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Visualizing Cell-Laden Fibrin-Based Hydrogels using Cryogenic Scanning Electron Microscopy and Confocal Microscopy
摘要： The present investigation explores the microscopic aspects of cell-laden hydrogels at high resolutions, using three-dimensional cell cultures in semi-synthetic constructs that are of very-high water content (>98% water). The study aims to provide an imaging strategy for these constructs, while minimizing artifacts. Constructs of PEG-fibrinogen (PEG-Fb) and fibrin hydrogels containing embedded mesenchymal cells (human dermal fibroblasts) were first imaged by confocal microscopy. Next, high resolution scanning electron microscopy (HR-SEM) was used to provide images of the cells within the hydrogels, at submicron resolutions. Because it was not possible to obtain artifact-free images of the hydrogels using room-temperature HR-SEM, a cryogenic HR-SEM (cryo-HR-SEM) imaging methodology was employed to visualize the sample while preserving the natural hydrated state of the hydrogel. The ultrastructural details of the constructs were observed at subcellular resolutions, revealing numerous cellular components, the biomaterial in its native configuration, and the uninterrupted cell membrane as it relates with the biomaterial in the hydrated state of the construct. Constructs containing microscopic albumin microbubbles were also imaged using these methodologies to reveal fine details of the interaction between the cells, the microbubbles and the hydrogel. Taken together with the confocal microscopy, this imaging strategy provides a more complete picture of the hydrated state of the hydrogel network with cells inside. As such, this methodology addresses some of the challenges of obtaining this information in amorphous hydrogel systems containing a very-high water content (>98%) with embedded cells. Such insight may lead to better hydrogel-based strategies for tissue engineering and regeneration.
关键词： Fibrin,Electron Microscopy,Hydrogel,Tissue Engineering,Scaffold,Confocal Microscopy
更新于2025-11-21 11:08:12
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Evaluation of various tissue-clearing techniques for the three-dimensional visualization of liposome distribution in mouse lungs at the alveolar scale
摘要： Purpose: To develop a three-dimensional visualization method for evaluating the distribution of pulmonary drug delivery systems and compare four tissue-clearing techniques (ClearT2, CUBIC, ScaleS, and SeeDB2) using intrapulmonary liposomes as drug carriers. Methods: Rhodamine B-labeled liposomes were administered intrapulmonarily to mice using a MicroSprayer, and then fluorescent-labeled tomato lectin was administered intravenously to visualize the general lung structure. Tissue-clearing treatment of the mouse lungs was performed using the standard protocols of the ClearT2, CUBIC, ScaleS, and SeeDB2 techniques. Lung clearing was clarified using laser-scanning confocal microscopy, and three-dimensional images were reconstructed. Results: Fluorescent-labeled tomato lectin was preserved using ClearT2 and SeeDB2 but not using CUBIC and ScaleS. In addition, the liposomes were stable in ClearT2 reagent, but they were mostly degraded in other reagents by surface-active agents. ClearT2 treatment enabled the three-dimensional visualization of intrapulmonary rhodamine B-labeled liposomes at the alveolar scale. Conclusions: These results suggest that the ClearT2 tissue-clearing technique was appropriate for the three-dimensional visualization of intrapulmonary liposomes at the alveolar scale. This study provides important information for selecting and optimizing suitable optical tissue-clearing techniques in lungs for evaluating the distribution of pulmonary drug delivery systems.
关键词： fluorescence preservation,Intrapulmonary distribution,inhalation,liposomes,drug delivery systems,laser-scanning confocal microscopy
更新于2025-11-21 11:08:12
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A robust and versatile platform for image scanning microscopy enabling super-resolution FLIM
摘要： Image scanning microscopy (ISM) can improve the effective spatial resolution of confocal microscopy to its theoretical limit. However, current implementations are not robust or versatile, and are incompatible with fluorescence lifetime imaging (FLIM). We describe an implementation of ISM based on a single-photon detector array that enables super-resolution FLIM and improves multicolor, live-cell and in-depth imaging, thereby paving the way for a massive transition from confocal microscopy to ISM.
