Search Results (4,691)

Formalin-fixed paraffin-embedded (FFPE) tissues represent an invaluable resource for both basic and clinical research due to their stable preservation of tissue architecture and molecular integrity. Multiplex immunofluorescence (mIF) using tyramide signal amplification (TSA) enables the simultaneous detection of multiple antigens within a single FFPE section. Here, we describe a kit-independent and customizable TSA-based mIF protocol that utilizes commercially available horseradish peroxidase (HRP)-conjugated secondary antibodies and tyramide–fluorophore reagents. The method was applied using FFPE endometriosis tissue, targeting estrogen receptor alpha (ERα), progesterone receptor (PR), α-smooth muscle actin (αSMA), CD20 and CD31. Each staining round was followed by heat-induced epitope removal (HIER) of the bound antibodies while preserving covalently deposited signals. Fluorescence imaging was performed using a multi-channel slide scanner with carefully selected fluorophores to enable optical separation between detection channels. Under the conditions described, the protocol enabled clear visualization of maker-specific staining patterns with preserved tissue morphology. This study provides a practical and flexible TSA-based mIF protocol as a qualitative proof of concept, offering an accessible alternative to commercial kit-based approaches. Further studies will be required to establish quantitative performance and a broader applicability across tissue types. Full article

Hair loss, particularly androgenetic alopecia (AGA) and alopecia areata (AA), is a prevalent condition with widespread psychosocial impact. Recently, low-level laser therapy (LLLT) has emerged as a promising non-invasive therapeutic alternative due to its bioregulatory effects and favorable safety profile compared to conventional pharmacological treatments. In this study, we employed fluorescence lifetime imaging microscopy (FLIM) to investigate the effects of red-light irradiation on hair follicle dynamics and the cutaneous microenvironment in a C57BL/6 mouse model. A hair regeneration model was established to evaluate the efficacy of 650 nm red-light irradiation (bandwidth ± 20 nm). Then, the skin tissue was stained with hematoxylin and eosin (H&E) and followed by FLIM analysis to provide a multidimensional assessment of tissue morphology and metabolic status. Results showed that red-light irradiation significantly increased hair follicle numbers and enhanced adenosine triphosphate (ATP) levels in the skin tissue. FLIM analysis further revealed prolonged fluorescence lifetime values across different epidermal and dermal layers in the irradiated group, indicating significant alterations in the skin metabolic microenvironment. Furthermore, phasor plot analysis enabled precise differentiation between hair follicles and their surrounding skin structures, highlighting FLIM’s high sensitivity and accuracy in evaluating hair growth. In conclusion, this study has provided novel imaging-based insights into the mechanisms of LLLT-induced hair regeneration, highlighting the potential of FLIM as a powerful tool for characterizing the cutaneous microenvironment and quantitatively evaluating phototherapeutic efficacy in future translational applications. Full article

Extrahepatic cholangiocarcinoma (eCCA) is a highly aggressive malignancy arising from the biliary epithelium, with surgical resection representing the only potentially curative treatment. The predominant periductal infiltrating growth pattern, characterized by subepithelial tumor spread and desmoplastic stromal reaction, severely limits the diagnostic sensitivity of conventional endoscopic sampling techniques, which primarily assess the luminal mucosal surface. This review provides a histomorphology-oriented diagnostic framework for indeterminate extrahepatic biliary strictures, integrating advanced endoscopic technologies with emerging optical diagnostic approaches. ERCP combined with cholangioscopy demonstrates superior sensitivity for perihilar strictures, while EUS-guided tissue acquisition shows higher diagnostic yield in distal cholangiocarcinoma, also providing locoregional staging. Advanced EUS technologies—including elastography, contrast harmonic EUS, and Detective Flow Imaging—further improve characterization of indeterminate strictures by evaluating tissue stiffness, microvascular architecture, and periductal infiltration. Ex vivo fluorescence confocal laser microscopy (FCM) enables real-time microscopic evaluation of biopsy specimens, reducing diagnostic turnaround time and minimizing inadequate sampling. A location-adapted diagnostic algorithm integrating cross-sectional imaging, ERCP, cholangioscopy, and EUS is proposed. An integrated, biology-informed endoscopic approach tailored to tumor location and ductal wall involvement may significantly improve early eCCA detection and guide patient selection for curative treatment. Full article

