Search Results (381)

Background: Often, neoadjuvant therapy, which relies on the induction of double-strand breaks (DSBs), is used prior to surgery to shrink tumors by inducing cancer cell apoptosis. However, recent studies have suggested that this treatment may also induce a fluctuating state between senescence and stemness in PA-1 embryonal carcinoma cells, potentially affecting therapeutic outcomes. Thus, the respective epigenetic pathways are up or downregulated over a time period of days. These fluctuations go hand in hand with changes in spatial DNA organization. Methods: By means of Single-Molecule Localization Microscopy in combination with mathematical evaluation tools for pointillist data sets, we investigated the organization of euchromatin and heterochromatin at the nanoscale on the third and fifth day after etoposide treatment. Results: Using fluorescently labeled antibodies against H3K9me3 (heterochromatin tri-methylation sites) and H3K4me3 (euchromatin tri-methylation sites), we found that the induction of DSBs led to the de-condensation of heterochromatin and compaction of euchromatin, with a peak effect on day 3 after the treatment. On day 3, we also observed the co-localization of euchromatin and heterochromatin, which have marks that usually occur in exclusive low-overlapping network-like compartments. The evaluation of the SMLM data using topological tools (persistent homology and persistent imaging) and principal component analysis, as well as the confocal microscopy analysis of H3K9me3- and H3K4me3-stained PA-1 cells, supported the findings that distinct shifts in euchromatin and heterochromatin organization took place in a subpopulation of these cells during the days after the treatment. Furthermore, by means of flow cytometry, it was shown that the rearrangements in chromatin organization coincided with the simultaneous upregulation of the stemness promotors OCT4A and SOX2 and senescence promotors p21Cip1 and p27. Conclusions: Our findings suggest potential applications to improve cancer therapy by inhibiting chromatin remodeling and preventing therapy-induced senescence. Full article

In prostate cancer (PCa) surgery, precise tumor margin identification remains challenging despite advances in surgical techniques. This study evaluates the combination of tumor-specific near-infrared imaging with the PSMA-targeting molecule PSMA-914 and optical endomicroscopy (NIR-pCLE) for single-cell-level tumor identification in a preclinical proof of concept. Methods: NIR-pCLE imaging of varying PSMA-914 concentrations was performed on PSMA-positive LNCaP and PSMA-negative PC-3 cells using Cellvizio^® 100 with pCLE Confocal Miniprobes™. To identify optimal PSMA-914 dosing for in vivo imaging, different doses (0–10 nmol) were evaluated using NIR-pCLE, Odyssey CLx imaging, and confocal microscopy in an LNCaP tumor-bearing xenograft model. A proof of concept mimicking a clinical workflow was performed using 5 nmol [⁶⁸Ga]Ga-PSMA-914 in LNCaP and PC-3 tumor xenografts, including PET/MRI, in/ex vivo NIR-pCLE imaging, and microscopic/macroscopic imaging. Results: NIR-pCLE detected PSMA-specific fluorescence at concentrations above 30 nM in vitro. The optimal dose was identified as 5 nmol PSMA-914 for NIR-pCLE imaging with cellular resolution in LNCaP xenografts. PET/MRI confirmed high tumor uptake and a favorable distribution profile of PSMA-914. NIR-pCLE imaging enabled real-time, single-cell-level detection of PSMA-positive tissue, visualizing tumor heterogeneity, confirmed by ex vivo microscopy and imaging. Conclusions: This preclinical proof of concept demonstrates the potential of intraoperative PSMA-specific NIR-pCLE imaging to visualize tissue structures in real time at cellular resolution. Clinical implementation could provide surgeons with valuable additional information, potentially advancing PCa patient care through improved surgical precision. Full article

