Spinning Disk Confocal Microscopy for Optimized and Quantified Live Imaging of 3D Mitochondrial Network.

Mitochondria are the energy factories of a cell, and depending on the metabolic requirements, the mitochondrial morphology, quantity, and membrane potential in a cell change. These changes are frequently assessed using commercially available probes. In this study, we tested the suitability of three commercially available probes-namely 5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolo-carbocyanine iodide (JC-1), MitoTracker Red CMX Rox (CMXRos), and tetramethylrhodamine methyl ester (TMRM)-for assessing the mitochondrial quantity, morphology, and membrane potential in living human mesoangioblasts in 3D with confocal laser scanning microscope (CLSM) and scanning disk confocal microscope (SDCM). Using CLSM, JC-1, and CMXRos-but not TMRM-uncovered considerable background and variation. Using SDCM, the background signal only remained apparent for the JC-1 monomer. Repetitive imaging of CMXRos and JC-1-but not TMRM-demonstrated a 1.5-2-fold variation in signal intensity between cells using CLSM. The use of SDCM drastically reduced this variation. The slope of the relative signal intensity upon repetitive imaging using CLSM was lowest for TMRM (-0.03) and highest for CMXRos (0.16). Upon repetitive imaging using SDCM, the slope varied from 0 (CMXRos) to a maximum of -0.27 (JC-1 C1). Conclusively, our data show that TMRM staining outperformed JC-1 and CMXRos dyes in a (repetitive) 3D analysis of the entire mitochondrial quantity, morphology, and membrane potential in living cells.

Focused Ultrasound as a Novel Non-Invasive Method for the Delivery of Gold Nanoparticles to Retinal Ganglion Cells.

Purpose: The blood-retinal barrier (BRB) restricts the delivery of intravenous therapeutics to the retina, necessitating innovative approaches for treating retinal disorders. This study sought to explore the potential of focused ultrasound (FUS) to non-invasively deliver intravenously administered gold nanoparticles (AuNPs) across the BRB. FUS-BRB modulation can offer a novel method for targeted retinal therapy. Methods: AuNPs of different sizes and shapes were characterized, and FUS parameters were optimized to permeate the BRB without causing retinal damage in a rodent model. The delivery of 70-kDa dextran and AuNPs to the retinal ganglion cell (RGC) layer was visualized using confocal and two-photon microscopy, respectively. Histological and statistical analyses were conducted to assess the effectiveness and safety of the procedure. Results: FUS-BRB modulation resulted in the delivery of dextran and AuNPs to the RGC and inner nuclear layer. Smaller AuNPs reached the retinal layers to a greater extent than larger ones. The delivery of dextran and AuNPs across the BRB with FUS was achieved without significant retinal damage. Conclusions: This investigation provides the first evidence, to our knowledge, of FUS-mediated AuNP delivery across the BRB, establishing a foundation for a targeted and non-invasive approach to retinal treatment. The results contribute to developing promising non-invasive therapeutic strategies in ophthalmology to treat retinal diseases. Translational Relevance: Modifying the BRB with ultrasound offers a targeted and non-invasive delivery strategy of intravenous therapeutics to the retina.

Effect of toothbrushing with conventional and whitening dentifrices on monolithic zirconia after finishing procedures.

PURPOSE: To evaluate the effect of toothbrushing with conventional and whitening dentifrices on the color difference (ΔE00), gloss (Δgloss), and surface roughness (SR) of stained stabilized zirconia with 5 mol% of yttrium oxide (5Y-TZP) after polishing or glazing. METHODS: Specimens were divided into four groups (n=20): C (control), S (staining), SG (staining and glazing) and SP (staining and polishing). 50,000 toothbrushing cycles were performed with conventional (n=10) and whitening (n= 10) dentifrice slurries. The ΔE00 and Δgloss were measured using a spectrophotometer and CIEDE2000 system while SR was measured by laser confocal microscope. The ΔE00 and Δgloss data were analyzed using 2-way ANOVA, and SR data were analyzed using the linear repeated measures model, with Bonferroni's complementary test (α= 0.05). RESULTS: The ΔE00 values were beyond the acceptability threshold and no differences were found among the groups. There was no difference among groups to Δgloss after toothbrushing with conventional dentifrice while SP presented the highest values of Δgloss after toothbrushing with whitening dentifrice. Conventional dentifrice decreased the SR of stained groups and whitening dentifrice decreased SR of S and SG. The toothbrushing with conventional and whitening dentifrices promoted color difference, but did not impair gloss and surface roughness of stained 5Y-TZP. CLINICAL SIGNIFICANCE: Monolithic zirconia has been routinely used for esthetic restorations, however the type of finishing procedures that is carried out on it must be taken into consideration, in addition to the fact that brushing can influence the color difference of the material as well as interfere with surface roughness and gloss.

