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Single-molecule localization microscopy (SMLM) is a powerful technique for elucidating structure and dynamics in the life- and material sciences with sub-50 nm spatial resolution. The simultaneous acquisition of spectral information (spectrally resolved SMLM, sSMLM) enables multiplexing using spectrally distinct fluorophores or enable the probing of local chemical environments by using solvachromatic fluorophores such as Nile Red. Until now, the widespread utilisation of sSMLM was hampered by several challenges: an increased complexity of the optical detection pathway, limited software solutions for data analysis, lower accessible emitter densities or smaller field-of-views, and overall compromised spatio-spectral resolution. Here, we present a low-cost implementation of sSMLM that addresses these challenges. Using a blazed, low-dispersion transmission grating positioned close to the image plane here represented by the camera sensor, the +1st diffraction order is minimally elongated compared to the point spread function of the 0th order and can therefore be analysed using common subpixel single-molecule localization algorithms. The distance between both PSFs provides accurate information on the spectral properties of the emitter. The minimal excess width of 1st order PSFs enables a fivefold higher emitter density compared to other sSMLM approaches whilst achieving a spatio-spectral localization accuracy sufficient to discriminate between fluorophores whose peak emission are less than 15 nm apart as demonstrated using dSTORM, DNA-PAINT and smFRET. We provide an ImageJ/Fiji plugin (sSMLMAnalyzer) and suitable Matlab scripts for data analysis. We envision that our approach will find widespread use in super-resolution applications that rely on distinguishing spectrally different fluorophores under low photon conditions.

The base of the cilium comprising the transition zone (TZ) and transition fibers (TF) acts as a selecting gate to regulate the intraflagellar transport (IFT)-dependent trafficking of proteins to and from cilia. Before entering the ciliary compartment, IFT complexes and transported cargoes accumulate at or near the base of the cilium. The spatial organization of IFT proteins at the cilia base is key for understanding cilia formation and function. Using stochastic optical reconstruction microscopy (STORM) and computational averaging, we show that seven TZ, nine IFT, three Bardet–Biedl syndrome (BBS), and one centrosomal protein, form 9-clustered rings at the cilium base of a ciliate Tetrahymena thermophila. In the axial dimension, analyzed TZ proteins localize to a narrow region of about 30 nm while IFT proteins dock approximately 80 nm proximal to TZ. Moreover, the IFT-A subcomplex is positioned peripheral to the IFT-B subcomplex and the investigated BBS proteins localize near the ciliary membrane. The positioning of the HA-tagged N- and C-termini of the selected proteins enabled the prediction of the spatial orientation of protein particles and likely cargo interaction sites. Based on the obtained data, we built a comprehensive 3D-model showing the arrangement of the investigated ciliary proteins.

Preclinical studies usually require high levels of morphological, functional, and biochemical information at subcellular resolution. This type of information cannot be obtained from clinical imaging techniques, such as MRI, PET/CT, or US. Luckily, many microscopy techniques exist that can offer this information, also for malignant tissues and therapeutic approaches. In this overview, we discuss the various advanced optical microscopy techniques and their applications in oncological research. After a short introduction in Sect. 16.1, we continue in Sect. 16.2 with a discussion on fluorescent labelling strategies, followed in Sect. 16.3 by an in-depth description of confocal, light-sheet, two-photon, and super-resolution microscopy. We end in Sect. 16.4 with a focus on the applications, specifically in oncology.

Preclinical studies usually require high levels of morphological, functional, and biochemical information at subcellular resolution. This type of information cannot be obtained from clinical imaging techniques, such as MRI, PET/CT, or US. Luckily, many microscopy techniques exist that can offer this information, also for malignant tissues and therapeutic approaches. In this overview, we discuss the various advanced optical microscopy techniques and their applications in oncological research. After a short introduction in Sect. 16.1, we continue in Sect. 16.2 with a discussion on fluorescent labelling strategies, followed in Sect. 16.3 by an in-depth description of confocal, light-sheet, two-photon, and super-resolution microscopy. We end in Sect. 16.4 with a focus on the applications, specifically in oncology.

Microscopy and microanalysis 24(5), 517-525 (2018). doi:10.1017/S1431927618015143, Published by Cambridge University Press, New York, NY

Examining cell behavior in its correct tissue context is a major challenge in cell biology. The recent development of mammalian stem cell-based organoid cultures offers exciting opportunities to visualize dynamic cellular events in a 3D tissue-like setting. We describe here an approach for live imaging of cell division processes in intestinal organoid cultures derived from human and mouse adult stem cells. These approaches can be extended to the analysis of cellular events in diseased tissue, such as patient-derived tumor organoids.