Images obtained from imaging instruments can endure issues such as high degradation, color distortion, and weak brightness. Effective systems for enhancing these images are critically required. To improve the image quality, herein, we propose two filters based on simple functions, including cosine, sine, hyperbolic secant, and the inverse of hyperbolic cosecant. These filters are used for enhancing the images obtained from a fluorescence imaging system, a near-infrared camera, and low-light condition. The contrast is increased while the image quality is improved. They perform better than a matched filter. Moreover, the combination of our filters with the filter based on the watershed algorithm or the matched filter can be used to extract the marginal features from images generated under water environment. Furthermore, their application in image fusion is explored. Our designed filters may be potentially used for future applications on target identification and tracking. Full article

The hERG potassium channel is critical for cardiac ventricular repolarization and a core target in pre-clinical drug safety screening. A robust, stable cell line with uniform, high hERG expression is essential for high-throughput assessments. In this study, we established a functional stable HEK293T cell line with high hERG expression. The hERG gene was subcloned into Lenti-HA-hERG-P2A-EGFP plasmid, in which GFP serves as a selection marker via a P2A self-cleaving peptide. GFP-positive monoclonal cells were isolated by fluorescence-activated cell sorting (FACS). Confocal imaging confirmed that hERG localized predominantly to the cell membrane, consistent with its physiological role. Manual patch-clamp revealed canonical hERG current properties: a small, stable current during depolarization to 20 mV, followed by a large outward tail current upon repolarization to −40 mV-a hallmark of hERG channel gating. Automated patch-clamp (APC)-based current profiling showed 93.5% of stable hERG cells exhibited peak tail currents >50 pA (87% > 100 pA, with 49.5% > 400 pA), whereas 100% of blank HEK293T cells showed peak tail currents < 50 pA. Pharmacological validation with E-4031 demonstrated concentration-dependent inhibition of hERG currents, with an IC₅₀ of 29.8 nM, which is consistent with literature-reported values. The stable hERG-expressing HEK293T cell line developed here exhibits consistent hERG expression, canonical channel function, and physiological sensitivity to hERG blockers. When paired with high-throughput APC systems, this cell model provides a robust, standardized platform for pre-clinical drug-induced hERG inhibition evaluation, aiding early detection of long QT syndrome risks and safer drug development. Full article

We present a reproducible pipeline that converts color images into quantitative fluorescence maps by combining spectral measurement with a linear mixture model. The method is designed specifically for quantitative comparisons of Glo Germ™ on images of hands taken under different experimental conditions with controlled illumination. The emission spectrum of Glo Germ is measured using a spectral photometer and normalized to obtain its spectral power density function. This spectrum is projected into CIE XYZ coordinates and incorporated into a linear mixture model in which each pixel contains contributions from white light, UV-illuminated skin reflectance, and fluorophore emission. Component magnitudes are estimated with non-negative least squares, yielding a grayscale image whose intensity is a monotonic proxy for local fluorophore density. Spatial integration provides an image-level summary proportional to total detected material. Compared with single-channel proxies, the observer suppresses background structure, improves contrast, and remains radiometrically interpretable. Because the method depends only on measurable spectra and linear transforms, it can be reproduced across cameras and extended to other fluorophores. Full article

Cigarette smoke (CS) is the primary risk factor for the development of chronic obstructive pulmonary disease (COPD). Investigating the impact of CS on human airway epithelium is important for understanding COPD development and combating its effects. While some studies show that long exposure to CS activates inflammasome formation in airway epithelium, leading to cytokines’ maturation and release, its acute effect on inflammation regulation requires further elucidation. Due to the importance of acute cellular responses in modulating cell survival and controlling inflammatory outcomes, we examined the effect of acute cigarette smoke extract exposure on human bronchial epithelial cells. Due to the high reactive oxygen species content in CS, we hypothesize that acute CS exposure activates the integrated stress response (ISR) pathway leading to stress granules (SG) formation to facilitate oxidative stress resolution and promote cell survival. Immunostaining, fluorescence confocal imaging, quantitative analyses, and immunoblotting were performed to test our hypothesis. We report here that acute exposure to CS extract triggers canonical SG formation by activating the ISR pathway via the PERK/eIF2α arm in a reactive oxygen species-dependent manner. SG formation is abolished upon inhibiting PERK or eIF2α function, or by scavenging oxidants prior to smoke exposure. Characterizing SG formation in terms of measuring SG size and abundance and the sequestration of the SG marker G3BP1 reveals that SG formation is maximal at 15% CS extract exposure for 2 h and undergoes gradual disassembly at longer exposure times. This is closely dependent on cytoplasmic p-eIF2α levels. These results demonstrate that acute exposure to CS activates the protective ISR pathway to potentially reduce the detrimental effects of CS and promote stress resolution and cell survival. Full article