Intrathoracic non-tuberculous mycobacteriosis with endobronchial lesion in a child aged 11 with HIV infection diagnosed by bronchoscopic biopsy, EBUS-TBNA and confocal laser endomicroscopy.

The diagnosis of intrathoracic non-tuberculous mycobacteriosis (NTM) is challenging. We report a case of a pediatric pulmonary NTM with endobronchial lesion and lymphadenitis in a child with HIV infection diagnosed by bronchoscopic biopsy, EBUS-TBNA and probe-based confocal laser endomicroscopy (pCLE). The pCLE showed a large number of highly fluorescent cells and zones of density and disorganized elastin fibers at alveolar areas. A combination of diagnostic endoscopic procedures is required to establish the diagnosis of NTM.

Spatial variations in the osteocyte lacuno-canalicular network density and analysis of the connectomic parameters.

Osteocyte lacuno-canalicular network (LCN) is comprised of micrometre-sized pores and submicrometric wide channels in bone. Accumulating evidence suggests multiple functions of this network in material transportation, mechanobiological signalling, mineral homeostasis and bone remodelling. Combining rhodamine staining and confocal laser scanning microscopy, the longitudinal cross-sections of six mouse tibiae were imaged, and the connectome of the network was quantified with a focus on the spatial heterogeneities of network density, connectivity and length of canaliculi. In-vivo loading and double calcein labelling on these tibiae allowed differentiating the newly formed bone from the pre-existing regions. The canalicular density of the murine cortical bone varied between 0.174 and 0.243 µm/µm3, and therefore is three times larger than the corresponding value for human femoral midshaft osteons. The spatial heterogeneity of the network was found distinctly more pronounced across the cortex than along the cortex. We found that in regions with a dense network, the LCN conserves its largely tree-like character, but increases the density by including shorter canaliculi. The current study on healthy mice should serve as a motivating starting point to study the connectome of genetically modified mice, including models of bone diseases and of reduced mechanoresponse.

Assessing the distribution of cancer stem cells in tumorspheres.

Cancer Stem Cells presumably drive tumor growth and resistance to conventional cancer treatments. From a previous computational model, we inferred that these cells are not uniformly distributed in the bulk of a tumorsphere. To confirm this result, we cultivated tumorspheres enriched in stem cells, and performed immunofluorescent detection of the stemness marker SOX2 using confocal microscopy. In this article, we present an image processing method that reconstructs the amount and location of the Cancer Stem Cells in the spheroids. Its advantage is the use of a statistical criterion to classify the cells in Stem and Differentiated, instead of setting an arbitrary threshold. Moreover, the analysis of the experimental images presented in this work agrees with the results from our computational models, thus enforcing the notion that the distribution of Cancer Stem Cells in a tumorsphere is non-homogeneous. Additionally, the method presented here provides a useful tool for analyzing any image in which different kinds of cells are stained with different markers.

A Pipeline for Dynamic Analysis of Mitochondrial Content in Developing T Cells: Bridging the Gap Between High-Throughput Flow Cytometry and Single-Cell Microscopy Analysis.

Analyzing the dynamics of mitochondrial content in developing T cells is crucial for understanding the metabolic state during T cell development. However, monitoring mitochondrial content in real-time needs a balance of cell viability and image resolution. In this chapter, we present experimental protocols for measuring mitochondrial content in developing T cells using three modalities: bulk analysis via flow cytometry, volumetric imaging in laser scanning confocal microscopy, and dynamic live-cell monitoring in spinning disc confocal microscopy. Next, we provide an image segmentation and centroid tracking-based analysis pipeline for automated quantification of a large number of microscopy images. These protocols together offer comprehensive approaches to investigate mitochondrial dynamics in developing T cells, enabling a deeper understanding of their metabolic processes.

