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Fluorescent boron Schiff bases dyes for staining silk fibroin: Green synthesis, structural characterization, DFT, and photophysical properties
摘要： Five novel organoboron complexes were synthesized in just 15 min via microware irradiation, by one pot multicomponent reactions between diverse aryl aldehydes with benzoylhydrazide, or 4‐nitrobenzoylhidrazine and diphenyl boronic acid, in a 1:1:1 ratio in benzene. The products were characterized by 1H, 13C, 11B NMR, UV, IR, spectroscopy and high‐resolution mass spectrometry (HRMS). The molecular structure was also determined by single‐crystal X‐ray diffraction for two complexes, which showed the tetra‐coordination of the boron atoms giving rise to distorted tetrahedral molecular geometry with a strong intermolecular C‐H···π interactions. In spite of the low quantum yields exhibited by the series in solution, some complexes stained uniformly the silk fibroins emitting enough fluorescence to allow its characterization by confocal microscopy. Boron as chelate center of the five complexes resulted not to be toxic for B16F10 cells, these compounds are appropriate for their used in medical applications.
关键词： organoboron,silk fibroin,cytotoxicity,confocal microscopy,fluorescent
更新于2025-09-09 09:28:46
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Ex Vivo Confocal Microscopy Of Basal Cell Carcinoma On A 3-Color Scale
摘要： Ex vivo confocal microscopy (CM) is capable of visualizing freshly excised tissue in real-time with cellular resolution without routine processing. Depending on the laser wavelength, either reflectance (RCM) or fluorescence (FCM) is utilized. Ex vivo CM is useful for the rapid evaluation of tumor margins during Mohs micrographic surgery (MMS). Initially, ex vivo RCM studies used acetic acid as contrast agent to enhance basal cell carcinoma (BCC) cell nuclei. However, thin strands of BCC were frequently missed. The use of fluorophores improves contrast, so that even small strands of BCC can be spotted in FCM mosaics. Acridine orange (AO) is the dye most widely used. AO binds specifically to DNA and emits fluorescence, so images of living cell nuclei can be enhanced and displayed as bright structures in FCM mosaics. Even though good resolution and morphological correlation are achieved with this standard technique, nowadays confocal mosaics are displayed in a grey scale format. However, dermato-pathologists are often neither familiar with nor comfortable assessing these black-and-white images. We herein report a new technique for obtaining 3-color scale confocal mosaics (3CS-FCM) with the simultaneous use of AO and ethidium bromide (EB) as fluorescent dyes. In this technique, the excised skin sample is first soaked with liquid nitrogen. The sample is then sectioned into 20-30 μm-thick slices using a cryostat and stained with the dye mixture (AO 0.1 mM + EB 0.25 mM) for about one minute. The sample is then placed in the confocal microscope plate for imaging (Nikon A1R+, NIKON CORPORATION?, Japan). The tissue is scanned simultaneously with two different wavelength lasers (405 and 488 nm) and the collected fluorescence displayed on the screen as a 3-color-scale mosaic. Around 10 to 15 minutes are required for completion of the tissue processing and for final mosaics to be developed. Unlike AO, EB binds specifically to the DNA of BCC cells that are damaged due to freezing. As a result, BCC nests are stained by EB and emit red fluorescence after laser stimulation; in contrast, the epidermis and dermis are stained by AO and emit green fluorescence. Blue color corresponds to the background tissue autofluorescence. All fluorescence is collected by the microscope displaying the final images in a 3-color scale format. AO and EB staining do not affect additional fixation or staining of the sample. Figure 1 shows completed BCC mosaics displayed with this new technique. Each color represents a different skin structure, making the mosaics easier to read. In this way, 3CS confocal mosaics are more user-friendly and can be interpreted by healthcare professionals without previous experience with FCM. Moreover, with frozen sample processing, the tissue is completely flattened and the entire sample can be displayed on the screen. These developments represent important advantages over previously described images obtained with CM. In conclusion, 3CS-FCM is an innovative technique that provides colored images, expanding significantly the applicability of FCM. Larger studies are nevertheless required to validate the technique for MMS and other applications.
