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Reconstruction of image sequences from ungated and scanning-aberrated laser scanning microscopy images of the beating heart
摘要： Fluorescence laser-scanning microscopy is a well-established imaging technique in biology, available in many imaging facilities to investigate structures within live animal embryos such as zebrafish. Laser scanning microscopes (LSM) are limited when used to study dynamic heart morphology or function. Despite their ability to resolve static cardiac structures, the fast motion of the beating heart introduces severe artifacts in the scanned images and gating the acquisitions to the heartbeat is difficult to implement on traditional microscopes. Furthermore, although alternative high-speed imaging instruments exist, they are not widely available (due to cost or hardware complications), putting dynamic cardio-vascular imaging off-limits for many researchers. Here, we propose a method that allows imaging the beating heart on conventional LSMs. Our approach takes a set of images containing scanning aberrations, each triggered at an arbitrary time in the cardiac cycle, and assembles an image sequence that covers a single cardiac heartbeat. The steps are: (i) frame sorting by solving a traveling salesman problem; (ii) heartbeat duration estimation; and (iii) scan-delay compensation via space-time resampling. We characterize the performance of our method on synthetic data under several light intensities and scanning speeds. We further illustrate our method's applicability on experimental images acquired in live zebrafish larvae, and show that the reconstruction quality approaches that of fast, state-of-the-art microscopes. Our technique opens the possibility of using LSMs to carry out studies of cardiac dynamics, without the need for prospective gating or fast microscopes.
关键词： cardiac imaging,scanning aberration compensation,laser scanning microscopy,zebrafish imaging,fast microscopy,combinatorial optimization,traveling salesman problem,image and video sampling,Computational microscopy,confocal microscopy
更新于2025-09-11 14:15:04
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[Methods in Molecular Biology] FOXO Transcription Factors Volume 1890 (Methods and Protocols) || Immunofluorescence Analysis by Confocal Microscopy for Detecting Endogenous FOXO
摘要： Cancer cells are known to inactivate tumor suppressor proteins by triggering their anomalous subcellular location. It has been well established that the aberrant location of FOXO proteins is linked to tumor formation, progression of the same, or resistance to anti-neoplastic treatment. Furthermore, the abnormal location of FOXO has also been considered a potential biomarker for diabetic complications or longevity in different organisms. Here, we describe the immunodetection of endogenous FOXO by confocal microscopy, which can be used as a chemical tool to quantify FOXO expression levels, its cellular location, and even its active/inactive forms with relevant antibodies.
关键词： Immunodetection,Nuclear translocation,FOXO proteins,Confocal microscopy,Biomarker
更新于2025-09-10 09:29:36
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Mapping of dendritic lesions in patients with herpes simplex keratitis using in vivo confocal microscopy
摘要： Purpose: To produce a two-dimensional reconstruction map of dendritic lesions in patients with herpes simplex keratitis (HSK) using in vivo confocal microscopy. Methods: Four eyes of four patients (mean 65.8 years) with HSK presenting with a dendritic lesion were enrolled. Slit-lamp biomicroscopy and in vivo laser confocal microscopy were performed. Acquired confocal images at the level of the epithelium were arranged and mapped into subconfluent montages. Changes in the shape and degree of light reflection of abnormal cells and deposits around dendritic lesions as well as other corneal layers were qualitatively evaluated. Results: Mapping of dendritic lesion was successful in all cases, and the subconfluent montages clearly showed the larger image of dendritic lesion. In all cases, the dendritic lesion consisted of hyperreflective irregular epithelial cells, and was surrounded by distorted and elongated epithelial cells. In three cases, hyperreflective deposits were noted at the midline of the lesion. The corneal stroma showed a hyperreflective honeycomb pattern. In two cases, inflammatory cells were observed at the level of endothelial cell layer. Conclusion: Mapping of dendritic lesions in patients with HSK was successful in all patients using in vivo confocal microscopy. Cellular level observation of dendritic lesion at a relatively larger magnification may help understand the in vivo morphological change of HSK. Further study in more patients with HSK and nonherpetic dendritic lesion is needed to utilize confocal microscopy images in differential diagnosis and follow-up of the epithelial lesions with dendrite.