Assessment of sealing efficacy, radiopacity, and surface topography of a bioinspired polymer for perforation repair.

Background: Root perforation repair presents a significant challenge in dentistry due to inherent limitations of existing materials. This study explored the potential of a novel polydopamine-based composite as a root repair material by evaluating its sealing efficacy, radiopacity, and surface topography. Methods: Confocal microscopy assessed sealing ability, comparing the polydopamine-based composite to the gold standard, mineral trioxide aggregate (MTA). Radiopacity was evaluated using the aluminium step wedge technique conforming to ISO standards. Surface roughness analysis utilized atomic force microscopy (AFM), while field emission scanning electron microscopy (FESEM) visualized morphology. Results: The polydopamine-based composite exhibited significantly superior sealing efficacy compared to MTA (P < 0.001). Radiopacity reached 3 mm aluminium equivalent, exceeding minimum clinical requirements. AFM analysis revealed a smooth surface topography, and FESEM confirmed successful composite synthesis. Conclusion: This study demonstrates promising properties of the polydopamine-based composite for root perforation repair, including superior sealing efficacy, clinically relevant radiopacity, and smooth surface topography. Further investigation is warranted to assess its clinical viability and potential translation to endodontic practice.

Fibrillogenesis in collagen hydrogels accelerated by carboxylated microbeads.

Collagen type I is a material widely used for 3D cell culture and tissue engineering. Different architectures, such as gels, sponges, membranes, and nanofibers, can be fabricated with it. In collagen hydrogels, the formation of fibrils and fibers depends on various parameters, such as the source of collagen, pH, temperature, concentration, age, etc. In this work, we study the fibrillogenesis process in collagen type I hydrogels with different types of microbeads embedded, using optical techniques such as turbidity assay and confocal reflectance microscopy. We observe that microbeads embedded in the collagen matrix hydrogels modify the fibrillogenesis. Our results show that carboxylated fluorescent microbeads accelerate 3.6 times the gelation, while silica microbeads slow down the formation of collagen fibrils by a factor of 1.9, both compared to pure collagen hydrogels. Our observations suggest that carboxylate microbeads act as nucleation sites and the early collagen fibrils bind to the microbeads.

Root Hair Imaging Using Confocal Microscopy.

Plant genetics plays a key role in determining root hair initiation and development. A complex network of genetic interactions therefore closely monitors and influences root hair phenotype and morphology. The significance of these genes can be studied by employing, for instance, loss-of-function mutants, overexpression plant lines, and fluorescently labeled constructs. Confocal laser scanning microscopy is a great tool to visually observe and document these morphological features. This chapter elaborates the techniques involved in handling of microscopic setup to acquire images displaying root hair distribution along the fully elongated zone of Arabidopsis thaliana roots. Additionally, we illustrate an approach to visualize early fate determination of epidermal cells in the root apical meristem, by describing a method for imaging YFP tagged transgenic plant lines.

Oral mucosa - an examination map for confocal laser endomicroscopy within the oral cavity: an experimental clinical study.

OBJECTIVES: Confocal laser endomicroscopy (CLE) is an optical method that enables microscopic visualization of oral mucosa. Previous studies have shown that it is possible to differentiate between physiological and malignant oral mucosa. However, differences in mucosal architecture were not taken into account. The objective was to map the different oral mucosal morphologies and to establish a "CLE map" of physiological mucosa as baseline for further application of this powerful technology. MATERIALS AND METHODS: The CLE database consisted of 27 patients. The following spots were examined: (1) upper lip (intraoral) (2) alveolar ridge (3) lateral tongue (4) floor of the mouth (5) hard palate (6) intercalary line. All sequences were examined by two CLE experts for morphological differences and video quality. RESULTS: Analysis revealed clear differences in image quality and possibility of depicting tissue morphologies between the various localizations of oral mucosa: imaging of the alveolar ridge and hard palate showed visually most discriminative tissue morphology. Labial mucosa was also visualized well using CLE. Here, typical morphological features such as uniform cells with regular intercellular gaps and vessels could be clearly depicted. Image generation and evaluation was particularly difficult in the area of the buccal mucosa, the lateral tongue and the floor of the mouth. CONCLUSION: A physiological "CLE map" for the entire oral cavity could be created for the first time. CLINICAL RELEVANCE: This will make it possible to take into account the existing physiological morphological features when differentiating between normal mucosa and oral squamous cell carcinoma in future work.