关键词： fluorescence confocal microscopy,3-color scale mosaics,basal cell carcinoma,ethidium bromide,acridine orange,ex vivo confocal microscopy,Mohs micrographic surgery
更新于2025-09-04 15:30:14
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Confocal Microscopy and Lentigo Maligna: An in vivo Pilot Study for the Assessment of Response to Imiquimod Therapy
摘要： Background: Reflectance confocal microscopy (RCM) is a noninvasive technique that provides real-time in vivo images of the epidermal layer. Imiquimod has been recommended as an alternative treatment in lentigo maligna (LM) when surgical excision is not the treatment of choice. In the present study we compare the results of in vivo RCM to the histopathological examination before and after treatment of LM with topical imiquimod. Methods: Thirty-four patients with confirmed LM were included. Imiquimod 5% was applied until a weeping erosion appeared in the LM-affected skin. Evaluation was performed by clinical examination, dermatoscopy, histopathology and RCM. Results: During the follow-up, 27 of 34 patients (79.42%) demonstrated a total tumor clearance by imiquimod treatment. In the treated area, a significant decrease of atypical cells was detected using RCM (p < 0.0001). Furthermore, a significant positive correlation in the detected atypical cells was shown using confocal microscopy and histology (p = 0.0001, r = 0.7335, respectively). Conclusion: In patients not suitable for surgical intervention imiquimod treatment is an appropriate treatment alternative. Thereby, in vivo RCM was demonstrated to be an excellent examining device, which not only allows diagnosis of LM, but also therapy and follow-up examinations. An important benefit of RCM, in contrast to conventional histopathology, is the simple handling with in vivo examination of epidermal skin without any pain for the patient.
关键词： Imiquimod therapy,Lentigo maligna,Noninvasive follow-up,Reflectance confocal microscopy
更新于2025-09-04 15:30:14
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Monitoring circulating tumor cells in vivo by a confocal microscopy system
摘要： Circulating tumor cells (CTCs) play a key role in cancer metastasis but are very difficult to detect. in vivo monitoring CTCs has been recognized as an important technique for cancer research and clinical diagnosis. Recently, a noninvasive method, in vivo flow cytometry (IVFC) has been developed to enable continuous, real-time, and long-duration detection of CTCs in animal models by detecting CTC fluorescence in blood vessels excited by lasers. In this study, we present a simple optical scheme for direct noninvasive CTC detection using confocal microscopes. We demonstrate that line scanning of confocal microscopy can provide effective and quantitative CTC detection in live mice during cancer development. Rare CTC signals can be acquired at the early stage of the tumor development after implantation of subcutaneous tumor and monitored continuously to the end. Signals from CTC clusters can also be acquired and distinguished from single CTCs. Our results suggest confocal microscopy is a simple and reliable method for biologists and doctors to use for cancer research.
关键词： line scanning,circulating tumor cells,confocal microscopy,CTC clusters
更新于2025-09-04 15:30:14
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Evaluation of the corneal epithelium in non-Sj?gren’s and Sj?gren’s dry eyes: an in vivo confocal microscopy study using HRT III RCM
摘要： Background: The corneal epithelium is directly affected in dry eye syndrome. Thus, we attempted to describe the morphological features and evaluate the cellular density within the corneal epithelial layers in patients with non-Sj?gren’s (NSDE) and Sj?gren’s syndrome dry eyes (SSDE) by in vivo confocal microscopy (IVCM). Methods: Central cornea was prospectively imaged by IVCM in 68 clinically diagnosed aqueous tear-deficient dry eyes and 10 healthy age-matched control eyes. Morphological characteristics of corneal epithelial layers and cellular densities were evaluated by four trained graders from the Doheny Eye Institute. Results: Corneal epithelium in dry eyes presents morphological changes such as areas of enlarged and irregular shaped cells. In comparison with controls, the density of superficial epithelial cells was decreased in both the NSDE (P < 0.05) and SSDE groups (P < 0.01); the density of the outer layer of wing cells was smaller but not significantly different in NSDE (P > 0.05), but was lower in the SSDE group (P < 0.01); the density of the inner layer of wing cells was decreased in both the NSDE (P < 0.05) and SSDE groups (P < 0.01) and the density of basal epithelial cells was lower in both the NSDE (P < 0.01) and SSDE groups (P = 0.01). For all cell counts, the interclass correlation coefficient showed good agreement between graders (ICC =0.75 to 0.93). Conclusions: IVCM represents a reliable technique for examining the corneal epithelial microstructural changes associated with dry eyes, as well as for objectively and reproducibly quantifying cell densities within all corneal epithelial layers.
