Recognition of Bulbilli Facilitates the Diagnosis of Cladorrhinum bulbillosum.

In vitro microfluidic experimentation holds great potential to reveal many insights into the microphysiological phenomena occurring in conditions such as acute respiratory distress syndrome (ARDS) and ventilator-induced lung injury (VILI). However, studies in microfluidic channels with dimensions physiologically relevant to the terminal bronchioles of the human lung currently face several challenges, especially due to difficulties in establishing appropriate cell culture conditions, including media flow rates, within a given culture environment. The presented protocol describes an image-based approach to evaluate the structure of NCI-H441 human lung epithelial cells cultured in an oxygen-impermeable microfluidic channel with dimensions physiologically relevant to the terminal bronchioles of the human lung. Using phalloidin-based filamentous-actin staining, the cytoskeletal structures of the cells are revealed by confocal laser scanning microscopy, allowing for the visualization of individual as well as layered cells. Subsequent quantification determines whether the cell culture conditions being employed are producing uniform monolayers suitable for further experimentation. The protocol describes cell culture and layer evaluation methods in microfluidic channels and traditional fixed-well environments. This includes channel construction, cell culture and requisite conditions, fixation, permeabilization and staining, confocal microscopic imaging, image processing, and data analysis.

Purpose To compare corneal endothelial changes in patients with Fuchs Uveitis Syndrome (FUS) undergoing phacoemulsification surgery using confocal and specular microscopy. Methods We included 14 patients with unilateral FUS and cataracts who underwent phacoemulsification surgery in a Mexican referral center for inflammatory eye diseases. Preoperative confocal and specular microscopies were conducted, establishing baseline images for subsequent analyses. Surgery on the FUS eye was performed by a single surgeon and an intraocular lens was implanted in all cases. Both specular and confocal microscopy were repeated 6 months after FUS eye surgery and compared with baseline images. We used Image J to do a manual segmentation of KP and determine their density for further analysis hence developing a new tool for confocal microscopy image analysis. Results All patients underwent uneventful phacoemulsification surgery in the FUS eye. There was no significant change in endothelial cell density (ECD) from 2257 (±508.2) cells/mm2 preoperatively to 2214 (±535.1) cells/mm2 postoperatively (p = 0.809). Confocal microscopy revealed a decrease in Keratic Precipitate Density (KPD) from a median of 1413 (±2809.7) KPs/mm2 preoperatively to a median of 685.5 (1527.9) KPs/mm2 postoperatively (p = 0.036). Conclusions Phacoemulsification surgery in patients with FUS produces no significant loss of endothelial cells and morphological changes that can be detected by confocal and specular microscopy. We found a reduction in KPD 6 months after surgery on confocal microscopy. Additionally, our manual segmentation technique for KPs utilizing Image J offers a novel and practical approach for confocal microscopy image analysis.

Three-dimensional Fourier optics of a thin lens confocal bright-field microscopy with a point detector confocal bright-field microscopy with a finite-sized detector confocal single-photon fluorescence microscopy confocal two-photon fluorescence microscopy fibre-optical confocal microscopy confocal microscopy under ultrashort pulse illumination confocal microscopy with high-aperture objectives significance of three-dimensional transfer functions.

