytotoxicity profile regarding the sun-damaged epidermis. Pre-clinical safety tests indicated that the HET-CAM assay was more appropriate than cytotoxicity assays to predict the irritant potential of the Ps. Based on cutaneous permeation assays associated with laser scanning confocal fluorescence microscopy, the Ps increased ten times the catalase percentage of skin permeation. Additionally, in vitro assays indicated that catalase-loaded Ps were able to eliminate UV-induced lipid peroxidation in deeper layers of the skin (viable epidermis + dermis) in comparison to free catalase. Penetration assays in 3D cell culture and efficacy assays confirmed the preferential delivery of catalase in the deeper layers of the skin owing to the permeation enhancer effect of Pluronic L121 and/or the membrane translocation capacity of the Ps. In conclusion, our findings indicate catalase-loaded Ps as an innovative nanomaterial for active sunscreens.

Intrapulpal Concentration of Hydrogen Peroxide of Teeth Restored With Bulk Fill and Conventional Bioactive Composites.

This study evaluated intrapulpal concentration and hydrogen peroxide (HP) penetration at the interface of teeth restored with bioactive composites, using conventional or bulk-fill composites. Cylindrical cavities were prepared on the buccal surface of bovine incisor crowns (n=20) and restored with: resin modified glass-ionomer (RMGI, Riva Light Cure, SDI), non-bioactive bulk-fill composite (FB, Filtek Bulk, 3M Oral Care), non-bioactive conventional composite (FZ, Filtek Z350, 3M Oral Care), bioactive bulk-fill composite (AC, Activa BioActive, Pulpedent), and bioactive conventional composite (BII, Beautifil II, Shofu). After 5,000 thermal cycles, restorations (n=10) were exposed to high (35% HP [4 applications of 8 min/session-4 sessions]) or low (9.5% HP [30 min/day-14 days]) concentration bleaching protocols. After the last bleaching application, the HP intrapulpal concentration was determined. Additional teeth were restored, pigmented with rhodamine B solution, and HP penetration around the interface was observed under laser scanning confocal fluorescence microscopy (LSCFM, n=3). The presence of gaps at the interface was observed on replicas of the cross-sectioned samples under scanning electron microscopy (SEM, n=5). Data were submitted to one-way (gap analysis) and twoway analysis of variance (ANOVA; HP intrapulpal concentration) and Tukey test (α=0.05). The LSCFM images were qualitatively analyzed. The restored teeth submitted to 35% HP presented higher HP intrapulpal concentration than teeth submitted to 9.5% HP (p<0.05). No differences in HP intrapulpal concentration was observed among groups (p>0.05) when exposed to 9.5% HP. Lower HP intrapulpal concentration was observed for teeth restored with RMGI exposed to HP 35%, when compared with teeth restored with nonbioactive conventional (FZ; p=0.004) and bulk-fill composites (FB; p=0.01). No gap formation was observed at the outer enamel adhesive interface for all restorative materials. LSCFM images showed that 35% HP promoted greater degradation of rhodamine B at the enamel, except for RMGI. In this context, RMGI promoted lower HP intrapulpal concentration than non-bioactive conventional and bulk-fill composites.

Limited-angle tomographic phase microscopy utilizing confocal scanning fluorescence microscopy.

We present a multimodal imaging technique, combining tomographic phase microscopy with limited angular projection range and number, and two-channel spinning-disk confocal scanning fluorescence microscopy. This technique allows high-accuracy 3D refractive index (RI) profiling of live cells in spite of the missing projections. The cellular outer shape and its interior organelles measured by the confocal fluorescence imaging not only specify the cell in molecular levels, but also provide the 3D distributions of the whole cell as well as its organelles. We take these additional 3D morphological details as constraints in Gerchberg-Papoulis-based optical diffraction tomography algorithm. We then obtain an accurate 3D RI tomogram, even with a sparse angular range having a small number of perspective projections, otherwise providing low-accuracy RI reconstruction. Then, we obtain both cellular molecular specificity and inner RI values of the cell and its organelles. We compare the reconstructed 3D RI profiles of various samples, demonstrating the superiority of the proposed technique.

Arrays of Si vacancies in 4H-SiC produced by focused Li ion beam implantation

Point defects in SiC are an attractive platform for quantum information and sensing applications because they provide relatively long spin coherence times, optical spin initialization, and spin-dependent fluorescence readout in a fabrication-friendly semiconductor. The ability to precisely place these defects at the optimal location in a host material with nano-scale accuracy is desirable for integration of these quantum systems with traditional electronic and photonic structures. Here, we demonstrate the precise spatial patterning of arrays of silicon vacancy ({V}_{Si}) emitters in an epitaxial 4H-SiC (0001) layer through mask-less focused ion beam implantation of Li+. We characterize these arrays with high-resolution scanning confocal fluorescence microscopy on the Si-face, observing sharp emission lines primarily coming from the {V1}^{{prime}} zero-phonon line (ZPL). The implantation dose is varied over 3 orders of magnitude, leading to {V}_{Si} densities from a few per implantation spot to thousands per spot, with a linear dependence between ZPL emission and implantation dose. Optically-detected magnetic resonance (ODMR) is also performed, confirming the presence of V2 {V}_{Si}. Our investigation reveals scalable and reproducible defect generation.