关键词： hyperreflective irregular epithelial cells,herpetic epithelial keratitis,in vivo confocal microscopy,fluorescein slit-lamp photograph,dendritic lesion
更新于2025-09-10 09:29:36
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Lesions Mimicking Melanoma at Dermoscopy Confirmed Basal Cell Carcinoma: Evaluation with Reflectance Confocal Microscopy
摘要： Background: Atypical basal cell carcinoma (BCC), characterized by equivocal dermoscopic features typical of malignant melanoma (MM), can be difficult to diagnose. Reflectance confocal microscopy (RCM) enables in vivo imaging at nearly histological resolution. Objectives: To evaluate with RCM atypical melanocytic lesions identified in dermoscopy, according to common RCM criteria for the differential diagnosis of BCC, and to identify representative RCM parameters for superficial (sBCCs) and nonsuperficial (nsBCCs) basal cell carcinomas (BCCs). Methods: A retrospective analysis of consecutive patients evaluated with RCM, selecting excised lesions classified at dermoscopy with ≥1 score from the revisited 7-point checklist, mimicking melanoma, registered between 2010 and 2016. Cluster analysis identified BCC subclassifications. Results: Of 178 atypical lesions, 34 lesions were diagnosed as BCCs with RCM. Lesions were confirmed BCCs with histopathology. Dermoscopic features included atypical network (55.9%) and regression structures (35.5%) associated with sBCCs, and an atypical vascular pattern (58.8%) and irregular blotches (58.8%) with nsBCCs. Hierarchical cluster analysis identified 2 clusters: cluster 1 (100% sBCCs) was characterized by the presence of cords connected to the epidermis (90%, p < 0.001), tumor islands located in the epidermis (100%, p < 0.001), smaller vascular diameter (100%, p < 0.001) and solar elastosis (90%, p = 0.017), and cluster 2 (nsBCCs 85%) was defined by the dermic location of tumor islands (87.5%, p < 0.001) with branch-like structures (70.8%, p = 0.007) and surrounding collagen (83.3%, p = 0.012), peripheral palisading (83.3%, p = 0.012) and coiled vascular morphology (79.2%, p < 0.001) with a larger vascular diameter (50%, p < 0.001). Conclusions: RCM is able to diagnose BCCs mimicking melanoma at dermoscopy and seems able to identify sBCCs and nsBCCs.
关键词： Melanoma,Basal cell carcinoma,Reflectance confocal microscopy,Dermoscopy,Noninvasive diagnosis
更新于2025-09-10 09:29:36
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Simplified, automated methods for assessing pixel intensities of fluorescently-tagged drugs in cells
摘要： Assessing the cytoplasmic uptake of fluorescently-tagged drugs in heterogeneous cell types currently involves time-consuming manual segmentation of confocal microscopy images. We developed a set of methods that incorporate map algebra techniques to facilitate and expedite image segmentation, particularly of the parenchyma of intermediate cells in the stria vascularis of the inner ear. Map algebra is used to apply a convolution kernel to pixel neighborhoods to create a masking image to select pixels in the original image for further operations. Here, we describe the utility of integrated intensity-based, percentile-based, and local autocorrelation-based methods to automate segmentation of images into putative morphological regions for pixel intensity analysis. Integrated intensity-based methods are variants of watershed segmentation tools that determine morphological boundaries from rates of change in integrated pixel intensity. Percentile- and local autocorrelation-based methods evolved out of the process of developing map algebra- and integrated intensity-based tools. We identified several simplifications that are surprisingly effective for image segmentation and pixel intensity analysis. These methods were empirically validated on three levels: first, the algorithms were developed based on iterations of inspected results; second, algorithms were tested for various types of robustness; and third, developed algorithms were validated against results from manually-segmented images. We conclude the key to automated segmentation is supervision of output data.
关键词： pixel intensity analysis,map algebra,fluorescently-tagged drugs,confocal microscopy,automated segmentation
更新于2025-09-10 09:29:36
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Clarification and confocal imaging of the nonhuman primate placental micro-anatomy
摘要： Geometry of the placental villous vasculature is a key determinant of maternal–fetal nutrient exchange for optimal fetal growth. Recent advances in tissue clarification techniques allow for deep high-resolution imaging with confocal microscopy; however, the methodology lacks a signal:noise ratio of sufficient magnitude to allow for quantitative analysis. Thus, we sought to develop a reproducible method to investigate the 3D vasculature of the nonhuman primate placenta for subsequent data analysis. Fresh placental tissue was dissected, formalin fixed, clarified using a modified Visikol? protocol and immunolabeled for CD31 (fetal endothelium) and cytokeratin-7 (villous trophoblast) for confocal imaging of the microanatomy. We present a detailed clarification and staining protocol augmented for imaging of nonhuman primate placental tissue. The image stacks generated by this refined staining method and our data acquisition parameters can be analyzed quantitatively to provide insights regarding the villous and vascular micro-anatomy of the placenta.
关键词： placenta,immunohistochemistry,tissue clarification,nonhuman primate,confocal microscopy
更新于2025-09-10 09:29:36
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Reflectance confocal microscopy features of labial melanotic macule: Report of three cases
摘要： The term labial melanotic macule of the lips (MML) is used to describe a flat-pigmented lesion on the vermilion border, with an estimated prevalence of up to 3% of the population. Most MMLs are located on the lower lip and are more common in female patients. MMLs are mostly associated with hyperpigmentation of the basal keratinocytes with normal to a slightly increased density of melanocytes; an infiltrate of melanophages in the superficial dermis can also contribute to the pigmented appearance of MMLs. These benign lesions may raise concern for melanoma or may be confused with other labial pigmentary disorders. Dermoscopy is widely used for the diagnosis of pigmented and nonpigmented lesions on the skin. Reflectance confocal microscopy (RCM) is an in vivo, noninvasive imaging technique that allows horizontal optical sectioning of skin at cellular-level resolution, from the surface to a depth of about 200 μm. Although the lips are amenable for hand-held RCM examination, publications about the RCM findings in MMLs are limited. To improve the recognition of MML under RCM, we report on 3 cases of biopsy-proven MMLs.