Electrophysiology of Ctenophore Smooth Muscle.

Unlike in the Cnidaria, where muscle cells are coupled together into an epithelium, ctenophore muscles are single, elongated, intramesogleal structures resembling vertebrate smooth muscle. Under voltage-clamp, these fibers can be separated into different classes with different sets of membrane ion channels. The ion channel makeup is related to the muscle's anatomical position and specific function. For example, Beroe ovata radial fibers, which are responsible for maintaining the rigidity of the body wall, generate sequences of brief action potentials whereas longitudinal fibers, which are concerned with mouth opening and body flexions, often produce single longer duration action potentials.Beroe muscle contractions depend on the influx of Ca2+. During an action potential the inward current is carried by Ca2+, and the increase in intracellular Ca2+ concentration generated can be monitored in FLUO-3-loaded cells. Confocal microscopy in line scan mode shows that the Ca2+ spreads from the outer membrane into the core of the fiber and is cleared from there relatively slowly. The rise in intracellular Ca2+ is linked to an increase in a Ca2+-activated K+ conductance (KCa), which can also be elicited by iontophoretic Ca2+ injection. Near the cell membrane, Ca2+ clearance monitored using FLUO3, matches the decline in the KCa conductance. For light loads, Ca2+ is cleared rapidly, but this fast system is insufficient when Ca2+ influx is maintained. Action potential frequency may be regulated by the slowly developing KCa conductance.

Diagnosis of invasive cutaneous squamous cell carcinoma, imaging and staging.

The assessment of patients with a lesion raising the suspicion of an invasive cutaneous squamous cell carcinoma (cSCC) is a frequent clinical scenario. The management of patients with cSCC is a multistep approach, starting with the correct diagnosis. The two main diagnostic goals are to differentiate from other possible diagnoses and correctly recognize the lesion as cSCC, and then to determine the tumor spread (perform staging), that is if the patient has a common primary cSCC or a locally advanced cSCC, or a metastatic cSCC (with in-transit, regional lymph nodal, or rarely distant metastasis). The multistep diagnostic approach begins with the clinical characteristics of the primary cSCC, it is complemented with features with dermoscopy and, if available, reflectance confocal microscopy and is confirmed with histopathology. The tumor spread is assessed by physical examination and, in some cases, ultrasound and/or computed tomography or magnetic resonance imaging, mainly to investigate for regional lymph node metastasis or for local infiltration into deeper structures. In the last step, the clinical, histologic and radiologic findings are incorporated into staging systems.

Evaluation of surface roughness, wettability and adhesion of multispecies biofilm on 3D-printed resins for the base and teeth of complete dentures.

OBJECTIVE: This study evaluated the surface roughness, wettability and adhesion of multispecies biofilms (Candida albicans, Staphylococcus aureus and Streptococcus mutans) on 3D-printed resins for complete denture bases and teeth compared to conventional resins (heat-polymerized acrylic resin; artificial pre-fabricated teeth). METHODOLOGY: Circular specimens (n=39; 6.0 mm Ø × 2.0 mm) of each group were subjected to roughness (n=30), wettability (n=30) and biofilm adhesion (n=9) tests. Three roughness measurements were taken by laser confocal microscopy and a mean value was calculated. Wettability was evaluated by the contact angle of sessile drop method, considering the mean of the three evaluations per specimen. In parallel, microorganism adhesion to resin surfaces was evaluated using a multispecies biofilm model. Microbial load was evaluated by determining the number of Colony Forming Units (CFU/mL) and by scanning electron microscopy (SEM). Data were subjected to the Wald test in a generalized linear model with multiple comparisons and Bonferroni adjustment, as well as two-way ANOVA (α=5%). RESULTS: The roughness of the conventional base resin (0.01±0.04) was lower than that of the conventional tooth (0.14±0.04) (p=0.023) and 3D-printed base (0.18±0.08) (p<0.001). For wettability, conventional resin (84.20±5.57) showed a higher contact angle than the 3D-printed resin (60.58±6.18) (p<0.001). Higher microbial loads of S. mutans (p=0.023) and S. aureus (p=0.010) were observed on the surface of the conventional resin (S. mutans: 5.48±1.55; S. aureus: 7.01±0.57) compared to the 3D-printed resin (S. mutans: 4.11±1.96; S. aureus: 6.42±0.78). The adhesion of C. albicans was not affected by surface characteristics. The conventional base resin showed less roughness than the conventional dental resin and the printed base resin. CONCLUSION: The 3D-printed resins for base and tooth showed less hydrophobicity and less adhesion of S. mutans and S. aureus than conventional resins.

