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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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Laser Scanning Confocal Microscopy Was Used to Validate the Presence of Burkholderia pseudomallei or B. mallei in Formalin-Fixed Paraffin Embedded Tissues
摘要： Burkholderia pseudomallei and B. mallei are Gram-negative, facultative intracellular bacteria that cause melioidosis and glanders, respectively. Currently, there are no vaccines for these two diseases. Animal models have been developed to evaluate vaccines and therapeutics. Tissues from infected animals, however, must be fixed in formalin and embedded in paraffin (FFPE) before analysis. A brownish staining material in infected tissues that represents the exopolysaccharide of the pathogen was seen by bright field microscopy but not the actual microorganism. Because of these results, FFPE tissue was examined by laser scanning confocal microscopy (LSCM) in an attempt to see the microorganism. Archival FFPE tissues were examined from ten mice, and five nonhuman primates after exposure to B. pseudomallei or B. mallei by LSCM. Additionally, a historical spleen biopsy from a human suspected of exposure to B. mallei was examined. B. pseudomallei was seen in many of the infected tissues from mice. Four out of five nonhuman primates were positive for the pathogen. In the human sample, B. mallei was seen in pyogranulomas in the spleen biopsy. Thus, the presence of the pathogen was validated by LSCM in murine, nonhuman primate, and human FFPE tissues.
关键词： melioidosis,Burkholderia pseudomallei,Burkholderia mallei,animal models,microorganism,formalin-fixed paraffin embedded tissue,laser scanning confocal microscopy,glanders
更新于2025-09-23 15:21:01
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Laser Scanning Confocal Microscopy 3D Surface Metrology Applications
摘要： Surface metrology, measurement of solid surfaces topography, has become an important topic for many material scientists and engineers. Characterization of the surfaces can help researchers find new functional materials, improve device performance and so forth. ‘Seeing is believing.’ Visualization of fine 3-dimensional (3D) details of surfaces is critical in surface metrology studies. Among various observation and measurement techniques, Laser Scanning Confocal Microscopy (LSCM) becomes more and more popular due to the fact that it is non-contact and non-destructive to the samples, requires minimal sample preparation, and efficient automations and provides single nanometer level resolution. In this paper, we will introduce these features in detail, by using Olympus LEXT OLS5000, the newest 3D laser confocal scanning microscope.
关键词： Olympus LEXT OLS5000,3D Surface Metrology,Laser Scanning Confocal Microscopy
更新于2025-09-23 15:19:57
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Use of the inferior whorl to detecting age-related changes in human corneal subbasal nerve plexus with laser-scanning confocal microscopy
摘要： Purpose: To determine the effect of aging on the corneal subbasal nerve plexus (SNP) by employing a wide-field mapping technique of composite images, scanned at the location of a distinctive spiraled subbasal nerve pattern located 1 to 2 mm inferior to the corneal apex (the inferior whorl) for SNP structural quantification. Material and methods: The central corneal tactile sensitivity (CCTS) and inferior whorl length (IWL) were compared among individuals in three age groups (20–39 years, 40–59 years, and 60–79 years). Statistical analyses constituted the Kruskal-Wallis test, one-way analysis of variance (with post hoc least significant difference test), Spearman correlation coefficient, and linear regression. Results: CCTS remained stable until the age of 50 years, when it began to decrease; the mean CCTS was 58.15±2.46 mm in the group aged 20–39, 55.74±3.85 mm in the group aged 40–59, and 50.23±3.27 mm in the group aged 60–79. IWL decreased with increasing age, with a corresponding linear decline of 0.2088 mm/mm2 per year, and the mean IWL was 25.43±4.50 mm/mm2 in the group aged 20–39, 22.71±6.19 mm/mm2 in the group aged 40–59, and 18.60±4.21 mm/mm2 in the group aged 60–79. Conclusion: Our work provided a more accurate and repeatable method for corneal nerve analysis using Laser-Scanning Confocal Microscopy (LSCM). By using this technique, we confirmed that aging is associated with progressive reduction in subbasal nerve length.
关键词： aging,subbasal nerve plexus,laser-scanning confocal microscopy,inferior whorl
更新于2025-09-19 17:13:59
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Optical Fluorescence Diagnostic of Wheat Leaf Rust with Laser Scanning Confocal Microscopy
摘要： Wheat is the most important grain crop and food source worldwide. The management of diseases and early detection of pathogens is a crucial step in diagnosis programs in wheat. In the primary stage, the symptoms of rust fungus are difficult to identify with visual monitoring and other conventional techniques. In this study, we intended to investigate the early stage leaf rust in wheat crop produced through rust fungus using light fluorescence from laser scanning confocal microscopy (LSCM). The leaf rust and normal samples were analyzed with an excitation of 488 nm wavelength of Ar+ laser without any marker or photosensitizer. The small dark pores instead of stomata appears in leaf due to fungus infection and can be observed after two week of leaf tillering. These spots are orange or brown in the beginning and become black, when plants reach maturity. In recent study, the potential of non-invasive techniques for the detection of plant diseases are demonstrated for the development of a rapid and less complex early stage detection procedure that can be utilized to evaluate the infection structures during fungus infection of wheat. The newly developed rapid procedure will be helpful for early stage detection and management fungal infection before proper development during wheat interaction.
关键词： Wheat leaf rust,Optical sensors,Laser scanning confocal microscopy,Fungus infection
更新于2025-09-19 17:13:59
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Correlated Secondary Ion Mass Spectrometry-Laser Scanning Confocal Microscopy Imaging for Single Cell-Principles and Applications
摘要： Secondary ion mass spectrometry (SIMS) as a powerful surface analysis technique has been widely applied in semiconductor industry and geology research. Recently, with the development of instrumental technology, SIMS is attracting more and more attention in life sciences. SIMS can provide surface MS spectra, 2D/3D chemical images and depth profiling of substances simultaneously. The minimal lateral resolution of 2D SIMS imaging is 80?100 nm, and the longitudinal resolution of 3D SIMS imaging is about 1–5 nm. However, owing to lack of specific ions to render the structures of organelles, SIMS imaging for single cells still have great challenges. Optical microscopy, in particular laser scanning confocal microscopy (LSCM), has been emerged to be an indispensable technique for single cell imaging and can obtain high spatial 2D/3D imaging to visualize the structures of organelles. Thus, the combinational use of SIMS and LSCM, which takes advantages of SIMS for molecular imaging and LSCM for morphological imaging, has greatly extended the application of SIMS imaging and ensured its accuracy at single cells level, providing novel insights into better understanding of the biological events inside cells. In this review, we focus on the development and application of SIMS imaging and the correlated SIMS and LSCM imaging in the research of cell biology and drug discovery. We anticipate that the combinational use of SIMS and LSCM imaging has promising future in biomedicine and life sciences.
关键词： Cell biology,Single cell imaging,Laser scanning confocal microscopy,Correlated secondary ion mass spectrometry and laser scanning confocal microscopy imaging,Secondary ion mass spectrometry,Review
更新于2025-09-16 10:30:52
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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