Robust scan synchronized force-fluorescence imaging

Simultaneous atomic force microscope (AFM) and sample scanning confocal fluorescence microscope measurements are widely used to obtain mechanistic and structural insights into protein dynamics in live cells. However, the absence of a robust technique to synchronously scan both AFM and confocal microscope piezo stages makes it difficult to visualize force-induced changes in fluorescent protein distribution in cells. To address this challenge, we have built an integrated AFM-confocal fluorescence microscope platform that implements a synchronous scanning method which eliminates image artifacts from piezo motion ramping, produces accurate pixel binning and enables the collection of a scanned image of a sample while applying force to a single point on the sample. As proof of principle, we use this instrument to monitor the redistribution of fluorescent E-cadherin, an essential transmembrane protein, in live cells, upon application of mechanical force.

Extending the depth of field in hybrid imaging system by subtracting different asymmetrical phase masks

The subtraction method has been proposed to improve resolution and contrast in confocal scanning fluorescent microscopy. In this paper, we propose applying the subtraction method based on the use of rotating two different asymmetric phase masks along three directions to extend the depth of field. The combining optical transfer function is generated by using three subtraction optical transfer functions. The proposed method can produce high-quality image, in which the digital processing by a deconvolution filter absents. A square root phase mask is used to demonstrate the proposed concept. Compared with the traditional imaging system with the clear aperture, the subtraction method can be used to attain sharp and invariant image over a wide range of defocus.

The Effects of Syndecan on Osteoblastic Cell Adhesion Onto Nano-Zirconia Surface.

PurposeZirconia is one of the most promising implant materials due to its favorable physical, mechanical and biological properties. However, until now, we know little about the mechanism of osseointegration on zirconia. The purpose of this study is to evaluate the effect of Syndecan (Sdc) on osteoblastic cell (MC3T3-E1) adhesion and proliferation onto zirconia materials.Materials and MethodsThe mirror-polished disks 15 mm in diameter and 1.5 mm in thick of commercial pure titanium (CpTi), 3mol% yttria-stabilized tetragonal zirconia polycrystalline (3Y-TZP) and nano-zirconia (NanoZr) are used in this study. MC3T3-E1 cells were seeded onto specimen surfaces and subjected to RNA interference (RNAi) for Syndecan-1, Syndecan-2, Syndecan-3, and Syndecan-4. At 48h post-transfection, the cell morphology, actin cytoskeleton, and focal adhesion were observed using scanning electron microscopy or laser scanning confocal fluorescence microscopy. At 24h and 48h post-transfection, cell counting kit-8 (CCK-8) assay was used to investigate cell proliferation.ResultsThe cell morphology of MC3T3-E1 cells on CpTi, 3Y-TZP, and NanoZr changed into abnormal shape after gene silencing of Syndecan. Among the Syndecan family, Sdc-2 is responsible for NanoZr-specific morphology regulation, via maintenance of cytoskeletal conformation without affecting cellular attachment. According to CCK-8 assay, Sdc-2 affects the osteoblastic cell proliferation onto NanoZr.ConclusionWithin the limitation of this study, we suggest that Syndecan affects osteoblastic cell adhesion on CpTi, 3Y-TZP, and NanoZr. Sdc-2 might be an important heparin-sensitive cell membrane regulator in osteoblastic cell adhesion, specifically on NanoZr, through the organization of actin cytoskeleton and affects osteoblastic cell proliferation.

Solid-State Donor-Acceptor Coaxial Heterojunction Nanowires via Living Crystallization-Driven Self-Assembly.

The creation of organic heterojunctions from conjugated polymers on the nanoscale has attracted recent attention as a consequence of their considerable potential in optoelectronic devices. Herein, we report proof-of-concept results on a versatile synthetic strategy to access various linearly segmented nanowire heterojunctions with controlled dimensions using the seeded growth "living crystallization-driven self-assembly" method followed by a secondary crystallization step. Specifically, we describe the creation of coaxial and also segmented coaxial B-A-B and A-B-A nanowires with a solvophilic poly(ethylene glycol) (PEG) corona, an inner crystalline core that consists of poly(di-n-hexylfluorene) (PDHF), which functions as a donor, and an outer crystalline core of poly(3-(2'-ethylhexyl)thiophene) (P3EHT), which acts as an acceptor. The latter is present either along the entire nanowire or solely in the central or terminal segments. These assemblies were created by seeded growth of two types of π-conjugated polymeric building blocks, the triblock copolymer PDHF-b-P3EHT-b-PEG and the diblock copolymer PDHF-b-PEG, by using fiber-like seeds derived from either material. The nanowires with both solid-state donor and acceptor blocks exhibit Förster resonance energy transfer (FRET) from the PDHF inner core to the P3EHT outer core which was characterized by fluorescence spectroscopy and laser confocal scanning fluorescence microscopy (LCSM). The FRET in the solid-state coaxial heterojunctions with an inner PDHF core and an outer P3EHT core was enhanced relative to the directly analogous system in which the P3EHT block was solvated.