关键词： Dry eye syndrome,corneal epithelium,Sj?gren’s syndrome,in vivo laser scanning confocal microscopy
更新于2025-09-04 15:30:14
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Feature-based Classification of Protein Networks using Confocal Microscopy Imaging and Machine Learning
摘要： Fluorescence imaging has become a powerful tool to investigate complex subcellular structures such as cytoskeletal filaments. Advanced microscopes generate 3D imaging data at high resolution, yet tools for quantification of the complex geometrical patterns are largely missing. Here we present a computational framework to classify protein network structures. We developed a machine-learning method that combines state-of-the-art morphological quantification with protein network classification through morphologically distinct structural features enabling live imaging–based screening. We demonstrate applicability in a confocal laser scanning microscopy (CLSM) study differentiating protein networks of the FtsZ (filamentous temperature sensitive Z) family inside plant organelles (Physcomitrella patens).
关键词： FtsZ,machine learning,classification,protein networks,confocal microscopy
更新于2025-09-04 15:30:14
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Nanoparticles at Biomimetic Interfaces: A Combined Experimental and Simulation Study on Charged Gold Nanoparticles/Lipid Bilayers Interfaces
摘要： The poor understanding of the interaction of nanomaterials with biologically relevant interfaces is recognized as one of the major issues currently limiting the development of nanomedicine. The central purpose of this study is to compare experimental (Confocal Microscopy, Fluorescence Correlation Spectroscopy, X-ray Reflectivity) and computational (Molecular Dynamics simulations) results to thoroughly describe the interaction of cationic gold nanoparticles (AuNP) with mixed zwitterionic/anionic lipid membranes. The adhesion of AuNPs to the lipid membrane is investigated on different length scales from a structural and dynamical point of view; with this approach, a series of complex phenomena, spanning from lipid extraction, localized membrane disruption, lateral phase separation and slaved diffusion, are characterized and interpreted from a molecular level to macroscopic observations.
关键词： Nanoparticles,Gold Nanoparticles,Molecular Dynamics,X-ray Reflectivity,Fluorescence Correlation Spectroscopy,Lipid Bilayers,Biomimetic Interfaces,Confocal Microscopy
更新于2025-09-04 15:30:14
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Application of In Vivo Confocal Microscopy in Dry Eye Disease
摘要： Confocal microscopy is a new, emerging, noninvasive technology that can aid in the in vivo assessment of structural changes in several ocular surface diseases at the cellular level. In the dry eye field, in vivo confocal microscopy has been applied to the examination of the cornea, bulbar and palpebral conjunctiva, Meibomian gland, and lacrimal gland. The device can assess the morphology, including superficial/wing/basal epithelial cell density, stromal keratocyte density, endothelial cell density, nerve fiber density, the number of beadings, nerve tortuosity, nerve reflectivity, and inflammatory cell density in the cornea. Furthermore, the device can not only assess epithelial cell density and area, goblet cell, microcyst, and inflammatory cell density but also the cellular architecture, including nucleocytoplasmic ratio in conjunctiva. The device also can disclose acinar unit density, acinar unit longest diameter, acinar unit shortest diameter, and inflammatory cell density in the Meibomian gland and lacrimal gland by other potential applications. Relevant research in Europe and the United States focused on the morphologic changes in the cornea in the dry eye field, while Japanese research focused on the conjunctival, Meibomian gland, and lacrimal gland alterations. The application of in vivo confocal microscopy in dry eye disease will be a powerful method to evaluate the morphologic change of the ocular surface around the world in the future.