Evaluation of linear dermatoses by reflectance confocal microscopy in children.

Lichen striatus (LS), linear psoriasis (LPs), linear cutaneous lupus erythematosus (LCLE) and linear lichen planus (LLP) often have similar clinical manifestations, which makes clinical diagnosis with the naked eye difficult; therefore, they are easily misdiagnosed. The purpose of this study was to determine whether reflectance confocal microscopy (RCM) is helpful in differentiating between these four linear dermatoses in children. This retrospective study included 14 patients with LS, nine with LPs, eight with LCLE and 12 with LLP. All patients were analysed using RCM, and biopsies were collected from lesions previously imaged by RCM. For LS, the dermal papillary rings were partially absent, but when present, manifested with small, homogeneously round, bright cells and occasionally highly refractive plump cellular structures, aggregated in clusters. LPs exhibited dark cyst-like structures with small, bright, round cells aggregated at the epidermal level; at the dermal-epidermal junction, homogeneously distributed, enlarged, faint dermal papillary rings and numerous enlarged low-refractive canalicular structures were observed in the superficial dermis. LCLE and LLP exhibited similar manifestations, including epidermal disarray, almost total absence of dermal papillary rings, and various sized refractive structures densely distributed in the dermis. The key distinguishing features of LCLE were the different sized structures mainly clustered around hair follicles, while LLP demonstrated dense structures with a scattered distribution. RCM may be used to distinguish between the key features of LS, LPs, LCLE and LLP in children.

Classification of Neural Stem Cell Activation State In Vitro using Autofluorescence.

Neural stem cells (NSCs) divide and produce newborn neurons in the adult brain through a process called adult neurogenesis. Adult NSCs are primarily quiescent, a reversible cell state where they have exited the cell cycle (G0) yet remain responsive to the environment. In the first step of adult neurogenesis, quiescent NSCs (qNSCs) receive a signal and activate, exiting quiescence and re-entering the cell cycle. Thus, understanding the regulators of NSC quiescence and quiescence exit is critical for future strategies targeting adult neurogenesis. However, our understanding of NSC quiescence is limited by technical constraints in identifying quiescent NSCs (qNSCs) and activated NSCs (aNSCs). This protocol describes a new approach to identify and enrich qNSCs and aNSCs generated in in vitro cultures by imaging NSC autofluorescence. First, this protocol describes how to use a confocal microscope to identify autofluorescent markers of qNSCs and aNSCs to classify NSC activation state using autofluorescence intensity. Second, this protocol describes how to use a fluorescent activated cell sorter (FACS) to classify NSC activation state and enrich samples for qNSCs or aNSCs using autofluorescence intensity. Third, this protocol describes how to use a multiphoton microscope to perform fluorescence lifetime imaging (FLIM) at single-cell resolution, classify NSC activation state, and track the dynamics of quiescent exit using both autofluorescence intensities and fluorescence lifetimes. Thus, this protocol provides a live-cell, label-free, single-cell resolution toolkit for studying NSC quiescence and quiescence exit.

Clinical features and comprehensive treatment of persistent corneal epithelial dysfunction after cataract surgery.