Robot-assisted surgery has transformed the landscape of genitourinary cancer treatment, offering enhanced precision, reduced morbidity, and improved recovery compared to open or conventional laparoscopic approaches. As the field matures, a new generation of technological innovations is redefining the boundaries of what robotic systems can achieve. This narrative review explores the integration of artificial intelligence, advanced imaging modalities, augmented reality, and connectivity in robotic urologic oncology. The applications of machine learning in surgical skill evaluation and postoperative outcome predictions are discussed, along with AI-enhanced haptic feedback systems that compensate for the lack of tactile sensation. The role of 3D virtual modeling, intraoperative augmented reality, and fluorescence-guided surgery in improving surgical planning and precision is examined for both kidney and prostate procedures. Emerging tools for real-time tissue recognition, including confocal microscopy and Raman spectroscopy, are evaluated for their potential to optimize margin assessment. This review also addresses the shift toward single-port systems and the rise of telesurgery enabled by 5G connectivity, highlighting global efforts to expand expert surgical care across geographic barriers. Collectively, these innovations represent a paradigm shift in robot-assisted urologic oncology, with the potential to enhance functional outcomes, surgical safety, and access to high-quality care. Full article

Currently, the focus of intraoperative imaging in brain tumor surgery is beginning to shift to optical methods such as optical coherence tomography (OCT), Raman spectroscopy, confocal laser endomicroscopy (CLE), and fluorescence lifetime imaging (FLIM). Optical imaging technologies provide in vivo and real-time high-resolution images of tissues. “Optical biopsy” can be considered as an alternative to traditional approaches for intraoperative histopathologic consultation. Intraoperative optical imaging can help to achieve precise intraoperative identification of tumor infiltrations within the surrounding brain parenchyma. Therefore, it can be considered as a complement to existing approaches based on wide-field imaging modalities such as MRI, US, or 5-ALA fluorescence. A promising future direction for intraoperative guidance during brain tumor surgery or stereotactic biopsy lies in the integration of optical imaging with machine learning techniques, enabling automated differentiation between tumor tissue and healthy brain parenchyma. We present this review to increase knowledge and form critical opinions in the field of using optical imaging in brain tumor surgery. Full article

Background/Objectives: Cutaneous squamous cell carcinoma (cSCC) is the second most common skin cancer, with diverse clinical presentations. This study aims to correlate findings from dermoscopy, ultrasonography, ex vivo confocal microscopy, and histology to improve diagnostic accuracy and guide better clinical management of cSCC. Methods: This cross-sectional study, conducted between July 2022 and December 2024, included 26 patients with 35 clinically suspicious cSCC tumors, analyzed through clinical, dermoscopic, high-frequency ultrasound (HFUS), ex vivo confocal fluorescence microscopy (FCM), and histopathology. Tumors were evaluated for various clinical, imaging, and histopathological criteria, such as tumor thickness, vascularization, differentiation degree, and invasion level, with FCM applied to 24 tumors for advanced microscopic analysis. Results: The study analyzed 35 cases of histopathologically confirmed cSCC, finding that invasive SCC was associated with greater tumor thickness, increased vascularization, and ulceration on both ultrasound and dermatoscopy, while in situ SCC showed homogeneous echogenicity and specific dermoscopic patterns like dotted vessels and white halos. Strong correlations were identified between ultrasound and histopathological measurements of tumor thickness and invasion depth, and confocal microscopy revealed that features like plump bright cells and nest-like structures were linked to invasive and poorly differentiated tumors. Conclusions: This study uniquely integrates advanced imaging techniques—dermatoscopy, skin ultrasound, and ex vivo confocal microscopy—with histopathological analysis to provide new insights into tumor grade, vascularity, and invasion depth in cSCC, enhancing non-invasive diagnosis. Full article