关键词： dry eye,conjunctiva,Meibomian gland,confocal microscopy,lacrimal gland,cornea
更新于2025-09-04 15:30:14
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<i>In Vivo</i> Confocal Microscopy Analysis of Corneal Microstructural Changes in Neurosurgically-Induced Neurotrophic Keratitis
摘要： Purpose: To investigate the changes of the corneal microstructure of neurosurgically-induced neurotrophic keratitis patients compared to normal human corneas using in vivo confocal microscope (IVCM). Methods: Ten eyes in the normal control group and 11 eyes in the neurosurgically-induced neurotrophic keratitis patient group were included in the present study. After corneal sensitivity tests were performed, thickness of each layer and number of endothelial cells and stromal keratocytes in the cornea were assessed using IVCM. Morphological characteristics of the corneal nerves were measured by ImageJ software. Results: After analysis of corneal thickness layer by layer, the Bowman’s layer was significantly reduced in the neurosurgically-induced neurotrophic keratitis patient group compared with the normal control group (p = 0.016) and the portion of Bowman’s layer was significantly reduced in the neurosurgically-induced neurotrophic keratitis patient group compared with the control group (p = 0.024). The nerve fiber length per square-millimeter became significantly shorter compared to the normal control group (p = 0.042). The nerve fiber length per square millimeter showed significant correlations with the number of fibers, number of beading, branching patterns, and nerve tortuosity (p = 0.002, 0.002, 0.013 and 0.034, respectively). The number of endothelial cells and stromal keratocytes, the number of nerve fibers and beading, and the pattern of branching and nerve tortuosity showed no significant differences between the normal and neurosurgically-induced neurotrophic keratitis patient groups. Conclusions: Our results showed that decreased thickness of Bowman’s layer may be related to the decreased corneal nerve distribution, secondary to the dysfunction of trigeminal nerve branch innervating the cornea. The microstructural changes of Bowman’s layer can help diagnose the disease and evaluate the current status in neurosurgically-induced neurotrophic keratitis patients.
关键词： Neurosurgically-induced neurotrophic keratitis,Corneal thickness,Corneal nerve,Confocal microscopy,Neurosurgery
更新于2025-09-04 15:30:14
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Corneal Fibroblast Migration Patterns During Intrastromal Wound Healing Correlate With ECM Structure and Alignment
摘要： PURPOSE. To assess keratocyte backscattering, alignment, morphology, and connectivity in vivo following a full-thickness corneal injury using the Heidelberg Retina Tomograph Rostock Cornea Module (HRT-RCM), and to correlate these findings with en bloc three-dimensional (3-D) confocal fluorescence and second harmonic generation (SHG) imaging. METHODS. Rabbit corneas were scanned in vivo both before and 3, 7, 14, and 28 days after transcorneal freeze injury (FI), which damages all corneal cell layers. Corneal tissue was also fixed and labeled for f-actin and nuclei en bloc, and imaged using 3-D confocal fluorescence microscopy and SHG imaging. RESULTS. Using the modified HRT-RCM, full-thickness scans of all cell layers were consistently obtained. Following FI, stromal cells repopulating the damaged tissue assumed an elongated fibroblastic morphology, and a significant increase in cellular light scattering was measured. This stromal haze gradually decreased as wound healing progressed. Parallel, interconnected streams of aligned corneal fibroblasts were observed both in vivo (from HRT-RCM reflection images) and ex vivo (from f-actin and nuclear labeling) during wound healing, particularly in the posterior cornea. Second harmonic generation imaging demonstrated that these cells were aligned parallel to the collagen lamellae. CONCLUSIONS. The modified HRT-RCM allows in vivo measurements of sublayer thickness, assessment of cell morphology, alignment and connectivity, and estimation of stromal backscatter during wound healing. In this study, these in vivo observations led to the novel finding that the pattern of corneal fibroblast alignment is highly correlated with lamellar organization, suggesting contact guidance of intrastromal migration that may facilitate more rapid wound repopulation.
关键词： SHG imaging,confocal microscopy,corneal wound healing,extracellular matrix
更新于2025-09-04 15:30:14