关键词： dermoscopy,labial melanotic macule,reflectance confocal microscopy
更新于2025-09-10 09:29:36
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Diagnostic accuracy of ex vivo fluorescence confocal microscopy for Mohs surgery of basal cell carcinomas: a prospective study on 753 margins
摘要： Background: Frozen histological sections are currently used for intra-operative margin assessment during Mohs surgery. Fluorescence confocal microscopy (FCM) is a new tool, which offers a promising and faster alternative to frozen histology. Objective: The aim of the present study is to prospectively evaluate in a clinical setting, the accuracy of FCM as compared to frozen sections in BCC’s margin assessment. Methods: Patients with BCC, scheduled for Mohs surgery were prospectively enrolled. Freshly-excised surgical specimens were first examined through FCM and then frozen sections were evaluated. Permanent sections were finally obtained, in order to validate our sample technique. A blind re-evaluation was also performed for discordant cases between FCM and frozen sections. Sensitivity and specificity levels, as well as positive and negative predicting values were calculated and ROC curves were generated. Results: We enrolled 127 BCCs in as many patients (40.2% females). A total number of 753 sections were examined. All BCCs were located on the head and neck area. When evaluating the performance of FCM as compared to frozen sections 79.8% sensitivity, 95.8% specificity, 80.5% positive predicting and 95.7% negative predicting values were found (Area Under the Curve: .88, 95%CI .84-.92; P<.001). A total of 49 discordant cases between FCM and frozen sections evaluations were blindly re-evaluated, of which 24 were false positive and 25 false negative. The performance of FCM and frozen sections was also evaluated according to the final histopathological assessment. Conclusions: We found high levels of accuracy for FCM as compared to frozen sections evaluation, in intra-operative BCC’s margin assessment during Mohs surgery. Some technical issues still prevent a wide use of this technique, but new upcoming devices promise to overcome these limitations.
关键词： basal cell carcinoma,fluorescence confocal microscopy,Mohs surgery,skin cancer,ex vivo confocal microscopy
更新于2025-09-09 09:28:46
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Efficient excitation of dye molecules for single photon generation
摘要： A reliable single photon source is required for many aspects of quantum technology. Organic molecules are attractive for this application because they can have high quantum yield and can be photostable, even at room temperature. To generate a photon with high probability, a laser must excite the molecule efficiently. We develop a simple model for that efficiency and discuss how to optimise it. We demonstrate the validity of our model through experiments on a single dibenzoterrylene (DBT) molecule in an anthracene crystal. We show that the excitation probability cannot exceed 75% at room temperature, but can increase to over 99% if the sample is cooled to liquid nitrogen temperature. The possibility of high photon generation efficiency with only modest cooling is a significant step towards a reliable photon source that is simple and practical.
关键词： single molecules,photon anti-bunching,single photon source,quantum optics,confocal microscopy
更新于2025-09-09 09:28:46
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Classification of Limbal Stem Cell Deficiency Using Clinical and Confocal Grading
摘要： To grade the severity of limbal stem cell deficiency (LSCD) based on the extent of clinical presentation and central corneal basal epithelial cell density (BCD). This is a retrospective observational comparative study of 48 eyes of 35 patients with LSCD and 9 eyes of 7 normal subjects (controls). Confocal images of the central cornea were acquired. A clinical scoring system was created based on the extent of limbal and corneal surface involvement. LSCD was graded as mild, moderate, and severe stages based on the clinical scores. The degree of BCD reduction was given a score of 0 to 3. Compared with BCD in controls, BCD decreased by 23.0%, 40.4%, and 69.5% in the mild, moderate, and severe stages of LSCD classified by the clinical scoring system, respectively. The degree of BCD reduction was positively correlated with larger limbal and corneal surface involvement and when the central visual axis was affected (all P # 0.0005). Mean corrected distance visual acuity logarithm of the minimum angle of resolution was 0.0 6 0.0 in control eyes, 0.2 6 0.5 in mild LSCD, 0.6 6 0.4 in moderate LSCD, and 1.6 6 1.1 in severe LSCD (P , 0.0001). There was a significant correlation between a higher clinical score and corrected distance visual acuity logarithm of the minimum angle of resolution (rho = 0.82; P , 0.0001) and a greater decrease in BCD (rho = 20.78; P , 0.0001). A clinical scoring system was developed to assess the extent of clinical presentation of LSCD. A classification system to grade the severity of LSCD can be established by combining the BCD score with the clinical score.
关键词： diagnosis,classification system,limbal stem cell,in vivo confocal microscopy,limbal stem cell deficiency
更新于2025-09-09 09:28:46