Purpose: The aim of this study was to investigate the protective effects of systemically administered tauroursodeoxycholic acid (TUDCA) in an optic nerve crush (ONC) mouse model of retinal ganglion cell (RGC) death. Methods: C57BL/6J mice were injected intraperitoneally (i.p.) three times per week with TUDCA (500 mg/kg) for two weeks, after which unilateral ONC was performed. A control cohort was identically treated with a drug vehicle (phosphate buffered saline; PBS). A separate cohort did not undergo any injections or surgeries (this was termed the “Naïve” group). Pattern electroretinography (PERG) was recorded 3 days after ONC. Retinas were harvested for whole-mount immunofluorescence staining with an antibody against RGC marker Brn3a and imaged by fluorescent confocal microscopy. Apoptotic cells in the ganglion cell layer (GCL) were detected by Terminal Deoxynucleotidyl Transferase-Mediated dUTP Nick End Labeling (TUNEL) performed on fixed retina sections. Glial fibrillary acidic protein (GFAP) immunostaining on fixed retina sections was conducted to detect the activation of Müller cells. Total RNA was extracted from retinas and expression of interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)-α, and IL-10 was determined by digital droplet PCR (ddPCR). Results: TUDCA treatment preserved visual function as assessed by PERG. P1 and N2 amplitudes from the PBS-treated ONC group were significantly diminished compared to those of the Naïve group (p < 0.001). TUDCA treatment prevented this diminution. The amplitudes of P1 and N2 in the TUDCA-treated ONC group were statistically indistinguishable from those of the Naïve group and were higher than the PBS-treated ONC group (TUDCA+ONC vs. PBS+ONC, P1: 6.99 ± 0.89 µV vs. 3.60 ± 0.69 µV, p < 0.01; N2: −9.30 (IQR: −13.43–−6.44) µV vs. −4.47 (IQR: −10.26–−2.17) µV). TUDCA treatment preserved RGCs. The ONC-vehicle-only group had 25% fewer RGCs (Brn3a-positive cells) than Naïve eyes (p < 0.0001). TUDCA treatment nearly completely prevented this loss, preserving all but 7.7% of the RGCs, and the number of RGCs in the TUDCA-treated ONC group was significantly higher than in the PBS-treated ONC group (TUDCA+ONC vs. PBS+ONC, 1738.00 ± 14.43 cells per field vs. 1454.00 ± 6.55 cells per field, p < 0.0001). The number of TUNEL-positive cells in the GCL (Naïve vs. PBS+ONC group: 1.00 (IQR: 0.00–2.00) % vs. 37.00 (IQR: 8.50–48.50) %, p < 0.05) and GFAP-positive fibers transversing retina sections (Naïve vs. PBS+ONC group: 33.00 ± 1.15 vs. 185.70 ± 42.37 fibers/retina, p < 0.05), and the expression of IL-6, TNF-α were significantly greater in the PBS-treated ONC group compared to that of the Naïve group (Naïve vs. PBS+ONC group, IL-6: 0.07 (IQR: 0.06–0.31) vs. 0.99 (IQR: 0.56–1.47), p < 0.05, TNF-α: 0.19 ± 0.069 vs. 1.39 ± 0.23; p < 0.01), an increase not observed with TUDCA treatment. Conclusions: Systemic TUDCA treatment significantly preserved RGC function and survival in the mouse ONC model of RGC damage. TUDCA treatment prevented RGC apoptosis, Müller glial cell activation, and retinal expression of several inflammatory cytokines. These data suggest that TUDCA is a promising therapeutic candidate for preserving RGC numbers and function. Full article

In homeostasis, the glial cells support pivotal functions, such as neuronal differentiation, neuroprotection, nutrition, drug metabolism, and immune response in the central nervous system (CNS). Among these cells, astrocytes and microglia have been highlighted due to their role in the pathogenesis of several diseases or due to their role in the defense against several insults (ex., chemicals, and pathogens). In Vitro cytological analysis of astrocytes and microglia has contributed to the understanding of the role of morphological changes in glial cells associated with a neuroprotective or neurotoxic phenotype. Currently, the main tools used for the investigation of glial cell morphology in culture are phase contrast microscopy or immunolabeling/fluorescence microscopy. However, generally, phase contrast microscopy does not generate images with high resolution and therefore does not contribute to visualizing a single cell morphology in confluent cell cultures. On the other hand, immunolabeling requires high-cost consumable antibodies, epifluorescence microscope or confocal microscope, and presents critical steps during the procedure. Therefore, identifying a fast, reproducible, low-cost alternative method that allows the evaluation of glial morphology is essential, especially for neuroscientists from low-income countries. This article aims to revise the use of Rosenfeld’s staining, as an alternative low-cost and easy-to-reproduce method to analyze astrocytic and microglial morphology in culture. Additionally, it shows Rosenfeld’s staining as a valuable tool to analyze changes in neural cell morphology in toxicological studies. Full article