关键词： super-resolution,SPAD array,fluorescence lifetime imaging,confocal microscopy,image scanning microscopy
更新于2025-09-23 15:23:52
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Identifying Experimental Tool Use Through Confocal Microscopy
摘要： Characterizing use-wear traces quantitatively is a valid way to improve the capacity of use-wear analysis. This aim has been on specialists’ agenda since the beginning of the discipline. Micropolish quantification is especially important, as this type of trace allows the identification of worked materials. During the last decade, confocal microscopy has been used as a promising approach to address this question. Following previous efforts in plant microwear characterization (Ibá?ez et al. Journal of Archaeological Science, 48, 96–103, 2014; Journal of Archaeological Science, 73, 62–81, 2016), here we test the capacity of the method for correctly grouping experimental tools used for working eight types of materials: bone, antler, wood, fresh hide, dry hide, wild cereals, domestic cereals, and reeds. We demonstrate, for the first time, that quantitative texture analysis of use-wear micropolish based on confocal microscopy can consistently identify tools used for working different contact materials. In this way, we are able to move toward using texture analysis as part of the standard functional analysis of prehistoric instruments.
关键词： Lithic tools,Use-wear,Confocal microscopy,Experimentation
更新于2025-09-23 15:23:52
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Research on Spatially Adaptive High-Order Total Variation Model for Weak Fluorescence Image Restoration
摘要： Confocal laser scanning microscopy (CLSM) has emerged as one of the most advanced ?uorescence cell imaging techniques in the ?eld of biomedicine. However, ?uorescence cell imaging is limited by spatial blur and additive white noise induced by the excitation light. In this paper, a spatially adaptive high-order total variation (SA-HOTV) model for weak ?uorescence image restoration is proposed to conduct image restoration. The method consists of two steps: optimizing the deconvolution model of the ?uorescence image by the generalized Lagrange equation and alternating direction method of multipliers (ADMM); using spatially adaptive parameters to balance the image ?delity and the staircase e?ect. Finally, an comparison of SA-HOTV model and Richardson-Lucy model with total variation (RL-TV model) indicates that the proposed method can preserve the image details ultimately, reduce the staircase e?ect substantially and further upgrade the quality of the restored weak ?uorescence image.
关键词： weak ?uorescence,spatially adaptive high-order total variation (SA-HOTV),image restoration,confocal microscopy
更新于2025-09-23 15:23:52
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Progrès dans les méthodes diagnostiques du déficit en cellules souches limbiques. Apport de la microscopie confocale et de la tomographie en cohérence optique
摘要： The limbus is the anatomical and functional barrier between corneal and conjunctival epithelia. It is characterized by presence of the limbal stem cell niche which allows corneal homeostasis to be maintained. Limbal stem cell deficiency is characterized by a dual process: insufficient regeneration of corneal epithelium, which cannot therefore assure its function of physiological support, associated with corneal invasion by conjunctival proliferation. Diagnosis is currently made via routine clinical examination, corneal impression cytology and in vivo confocal microscopy (IVCM). Slit lamp examination shows abnormal limbal anatomy, thin and irregular epithelium with late fluorescein staining, and superficial vascularization. With its high resolution, IVCM allows identification of limbal and corneal epithelial changes at a cellular level in en face views, parallel to the corneal surface, but with a restricted viewing field of the corneal surface. It shows a poor transition between the corneal and conjunctival epithelia, associated with a loss of the normal corneal epithelial stratification, low basal cell and sub-basal nerve plexus densities, even with sub-epithelial fibrosis. Optical coherence tomography in central cornea and at the limbus, with scans in different orientations, allows a quick, global and non-invasive analysis of normal eyes and those with limbal stem cell deficiency. It shows a thin limbal epithelium, lacking normal thickening, featuring absence of stromal undulations and limbal crypts in cross-sections and sections parallel to the limbus, lack of visible limbal crypts in en face sections, loss of clear transition between the hyporeflective corneal epithelium and the hyperreflective conjunctival epithelium, and hyperreflective sub-epithelial fibrosis.
关键词： Limbal stem cell deficiency,Limbus,Optical coherence tomography,In vivo confocal microscopy
更新于2025-09-23 15:23:52
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Reflectance confocal microscopy margin mapping and monitoring of an amelanotic melanoma <i>in situ</i> of the ear
摘要： In situ amelanotic melanoma represents a diagnostic challenge for clinicians. Poor demarcation of these lesions often results in repeated therapeutic intervention until appropriate clearance has been achieved. Reflectance confocal microscopy (RCM) is a noninvasive bedside imaging modality which allows real-time visualisation, to a near-histological level, of the epidermis and reticular dermis. We present a case of an amelanotic melanoma in situ in which reflectance confocal microscopy margin mapping allowed for demarcation of the melanocytic proliferation and targeted therapeutic intervention with topical imiquimod. Reflectance confocal microscopy was further utilised for noninvasive assessment of therapeutic response.