Time-lapse confocal fluorescence microscopy images from mouse embryonic stem cells (ESCs) carrying reporter genes, histone H2B-mCherry and Mvh-Venus, have been used to monitor dynamic changes in cellular/differentiation characteristics of live ESCs. Accurate cell nucleus segmentation is required to analyse the ESC dynamics and differentiation at a single cell resolution. Several methods used concavities on nucleus contours to segment overlapping cell nuclei. Our proposed method evaluates not only the concavities but also the size and shape of every 2D nucleus region to determine if any of the strait, extrusion, convexity and large diameter criteria is satisfied to segment overlapping nuclei inside the region. We then use a 3D segmentation method to reconstruct simple, convex, and reasonably sized 3D nuclei along the image stacking direction using the radius and centre of every segmented region in respective microscopy images.To avoid false concavities on nucleus boundaries, fluorescence images of the H2B-mCherry reporter are used for localisation of cell nuclei and Venus fluorescence images are used for determining the cell colony ranges. We use a series of image preprocessing procedures to remove noise outside and inside cell colonies, and in respective nuclei, and to smooth nucleus boundaries based on the colony ranges. We propose dynamic data structures to record every segmented nucleus region and solid in sets (volumes) of 3D confocal images. The experimental results show that the proposed image preprocessing method preserves the areas of mouse ESC nuclei on microscopy images and that the segmentation method effectively segment out every nucleus with a reasonable size and shape. All 3D nuclei in a set (volume) of confocal microscopy images can be accessed by the dynamic data structures for 3D reconstruction. The 3D nuclei in time-lapse confocal microscopy images can be tracked to calculate cell movement and proliferation in consecutive volumes for understanding the dynamics of the differentiation characteristics about ESCs. LAY DESCRIPTION: Embryonic stem cells (ESCs) are considered as an ideal source for basic cell biology study and producing medically useful cells in vitro.This study uses time-lapse confocal fluorescence microscopy images from mouse ESCs carrying reporter gene to monitor dynamic changes in cellular/differentiation characteristics of live ESCs. To automate analyses of ESC differentiation behaviours, accurate cell nucleus segmentation to distinguish respective cells are required. A series of image preprocessing procedures are implemented to remove noise in live-cell fluorescence images but yield overlapping cell nuclei. A segmentation method that evaluates boundary concavities and the size and shape of every nucleus is then used to determine if any of the strait, extrusion, convexity, large and local minimum diameter criteria satisfied to segment overlapping nuclei. We propose a dynamic data structure to record every newly segmented nucleus. The experimental results show that the proposed image preprocessing method preserves the areas of mouse ESC nuclei and that the segmentation method effectively detects overlapping nuclei. All segmented nuclei in confocal images can be accessed using the dynamic data structures to be visualised and manipulated for quantitative analyses of the ESC differentiation behaviours. The manipulation can be tracking of segmented 3D cell nuclei in time-lapse images to calculate their dynamics of differentiation characteristics.

Confocal Microscopy and Three-Dimensional Image Analysis Methods for Recognition and Fluorometric Measurements: An Application to Inhibitory Glycine Receptor.

Infectious keratitis refers to a group of corneal disorders in which corneal tissues suffer inflammation and damage caused by pathogenic infections. Among these disorders, fungal keratitis (FK) and acanthamoeba keratitis (AK) are particularly severe and can cause permanent blindness if not diagnosed early and accurately. In Vivo Confocal Microscopy (IVCM) allows for imaging of different corneal layers and provides an important tool for an early and accurate diagnosis. In this paper, we introduce the IVCM-Keratitis dataset, which comprises of a total of 4001 sample images of AK and FK, as well as non-specific keratitis (NSK) and healthy corneas classes. We use this dataset to develop multiple deep-learning models based on Convolutional Neural Networks (CNNs) to provide automated assistance in enhancing the diagnostic accuracy of confocal microscopy in infectious keratitis. Densenet161 had the best performance among these models, with an accuracy, precision, recall, and F1 score of 93.55%, 92.52%, 94.77%, and 96.93%, respectively. Our study highlights the potential of deep learning models to provide automated diagnostic assistance for infectious keratitis via confocal microscopy images, particularly in the early detection of AK and FK. The proposed model can provide valuable support to both experienced and inexperienced eye-care practitioners in confocal microscopy image analysis, by suggesting the most likely diagnosis. We further demonstrate that these models can highlight the areas of infection in the IVCM images and explain the reasons behind their diagnosis by utilizing saliency maps, a technique used in eXplainable Artificial Intelligence (XAI) to interpret these models.

Genetic instability (GI) is frequently associated with solid tumors including cancers of the breast. We hypothesize that the degree of GI, as evidenced by the cell-to-cell variations in the copy number of specific DNA loci, can be used as a marker for cancer prognosis. We present a computer-aided method for GI measurement in thick tissue sections based on confocal microscopy and 3D image analysis and visualization. The results, based on the analysis of breast tissue sections show considerable cell-to-cell variability in the copy number of the centromere of chromosome 1 and the 20q13 locus in invasive cancer but the normal 2 copies per cell in histologically normal tissue.