The Rab family of small guanosine triphosphatases (GTPases) are nucleotide-dependent switches. Mutations in Rabs can result in human diseases. Rab7a and Rab7b transition from early endosomes to lysosomes and are presumed to function similarly. Most studies look at Rab7a, less on Rab7b, with the underlying assumption they function similarly. There have yet to be articles comparing them side by side. Whilst cloning Rab7 homologues, we identified splice isoforms for Rab7b only. These splice isoforms, Rab7b2 and Rab7bx8 lacking different exons, have not been previously characterized but suggest alternative function(s) for Rab7b. Thus, we hypothesize that Rab7 homologues have distinct functions. Here, we compare Rab7a and Rab7b nucleotide mutants locked in GDP-bound (Rab7T22N), GTP-bound (Rab7Q67L), nucleotide-free (Rab7aN125I/Rab7bN124I) states and characterized localization of the Rab7b splice isoforms. HeLa cells were transiently transfected with fluorescently tagged Rab7 reporters. Confocal images were processed with ImageJ and analyzed with SPSS. Rab7a and Rab7b nucleotide mutants were significantly different to one another. Approximately 50% of Rab7b splice isoform-expressing cells had aggregated vesicles, which were phenotypically different from Rab7b vesicles. Rab7a and Rab7b vesicles shared approximately 60% colocalization with each other, while Rab7b vesicles preferentially localized to the Trans Golgi Network. Our results suggest Rab7b is distinct from Rab7a, and Rab7b splice isoforms have different biological functions. Full article

Protein S-nitrosation, a redox post-translational modification elicited by nitric oxide (NO), is essential for modulating diverse protein functions and signaling pathways. Dysregulation of S-nitrosation is implicated in various pathological processes, including oxygen-glucose deprivation/reperfusion (OGD/R) injury, a widely used model for ischemia-reperfusion diseases. The dynamic changes in S-nitrosothiols (SNOs) during ischemia-reperfusion highlight the need for theranostic strategies to monitor and modulate SNO levels based on pathological progression. However, to date, no theranostic strategies have been reported for addressing dysregulated SNO in disease models, particularly in OGD/R conditions. Here, we report the development of a selective probe P-EHC, which could specifically react with SNOs to release EHC, not only exhibiting turn-on fluorescence with high quantum yield and good water solubility but also demonstrating macrophage migration inhibitory factor (MIF) inhibitory activity. In an OGD/R model of SH-SY5Y cells, we observed elevated SNO levels by using live-cell confocal imaging. Treatment of P-EHC significantly reduced intracellular reactive oxygen species (ROS), lowered total NO_x species, and improved cell viability in the OGD/R model. In summary, the simplicity and versatility of P-EHC suggest its broad applicability for monitoring SNO in various biological models and therapeutic contexts, particularly in ischemia-reperfusion diseases. Full article

Background: Multiphoton tomography (MPT) is a femtosecond laser imaging technique that enables high-resolution virtual biopsies of human skin. It provides a non-invasive method for analyzing cellular metabolism, structural changes, and responses to cosmetic products, providing insights into cell–cosmetic interactions. This review explores the principles, historical development, and key applications of MPT in cosmetic research. Methods: The latest MPT device combines five modalities: (i) two-photon fluorescence: visualizes cells, elastin, and cosmetic ingredients; (ii) second harmonic generation (SHG): maps the collagen network; (iii) fluorescence lifetime imaging (FLIM): differentiates eumelanin from pheomelanin and evaluates the impact of cosmetics on cellular metabolic activity; (iv) reflectance confocal microscopy (RCM): images cell membranes and cosmetic particles; and (v) white LED imaging for dermoscopy. Results: MPT enables in-depth examination of extracellular matrix changes, cellular metabolism, and melanin production. It identifies skin responses to cosmetic products and tracks the intratissue distribution of sunscreen nanoparticles, nano- and microplastics, and other cosmetic components. Quantitative measurements, such as the elastin-to-collagen ratio, provide insights into anti-aging effects. Conclusions: MPT is a powerful in vivo imaging tool for the cosmetic industry. Its superior resolution and metabolic information facilitate the evaluation of product efficacy and support the development of personalized skincare solutions. Full article