关键词： margin mapping,reflectance confocal microscopy,amelanotic melanoma
更新于2025-09-23 15:23:52
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A miniaturized, tethered, spectrally-encoded confocal endomicroscopy capsule
摘要： The tethered spectrally-encoded confocal endomicroscopy (SECM) capsule is an imaging device that once swallowed by an unsedated patient can visualize cellular morphologic changes associated with gastrointestinal (GI) tract diseases in vivo. Recently, we demonstrated a tethered SECM capsule for counting esophageal eosinophils in patients with eosinophilic esophagitis (EoE) in vivo. Yet, the current tethered SECM capsule is far too long to be widely utilized for imaging pediatric patients, who constitute a major portion of the EoE patient population. In this paper, we present a new tethered SECM capsule that is 33% shorter, has an easier and repeatable fabrication process, and produces images with reduced speckle noise.
关键词： spectrally encoded confocal microscopy,confocal endomicroscopy,eosinophilic esophagitis,reflectance confocal microscopy,esophageal imaging,tethered capsule endomicroscopy
更新于2025-09-23 15:22:29
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[Methods in Molecular Biology] T-Cell Motility Volume 1930 (Methods and Protocols) || Live Imaging of Resident T-Cell Migration in Human Lymphoid Tissue Slices Using Confocal Microscopy
摘要： In order to mount a potent immune response, immune cells must move actively through tissues. As an example, T-cell need to migrate within lymph nodes in order to scan the surface of many dendritic cells and recognize rare expressed antigens. The recent development of improved imaging approaches, such as two-photon microscopy, and the use of powerful mouse models have shed light on some of the mechanisms that regulate the migration of immune cells in many organs. Whereas such systems have provided valuable insights, they do not always predict human responses. In human, our knowledge in the field mainly comes from a description of fixed tissue samples. However, these studies lack a temporal dimension since samples have been fixed. In order to overcome some of these limitations, we describe, in this methodology chapter, an experimental system of fresh human adenoid slices to monitor the dynamics of resident T-lymphocytes that have been stained with directly-coupled fluorescent antibodies. Combined with confocal fluorescent imaging, this preparation offers an effective approach to imaging immune cells in a three-dimensional (3D) human lymphoid tissue environment.
关键词： Immunostaining,Confocal microscopy,Motility,Vibratome,T-cells
更新于2025-09-23 15:22:29
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In vivo confocal microscopy morphometric analysis of corneal subbasal nerve plexus in dry eye disease using newly developed fully automated system
摘要： Purpose To evaluate in vivo confocal microscopy (IVCM) features of corneal subbasal nerve plexus (SNP) in the setting of dry eye disease (DED) using fully automated software BACCMetrics,^ and to further investigate its diagnostic performance in discriminating DED patients. Methods IVCM exams of SNP in DED patients and matched control subjects were performed using Heidelberg Retina Tomograph with the Rostock Cornea Module. The following parameters were obtained with ACCMetrics: corneal nerve fiber density (CNFD), corneal nerve branch density (CNBD), corneal nerve fiber length (CNFL), corneal nerve total branch density (CTBD), corneal nerve fiber area (CNFA), corneal nerve fiber width (CNFW), and corneal nerve fractal dimension (CNFrD). The Mann–Whitney U test was used to compare variables. Receiver operating characteristic curves with calculations of the area under the curve (AUC) were used to describe the accuracy of IVCM parameters for discriminating DED patients from controls. Results Thirty-nine DED patients and 30 control subjects were included. Significantly, lower values of CNFD, CNBD, and CNFL and higher value of CNFW were found in DED patients compared to controls (respectively, 20.5 ± 8.7 vs 25.4 ± 6.7 n/ mm2; 25.6 ± 20.1 vs 37.6 ± 21.5 n/mm2; 12.6 ± 4.4 vs 14.5 ± 2.9 mm/mm2; 0.021 ± 0.001 vs 0.019 ± 0.001 mm/mm2; always p < 0.024). CNFW value had the highest diagnostic power in discriminating DED patients (AUC = 0.828). When the diagnosis of DED was made based on either CNFW or CNBD, the sensitivity was 97.4% and the specificity 46.7%. Conclusions The software ACCMetrics was able to rapidly detect SNP alterations occurring in the setting of DED and showed good diagnostic performance in discriminating DED patients.
关键词： Dry eye,Sub-basal nerve plexus,Automated analysis,ACCMetrics,In vivo confocal microscopy
更新于2025-09-23 15:22:29