The protozoan parasite Leishmania mexicana serves as a widely used model for studying trypanosomatid biology, yet the demand for stable, high-intensity fluorescent tools for precise subcellular protein localization remains unmet. In this study, we developed a versatile molecular toolbox by re-engineering the pLEXSY-hyg2.1 vector to express mNeonGreen (mNG), a next-generation fluorophore with superior brightness and photostability. Using a modular cloning strategy, we introduced a customized multiple cloning site (MCS) upstream of the mNG sequence to facilitate seamless N-terminal tagging of target proteins. The construct was integrated into the 18S rRNA locus via homologous recombination to ensure stable, constitutive expression. As a proof-of-concept, we fused a flagellar marker to the mNG reporter, resulting in a transgenic line exhibiting robust and specific subcellular fluorescence without compromising cellular fitness. Our results demonstrate that this integration-based system provides a highly efficient and stable platform for visualizing protein distribution within Leishmania. This tool significantly simplifies the generation of reporter strains and will facilitate high-resolution imaging studies of parasite organelle dynamics and functional genomics. Full article

Background/Objectives: We developed a telehealth-enabled fiber-bundle endomicroscopy platform and evaluated its preclinical feasibility for targeted fluorescence imaging in cervical cancer models. Methods: The platform integrates a portable fiber-bundle endomicroscopy (FBE) system, fluorescein isothiocyanate (FITC)-labeled candidate peptides, and a secure web-based telehealth platform for remote consultation. The FBE probe achieved a field of view of 1,700 µm and a lateral resolution of 4 µm, enabling cellular-level fluorescence imaging in a compact, portable format. Four FITC-labeled peptides (SHS1*, SHS2*, FPP*, and CRL*) were evaluated in A549, SiHa, and CaSki cell lines. Ex vivo testing was performed on commercial cervical tissue-array samples. The telehealth platform was assessed for secure medical-image/video transmission and end-to-end latency in a simulated remote-consultation setting. Results: Among the tested probes, FPP*-FITC and CRL*-FITC showed higher fluorescence-positive fractions in the p16-overexpressing cervical cancer cell lines than in the A549 comparator line, with the strongest signals observed in CaSki cells. In ex vivo testing, CRL*-FITC generated higher fluorescence intensity in malignant cervical tissue-array samples than in non-malignant comparator tissues, with a reported 4.6- to 7.4-fold difference in mean signal intensity (p < 0.001). The telehealth platform supported the secure transmission of medical images and video and demonstrated an end-to-end latency of <500 ms in a simulated remote consultation setting. Conclusions: These results support the technical and preclinical feasibility of integrating targeted fluorescence imaging, portable fiber-bundle endomicroscopy, and telehealth into a single platform. This study should therefore be interpreted as a preclinical feasibility study evaluating optical, molecular, and telehealth integration, rather than as a clinically validated cervical cancer screening test. Full article

Background/Objectives: Bone-targeted drug delivery systems hold great promise for treating skeletal diseases, yet the optimal strategy for functionalizing lipid nanoparticles (LNPs) with bone-homing ligands remains insufficiently explored. Herein, we compared two alendronate sodium (Alen) modification approaches (pre-conjugation and post-conjugation) for constructing bone-targeted LNPs capable of delivering mRNA to skeletal tissues. Methods: LNPs were fabricated via microfluidic mixing, and the 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol-alendronate conjugate (DSPE-PEG-Alen) required for the pre-conjugation method was synthesized. The bone-targeting ability of LNPs prepared by the two Alen modification strategies was evaluated using an in vitro hydroxyapatite (HAP) binding assay. Furthermore, the physicochemical properties, bone-targeting performance, mRNA delivery efficiency, and biosafety of the LNPs prepared by the post-conjugation method were assessed through cellular uptake, in vivo imaging, and other methods. Results: Hydroxyapatite binding assays revealed that the post-conjugation strategy afforded significantly superior bone affinity compared to the pre-conjugation approach. In addition, ex vivo bone fragment binding experiments further confirmed that the bone-targeting LNPs prepared by the post-conjugation method exhibited stronger bone-binding capability compared to unmodified LNPs. The optimized Alen-LNPs demonstrated efficient cellular uptake and functional mRNA translation in bone marrow mesenchymal stem cells with negligible cytotoxicity. In vivo studies in mice confirmed the preferential accumulation of Alen-LNPs in bone tissues, with successful green fluorescent protein (GFP) mRNA translation detected in bone tissue sections. Histopathological analysis confirmed the biosafety of the formulation. Conclusions: This study establishes the post-conjugation strategy as the superior approach for Alen functionalization of LNPs, providing a robust and reproducible platform for bone-targeted mRNA therapeutics. Full article