The adductor muscle of the Bermuda scallop, Pecten ziczac, is composed of two different types of muscle: cross-striated and smooth. The major portion consists of ribbon-shaped cross-striated muscle cells averaging about ten µm by 1.5 µm in cross section. Each cell contains only one myofibril. In some of the wider cells, an extra sarcomere sometimes is inserted in the lateral part of the myofibril creating a vernier. The individual striated muscle cells do not span the entire length of the adductor but are connected at their ends via junctions similar to intercalated discs. The minor portion of the adductor muscle consists of smooth muscle cells which are fusiform averaging 6 µm in diameter. There are no specialized cell surface invaginations in either muscle type that correspond to a T-tubule or caveolae system. The sarcoplasmic reticulum in both muscles is confined to the area just beneath the cell surface. In both muscle types, the sarcoplasmic reticulum systems have distended vesicles connected to the cell membrane via surface couplings. These vesicles are interconnected by one to seven tubular elements of the sarcoplasmic reticulum to form a closed and continuous system. The tubular elements of the sarcoplasmic reticulum in the cross-striated muscle are fairly uniform in bore, 40 nm in diameter, as compared to the irregular bore in smooth muscle, 15-40 nm. The striated muscle has twice as much of its surface covered with sarcoplasmic reticulum as does the smooth muscle. Moreover, the striated muscle cells have about 4% of their cross-sectional area devoted to sarcoplasmic reticulum while the smooth muscle cells have only 0.5%. This eight-fold difference in the amount of sarcoplasmic reticulum in the striated muscle is consistent with its reported 50-fold faster contraction rate and 128-fold faster relaxation time over that of the scallop adductor smooth muscle.

A series of aluminum melts has been refined with respect to inclusions by use of ALCOA 469, FILD, or SNIF. The content and size distribution of inclusions in the original-and the refined melts-have been measured by use of neutron activation (oxygen content), gas chromatography (carbide content), sedimentation analysis, and dissolution of metal in hydrochloric acid and subsequent analysis of oxides by means of a Coulter Counter. All the units tested have a beneficial effect and decrease the inclusion content, but the number of analyses are too few to make general conclusions. However, for melts cleaned by use of SNIF, it was found that oxides larger than 50 μm in cross section and borides larger than 20 μm in diameter were removed, while the smaller borides were agglomerated only. The effect of FILD and ALCOA 469 upon the melt tested was removal of borides larger than 5-10 μ m and oxides larger than 15μm in diameter, respectively.

Visual behaviour and the structure of dark and light-adapted larval and adult eyes of the New Zealand glowworm Arachnocampa luminosa (Mycetophilidae: Diptera)

We describe several unusual forms of natural graphite from an alkaline pegmatite that cross-cuts rischorrite in the Hackman Valley, Khibiny Massif, Kola Peninsula, Russia. The graphite occurs macroscopically in two forms: as spherical aggregates up to 2 cm in diameter of friable, radially aligned fibers ~20 μm in cross section, and as fine-grained surface coatings in cavities covering aegirine, strontian fluorapatite and K-feldspar. Optical microscopy and field emission scanning electron microscopy (FESEM) show that the fibers are actually hollow channels whose walls are composed of tabular crystals of graphite greatly elongate in the direction of the fiber axis and with their basal planes oriented parallel to the channel walls. Inside and among the channels occur rolled graphitic structures (RGS): scrolls, tubes, and cones up to 2 μm in diameter and up to 15 μm in length. The fine-grained graphite coatings on the surfaces of cavities, on the other hand, consist almost solely of micro- and nanoscale RGS. The largest of the RGS are hollow scrolls, with the c axis predominantly perpendicular to the scroll axis. These are usually cigar-shaped but can also be more tubular. Conical RGS occur at the micro- and nanoscales. The nanoscale cones tend not to be hollow and may have a cone–helix structure. Transmission electron microscopy (TEM), Raman spectroscopy, and FESEM indicate that the RGS are composed of well-ordered graphitic layers but are commonly coated by amorphous carbon. The morphologies and paragenesis of these unusual graphite forms suggest a possible hydrothermal origin.