Microscale systems have been underexplored in contemporary regenerative therapies developed to treat vision loss. The pairing of in vitro cell systems with optical fluorescent imaging provides unique opportunities to examine the infiltration of donor stem cells needed for successful transplantation therapies. A parallel eye device was developed to provide electric field (EF) stimulation to guide the migration of cells within 3D eye facsimiles synthesized from different ocular biomaterials. Cell infiltration within facsimiles was rapidly resolved using confocal microscopy to eliminate dependence on the cryostat sectioning commonly used for cell study. Moreover, EF stimulated galvanotaxis of donor cells within different depths of eye facsimiles. Optical imaging provided rapid resolution of z-stack images at physiologically appropriate depths below 500 microns. This study demonstrates that paired microscale–optical systems can be developed to elucidate understudied transplantation processes and improve future outcomes in patients. Full article

Background Despite many studies on polymer-incorporated nanocarriers for ophthalmic drug delivery, few have thoroughly explored the relationship between coating composition and performance. This study aimed to evaluate the effects of three commonly used cationic polymers—distearoyl phosphatidylethanolamine-polyethylene glycol 1000-poly(amidoamine) (DSPE-PEG1000-PAMAM), trimethyl chitosan (TMC), and (2,3-dioleoyloxypropyl) trimethylammonium chloride (DOTAP)—on the corneal behaviors and anti-cataract efficacy of diosmetin (DIO)-loaded micelles (D-M-P, D-M-T, and D-M-D, respectively). Methods The DIO-loaded micelles were prepared using the thin-film dispersion method and incorporated with the three polymers through hydrophobic interactions and electrostatic adsorption. Structural characterization was demonstrated by TEM imaging and particle size analyzer. In vitro release behavior was detected by the dialysis method. Cell viability of D-M-P, D-M-T, and D-M-D on L929 cells was detected by CCK-8 assays, with cellular uptake performed using coumarin 6 as the fluorescence indicator. Precorneal retention behaviors of these three vesicles were observed by In Vivo Imaging System. Transcorneal permeability was determined by modified Franz diffusion method and the permeation routes of the vesicles are investigated. Selenite-induced cataract model was established. The anti-cataract effects of three different DIO-loaded micelles were evaluated by the observation of lens opacity and antioxidant enzyme activities. Eye Irritation of the DIO in different preparations was estimated using the Draize test, along with H&E staining of the corneas. Results Structural characterization of DIO-loaded micelles revealed that the vesicles were spherical, with a uniform size distribution of around 28 nm, a similar surface potential of approximately 6.0 mV, and a high DIO entrapment efficiency of about 95%. Compared to the DIO suspension, all three formulations exhibited a significant sustained-release effect. They showed no signs of irritation and demonstrated increased IC50 values in L929 cells, indicating improved biocompatibility. Cellular uptake in human lens epithelial cells (HLECs) was assessed using confocal laser scanning microscopy. C-M-T displayed the highest fluorescence signals, with a cellular internalization 3.2 times greater than that of the solution group. Both C-M-T and C-M-P enhanced vesicle retention on the corneal surface by at least 47.8% compared to the Cou-6 solution. Furthermore, TMC facilitated the paracellular transport of vesicles into the deepest layers of the cornea and delivered DIO across the cornea, with a P_app value 3.11 times and 1.49 times those of D-M-D and D-M-P, respectively. In terms of therapeutic efficacy, D-M-T demonstrated the most significant attenuation of lens opacity, along with enhanced antioxidant enzyme activities and inhibition of lipid peroxidation. Conclusion The modification of micelle vesicles with different cationic polymers significantly influences their performance in ocular drug delivery. Among the tested formulations, D-M-T stands out due to its multiple advantages, including enhanced transcorneal drug delivery, therapeutic efficacy for DIO, and safety, making it the most promising candidate for ophthalmic applications. Full article