Soil salinity is a major abiotic stressor that constrains plant growth and development, yet the coordinated regulatory mechanisms underlying salt stress impacts on plant cell mechanical properties and the cytoskeleton remain elusive. In this study, tobacco suspension cells were employed as a model system. Combining mechanical measurements, fluorescence microscopy imaging, and bright-field morphological observation, we systematically characterized the dynamic response patterns of cell-generated surface stress (ΔS), cell viscoelastic index (CVI), microfilament cytoskeleton structure, as well as cell morphology and plasmolysis under NaCl stress ranging from 50 to 150 mmol/L. The results revealed three distinct response thresholds: 50 mmol/L NaCl treatment induced only transient ΔS fluctuations and mild plasmolysis, with no significant changes in CVI or microfilament fluorescence intensity, suggesting a safe tolerance threshold. The 75–100 mmol/L NaCl treatments triggered reversible “rise–recovery” mechanical responses in ΔS and CVI. The microfilament cytoskeleton showed minor structural adjustments, and plasmolysis increased gradually but remained reversible, defining this range as a reversible acclimation phase. The 125–150 mmol/L NaCl treatment caused an irreversible decline in ΔS (with a sharp instantaneous drop at 150 mmol/L). CVI variations diminished and stabilized after 6 h. The microfilament cytoskeleton suffered progressive disruption, as fluorescence intensity dropped to 1% of the control group at 150 mmol/L, accompanied by severe plasmolysis and protoplast shrinkage, indicating irreversible cellular damage. These findings demonstrate a concentration-dependent gradient effect of NaCl stress, highlighting tight coordination between mechanical properties, cytoskeletal integrity, and morphological adaptation. This work provides critical cytological insights into the molecular regulation of plant salt stress responses. Full article

Efficient mixing is a persistent bottleneck in agricultural and agrochemical processing, where rapid and uniform mixing must be achieved under laminar flow with low energy input and gentle shear. Inspired by peristaltic transport in biological systems, this study investigates a bio-inspired flexible-wall squeezing mixer and establishes a two-dimensional computational framework to quantify how periodic wall deformation governs scalar homogenization in a flexible conduit. An Arbitrary Lagrangian–Eulerian dynamic mesh approach is implemented to resolve moving boundaries and to prescribe actuation, enabling the systematic evaluation of the separate and coupled effects of peak wall-normal velocity amplitude A and actuation frequency f on mixing performance. Mixing effectiveness is quantified using a variance-based mixing index MI and a sustained-threshold mixing time ts, and response surface methodology is employed to map the A–f design space and interpret the roles of time-dependent shear, interfacial stretching and folding, and vortex intensification. Relative to a non-actuated baseline, a peak wall-normal velocity amplitude of 3 × 10⁻³ m s⁻¹ at 2 Hz reduces t_s by 21.3%. At fixed f = 3 Hz, increasing A from 1 × 10⁻³ to 4 × 10⁻³ m s⁻¹ shortens t_s by 10.2%, while at fixed A = 3 × 10⁻³ m s⁻¹, raising f from 1 to 5 Hz further decreases t_s by 6.6% with diminishing gains at the lowest frequencies. The response surface identifies an operating optimum at A = 4 × 10⁻³ m s⁻¹ and f = 5 Hz, achieving a peak MI of 0.9557 and a minimum t_s of 7.81 s. A periodically squeezed physical mixing loop was further examined using fluorescence imaging to assess outlet homogeneity trends. The stabilized outlet coefficient of variation (CV) decreased from about 0.65 without squeezing to 0.60 at 1 Hz and 10 mm s⁻¹, 0.58 at 2 Hz and 10 mm s⁻¹, and 0.54 at 2 Hz and 30 mm s⁻¹, indicating that stronger and faster actuation improves outlet uniformity. The numerical and experimental results are therefore interpreted jointly as mechanistic and trend-level evidence, while a rigorous quantitative prediction for the cylindrical compliant device will require future three-dimensional, compliance-resolved simulations and broader experimental benchmarking. Full article