Fluorescence imaging has become an indispensable technique in cancer research because it can reveal informative molecular, cellular, anatomical, and functional insights. Development of advanced fluorescent probes with superior sensitivity and biological selectivity for fluorescence imaging is thus imperative. To move forward in this direction, we developed an easy self-assembly method for fabricating aptamer- anchored rubrene-loaded organic fluorescent nanoprobes. The aptamer-modified organic nanoprobes integrated the best features of the organic light-emitting materials and the aptamers, thus endowing them with excellent cell-targeting capability, high stability, and good biocompatibility. By using this general method, a variety of biocompatible and highly bright organic fluorescent nanoprobes based on novel organic light-emitting materials with specific recognition could be easily constructed for real-time biosensing and long-term biomedical imaging. © 2019 Chinese Chemical Society.

Objective Keratomycosis is a suppurative, usually ulcerative corneal disease. Members of the filamentous fungal genus Fusarium are the most frequently cause of the ketatomycosis in humans, This study was to characterize restrospectively the clinical features and confocal microscopic imaging features of Fusarium keratitis. Methods Forty-three cases with Fusarium keratitis determined by fungal cultured were reviewed. Results Of the 43 patients analyzed in this study, 19（44. 2% ） were male,and 24（55.8% ） were female. The average age of patients was 44.9 ± 12.4 years. The tough elevated surface was seen in the cornea under the slit lamp biomicroscope in 23 of 43 patients. The feathery border appeared in 20 patients. The bright hyper-reflective branching structures were revealed under the confocal microscope, and the hypha ranged 2 -5 μm in diameter and 50 -200μm in length in 19 patients. In 16 eyes, bright hyper-refleetive line structures appeared under the confocal microscope,and the hypha ranged 3 to 7 μm in diameter and 150 -300μm in length. Conclusion The tough elevated surface and feathery border are believed to be the primary clinical features of keratitis infected by Fusarium. The hypha present two kinds of configuration in Fusarium keratitis. The keratitis caused by Fusarium has the similar imaging characteristics Key words: Fusarium; fungal keratitis; confocal microscopy; eye infection

There is considerable interest in tumor angiogenesis and its inhibition, because tumor angiogenesis plays an important role in tumor growth and distant metastasis, and is highly associated with overall survival and relapse free-survival in cancer patients. This study was performed to determine the mechanisms underlying tumor angiogenesis and its inhibition using the angiogenesis inhibitor TNP 470 and antimitotic drug, docetaxel (Taxotere). Variable 3D image analysis of tumor vasculature after chemotherapy was performed by CLSM using the DMBA-induced rat breast cancer model. 3D images of the vasculature in DMBA-induced rat breast cancer were characterized by increased microvessel density and anastomosing, irregularly branched microvessel networks. After administration of TNP-470 or docetaxel, tumor microvessel density was markedly decreased and tumor microvessel networks were markedly destroyed. Furthermore, tumor microvessels less than 30 μm in diameter were not found. The remaining tumor microvessels (30-60 μm in diameter) were mainly localized in the periphery of the tumor. These observations suggest that docetaxel might have the ability to inhibit tumor angiogenesis as well as TNP-470. CLSM with the FI method is a valuable technique for estimation of the efficacy of anti-angiogenic chemotherapy.

Porosity over a broad range (typically 0.001–300 μm in diameter) of tissue scaffolds provides appropriate conditions for diffusion and adsorption of small molecules and macromolecules, migration of cells through the scaffold, and adequate cell proliferative capacity. Characterisation of pores over this large range poses a problem especially when analysing soft polymer hydrogels, as no one methodology can adequately cover the entire range. This work describes a combined technique used for evaluation of the porous structure of a collagen hydrogel (dermal substitute Integra®) on the basis of NMR-cryoporometry (sensitive to nanopores) and confocal laser scanning microscopy (CLSM) imaging (sensitive to macropores). Thermodesorption of water, diffusion of proteins through a collagen membrane, migration and growth of normal primary human skin fibroblasts, and the interaction kinetics of 3T3 mouse fibroblast cells (using a quartz crystal microbalance) with collagen were analysed with respect to the porous structure of the material. The contribution to the total porosity of pores with a diameter of less than 100 nm is low, at approximately 3–5%, a figure estimated using the methods described above. However, these pores are the main contributor to the specific surface area (S ≈ 120 m2 g−1) as larger diameter macropores, with diameters of 20–200 μm, have a much lower surface area at S ≈ 0.4 m2 g−1 relative to their large pore volume V = 14.4 cm3 g−1.