OBJECTIVE: Evaluation of clinical efficacy and safety of tobramycin/dexamethasone eye ointment in treating persistent corneal epithelial dysfunction (PED) after cataract surgery. METHODS: 26 cases diagnosed as PED after cataract surgery accept the tobramycin/dexamethasone ophthalmic ointment and intense pulse light treatment in the Xiamen University of Xiamen eye center between September 2016 and April 2022 were retrospectively analyzed, mainly including clinical manifestations, characteristics of morphological changes imaged by in vivo confocal microscopy, meibomian glands infrared photography, lipid layer thickness (LLT), management and therapeutic effects. RESULTS: There were 26 eyes, include 8(35%) males and 15(65%) females with an average age of 69.6 ± 5.2 years(50 to 78 years). The mean hospitalization time was (18.4 ± 7.5) days after cataract surgery. Twenty patients had meibomian gland dysfunction. Infrared photography revealed varying loss in the meibomian glands, with a mean score of 3.8 ± 1.2 for gland loss. The mean LLT was 61.6 ± 8.4 nm. After treatment, 20 patients were cured, and 3 received amniotic membrane transplantation. After treatment, the uncorrected visual acuity (UCVA) and best-corrected vision activity (BCVA) improved (P < 0.001), and there was no significant difference in intraocular pressure (IOP) before and after treatment (P > 0.05). CONCLUSIONS: The early manifestation of PED after surgery is punctate staining of the corneal epithelium. Tobramycin and dexamethasone eye ointment bandages have a good repair effect. The meibomian gland massage combined with intense pulse light treatment can effectively shorten the course of the disease.

Combining sophisticated fast FLIM, confocal microscopy, and STED nanoscopy for live-cell imaging of tunneling nanotubes.

Cell-to-cell communication via tunneling nanotubes (TNTs) is a challenging topic with a growing interest. In this work, we proposed several innovative tools that use red/near-infrared dye labeling and employ lifetime-based imaging strategies to investigate the dynamics of TNTs in a living mesothelial H28 cell line that exhibits spontaneously TNT1 and TNT2 subtypes. Thanks to a fluorescence lifetime imaging microscopy module being integrated into confocal microscopy and stimulated emission depletion nanoscopy, we applied lifetime imaging, lifetime dye unmixing, and lifetime denoising techniques to perform multiplexing experiments and time-lapses of tens of minutes, revealing therefore structural and functional characteristics of living TNTs that were preserved from light exposure. In these conditions, vesicle-like structures, and tubular- and round-shaped mitochondria were identified within living TNT1. In addition, mitochondrial dynamic studies revealed linear and stepwise mitochondrial migrations, bidirectional movements, transient backtracking, and fission events in TNT1. Transfer of Nile Red-positive puncta via both TNT1 and TNT2 was also detected between living H28 cells.

Spatial and Temporal Localization of Connexins in Cells Using Confocal Microscopy.

The 21-member connexin family found in humans is the building block of both single-membrane spanning channels (hemichannels) and double-membrane spanning intercellular channels. These large-pore channels are dynamic and typically have a short life span of only a few hours. Imaging connexins from the time of synthesis in the endoplasmic reticulum through to their degradation can be challenging given their distinct assembly states and transient residences in many subcellular compartments. Here, we describe how connexins can be effectively imaged on a confocal microscope in living cells when tagged with fluorescent proteins and when immunolabeled with high affinity anti-connexin antibodies in fixed cells. Temporal and spatial localization of multiple connexins and disease-linked connexin mutants at the subcellular level extensively informs on the mechanisms governing connexin regulation in health and disease.

In-situ investigation of supercooling behaviour during high-pressure shift freezing of pure water and sucrose solution.

Supercooling is a main controllable factor for the fundamental understanding the high-pressure shift freezing (HPSF). In the study, a self-developed device based on the diamond anvil cell (DAC) and confocal Raman microscopy was utilized to realize an in-situ investigation of supercooling behaviour during HPSF of the pure water and sucrose solution. The spectra were used to determine the freezing point which is shown as a spectral phase marker (SD). The hydrogen bond strengths of water and sucrose solution under supercooling states were estimated by peak position and peak area ratio of sub-peaks. The results showed that the OH stretching bands had redshift under supercooling states. Moreover, the addition of sucrose molecules could strengthen the hydrogen bonding strength of water molecules under supercooling states. Thus, the DAC combined with Raman spectroscopy could be considered a novel strategy for a deep understanding of the supercooling behaviour during HPSF.