The controlled functionalization of graphene is critical for tuning and enhancing its properties, thereby expanding its potential applications. Covalent functionalization offers a deeper tuning of the geometric and electronic structure of graphene compared to non-covalent methods; however, the existing techniques involve side reactions and spatially uncontrolled functionalization, pushing research toward more selective and controlled methods. A promising approach is 1,3-dipolar cycloaddition, successfully utilized with carbon nanotubes. In the present work, this method has been extended to graphene flakes with low defect concentration. A key innovation is the use of a custom-synthesized ylide with a protected amine group (Boc), facilitating subsequent attachment of functional molecules. Indeed, after Boc cleavage, fluorescent dyes (Atto 425, 465, and 633) were covalently linked via NHS ester derivatization. This approach represents a highly selective method of minimizing structural damage. Successful functionalization was demonstrated by Raman spectroscopy, photoluminescence spectroscopy, and confocal microscopy, confirming the effectiveness of the method. This novel approach offers a versatile platform, enabling its use in biological imaging, sensing, and advanced nanodevices. The method paves the way for the development of sensors and devices capable of anchoring a wide range of molecules, including quantum dots and nanoparticles. Therefore, it represents a significant advancement in graphene-based technologies. Full article

Brain tumors, both primary and metastatic, represent a significant global health burden due to their high incidence, mortality, and the severe neurological deficits they frequently cause. Gliomas, especially high-grade gliomas (HGGs), rank among the most aggressive and lethal neoplasms, with only modest gains in long-term survival despite extensive molecular research and established standard therapies. In neurosurgical practice, maximizing the extent of safe resection is a principal strategy for improving clinical outcomes. Yet, the infiltrative nature of gliomas often complicates the accurate delineation of tumor margins. Confocal laser endomicroscopy (CLE), originally introduced in gastroenterology, has recently gained prominence in neuro-oncology by enabling real-time, high-resolution cellular imaging during surgery. This technique allows for intraoperative tumor characterization and reduces dependence on time-consuming frozen-section analyses. Recent technological advances, including device miniaturization and second-generation CLE systems, have substantially improved image quality and diagnostic utility. Furthermore, integration with deep learning algorithms and telepathology platforms fosters automated image interpretation and remote expert consultations, thereby accelerating surgical decision making and enhancing diagnostic consistency. Future work should address remaining challenges, such as mitigating motion artifacts, refining training protocols, and broadening the range of applicable fluorescent probes, to solidify CLE’s role as a critical intraoperative adjunct in neurosurgical oncology. Full article

The dynamic nature of mitochondria makes live cell imaging an important tool in mitochondrial research. Although imaging using fluorescent probes is the golden standard in studying mitochondrial morphology, these probes might introduce aspecific features. In this study, live cell fluorescent imaging was applied to investigate a pearl-necklace-shaped mitochondrial phenotype that arises when mitochondrial fission is restricted. In this fibroblast-specific pearl-necklace phenotype, constricted and expanded mitochondrial regions alternate. Imaging studies revealed that the formation time of this pearl-necklace phenotype differs between laser scanning confocal, widefield and spinning disk confocal microscopy. We found that the phenotype formation correlates with the excitation of the fluorescent probe and is the result of phototoxicity. Interestingly, the phenotype only arises in cells stained with red mitochondrial dyes. Serial section electron tomography of the pearl-necklace mitochondria revealed that the mitochondrial membranes remained intact, while the cristae structure was altered. Furthermore, filaments and ER were present at the constricted sites. This study illustrates the importance of considering experimental conditions for live cell imaging to prevent imaging artifacts that can have a major impact on the obtained results. Full article