Background: Early detection and monitoring of enamel changes during caries lesion formation are essential for preventive management. This study aimed to evaluate time-dependent enamel demineralization using micro-computed tomography (micro-CT) and to compare its diagnostic performance with laser fluorescence and digital colorimetric image analysis. Methods: Twelve sound human permanent teeth were subjected to a gel-based lactic acid demineralization for 14 days. Assessments were performed at baseline and after 3, 7, and 14 days. Enamel mineral density (MD) and demineralization depth (DD) were measured using micro-CT. Laser fluorescence was evaluated using DIAGNOdent, while colorimetric changes were analyzed through standardized digital imaging using the CIE Lab* system, including ΔE and Whiteness Index (WI). Statistical analysis included repeated measures ANOVA and Pearson correlation (p < 0.05). Results: A significant time-dependent progression of enamel demineralization was observed. Demineralization depth increased from 0.0828 mm (3 days) to 0.234 mm (14 days) (p < 0.001), while mineral density decreased significantly over time (p < 0.001). DIAGNOdent values showed significant increases after 7 and 14 days (p = 0.002). Colorimetric analysis revealed early detectable changes, with ΔE exceeding clinically perceptible thresholds as early as day 3. WI values increased progressively, indicating enhanced enamel opacity. A weak but significant negative correlation between MD and DD was found (p = 0.04). Conclusions: Enamel demineralization progresses in a time-dependent manner and can be effectively monitored using micro-CT, laser fluorescence, and colorimetric analysis. Digital colorimetric analysis may serve as a valuable adjunctive tool in caries diagnostics. Full article

Accurate delineation of tumor margins is critical for complete resection and minimizing recurrence, yet existing imaging modalities such as MRI, CT, and fluorescence imaging suffer from limitations including high cost, limited accessibility, and intraoperative constraints. In this study, we propose a low-cost, non-invasive approach for subsurface imaging based on near-infrared (NIR) illumination combined with deep learning. A controlled experimental setup was developed in which structured patterns displayed on an electronic paper screen were concealed beneath a tissue-mimicking chicken phantom and imaged using a NIR-sensitive camera under halogen illumination. A convolutional autoencoder based on a U-Net architecture was trained on approximately 10,000 paired samples to reconstruct hidden structures from highly scattered surface images. The proposed method achieved strong reconstruction performance, with the best model reaching a peak signal-to-noise ratio (PSNR) of 20.14 dB, structural similarity index (SSIM) of 0.92, and feature similarity index (FSIM) of 0.94, significantly outperforming conventional Wiener filtering. Qualitative results demonstrated accurate recovery of subsurface shapes with minor smoothing artifacts. While generalization to out-of-distribution samples remains limited, the findings highlight the potential of combining NIR imaging and deep learning for safe, rapid, and cost-effective subsurface visualization. This work establishes a foundation for future development toward clinically relevant tumor margin detection. Full article

Background/Objectives: Peritoneal micrometastases (micromets) remain a major barrier to durable cytoreduction in ovarian and other intra-abdominal cancers because lesions are difficult to visualize and are often resistant to systemic therapy. Liposomal doxorubicin (Dox) improves pharmacokinetics but can be limited by slow intratumoral release. Porphyrin-phospholipid (PoP) liposomes enable near-infrared light–triggered release of Dox (chemophototherapy (CPT)), creating an opportunity for intraoperative fluorescence-guided treatment planning and monitoring. Here, we evaluate a laparoscopic fluorescence imaging platform for quantifying light-triggered drug delivery. Methods: LC-Dox-PoP was applied to SCC2095sc and SKOV-3 cultures in 2D monolayers and 3D spheroid clusters. Dox fluorescence was quantified using a laparoscopic fluorescence imaging system over 1–9 μg/mL concentrations and compared with standard well-plate reader measurements. Porphyrin fluorescence was monitored to assess spheroid localization and photobleaching after activation light exposure. Results: For both cell lines, Dox fluorescence exhibited an approximate 4-fold increase at the maximum administered LC-Dox-PoP concentration, following a linear trend in both SCC2095sc and SKOV-3 cultures (R² = 0.97, 0.98 for 2D and R² = 0.98, 0.98 for spheroids). Laparoscope-derived fluorescence measurements agreed with well-plate reader measurements (R² = 0.89–0.96). Porphyrin fluorescence provided stronger complementary contrast for localizing spheroid constructs and decreased after activation light exposure, consistent with photobleaching during triggered release. Conclusions: These results support a quantitative imaging framework for fluorescence-guided monitoring of light-triggered liposomal drug release and may enable individualized CPT dosimetry for peritoneal micrometastases. Findings in SCC2095sc additionally suggest potential relevance of fluorescence-guided CPT for head and neck/oral cancer, where localized post-resection adjuvant treatment may improve control of residual disease. Full article