The diagnosis of mycobacterial infections has been dramatically improved by the introduction of molecular methods aimed to reduce the time to diagnosis as compared with culture. The broad range pan-mycobacterial PCR can detect all the mycobacterial species directly from clinical specimens. We aimed to evaluate its usefulness and its clinical added value for the diagnosis of nontuberculous mycobacterial (NTM) infections. We performed a retrospective study (2003–2013) including 952 samples taken from 639 patients with clinical suspicion of NTM infection. The performance of smear microscopy, PCR and culture was established using clinical data to investigate discrepant results. We also compared the time to microbial diagnosis between the direct PCR and culture. The sensitivity, specificity, positive and negative predictive values of the PCR were 61.6% (53.5–69.1), 99.1% (98.2–99.6), 92.8% (85.8–96.5) and 93.4% (91.6–94.9), respectively, when considering all specimens. When considering smear-positive specimens and smear-negative specimens, the sensitivity was 81.6% and 40%, respectively. The sensitivity for pulmonary and extra-pulmonary smear-positive specimens was 85.2% versus 72.7%. The median time to identification at species level was 35 days (SD, 17.67) for culture and 6 days (SD, 2.67) for the PCR (when positive), which represents a 29-day shorter time to results (p

Leishmania parasites cause a set of neglected tropical diseases with considerable public health impact, the leishmaniases, which are often fatal if left untreated. Since current treatments for the leishmaniases exhibit high toxicity, low efficacy and prohibitive prices, many laboratories throughout the world are engaged in research for the discovery of novel chemotherapeutics. This entails the necessity of screening large numbers of compounds against the clinically relevant form of the parasite, the obligatory intracellular amastigote, a procedure that in many laboratories is still carried out by manual inspection. To overcome this well-known bottleneck in Leishmania drug development, several studies have recently attempted to automate this process. Here we implemented an image-based high content triage assay for Leishmania which has the added advantages of using primary macrophages instead of macrophage cell lines and of enabling identification of active compounds against parasite species developing both in small individual phagolysosomes (such as L. infantum) and in large communal vacuoles (such as L. amazonensis). The automated image analysis protocol is made available for IN Cell Analyzer systems, and, importantly, also for the open-source CellProfiler software, in this way extending its implementation to any laboratory involved in drug development as well as in other aspects of Leishmania research requiring analysis of in vitro infected macrophages., Project Norte-01-0145-FEDER-000012Structured program on bioengineered therapies for infectiousdiseases and tissue regeneration, supportedby Norte Portugal Regional 17 Operational Programme (NORTE 2020), under the PORTUGAL 2020 PartnershipAgreement,through the European Regional Development Fund (FEDER). AGGA and TC were supported by contracts SFRH/BD/93766/2013,financed by POPH—QREN, and subsidized by Fundo Social Europeu and Ministe ´rio da Ciência,Tecnologia e Ensino Superior, and PTDC/QEQ-MED/7097/2014, funded by National Funds throughFCT, respectively. HC is fundedby FCT under the “Investigador FCT” Programme., info:eu-repo/semantics/publishedVersion

Objective Cardiomyocytes derived from human-induced pluripotent stem cells are a powerful platform for high-throughput drug screening in vitro. However, current modalities for drug testing, such as electrophysiology and fluorescence imaging have inherent drawbacks. To circumvent these problems, we report the development of a bioluminescent Ca2+ indicator GmNL(Ca2+), and its application in a customized microscope for high-throughput drug screening. Results GmNL(Ca2+) gives a 140% signal change with Ca2+, and can image drug-induced changes of Ca2+ dynamics in cultured cells. Since bioluminescence requires application of a chemical substrate, which is consumed over ~ 30 min we made a dedicated microscope with automated drug dispensing inside a light-tight box, to control drug addition. To overcome thermal instability of the luminescent substrate, or small molecule, dual climate control enables distinct temperature settings in the drug reservoir and the biological sample. By combining GmNL(Ca2+) with this adaptation, we could image spontaneous Ca2+ transients in cultured cardiomyocytes and phenotype their response to well-known drugs without accessing the sample directly. In addition, the bioluminescent strategy demonstrates minimal perturbation of contractile parameters and long-term observation attributable to lack of phototoxicity and photobleaching. Overall, bioluminescence may enable more accurate drug screening in a high-throughput manner.

Background: Cancers are heterogeneous and contain various types of irregular structures that can go undetected when examining them with standard two-dimensional microscopes. Studies of intricate networks of vasculature systems, e.g., the tumour lymphatic microvessels, benefit largely from three-dimensional imaging data analysis. Methods: The new DIPCO (Diagnosing Immunolabeled Paraffin-Embedded Cleared Organs) imaging platform uses three-dimensional light-sheet microscopy and whole-mount immunolabelling of cleared samples to study proteins and micro-anatomies deep inside of tumours. Results: Here, we uncovered the whole three-dimensional lymphatic microvasculature of formalin-fixed paraffin-embedded (FFPE) tumours from a cohort of 30 patients with bladder cancer. Our results revealed more heterogeneous spatial deviations in more advanced bladder tumours. We also showed that three-dimensional imaging could determine tumour stage and identify vascular or lymphatic system invasion with higher accuracy than standard two-dimensional histological diagnostic methods. There was no association between sample storage times and outcomes, demonstrating that the DIPCO pipeline could be successfully applied on old FFPE samples. Conclusions: Studying tumour samples with three-dimensional imaging could help us understand the pathological nature of cancers and provide essential information that might improve the accuracy of cancer staging.

A robust and reproducible method for culturing monolayers of adherent and well-spread primary islet cells on glass coverslips is required for detailed imaging studies by super-resolution and live-cell microscopy. Guided by an observation that dispersed islet cells spread and adhere well on glass surfaces in neuronal co-culture and form a monolayer of connected cells, we demonstrate that in the absence of neurons, well-defined surface coatings combined with components of neuronal culture media collectively support robust attachment and growth of primary human or rat islet cells as monolayers on glass surfaces. The islet cell monolayer cultures on glass stably maintain distinct mono-hormonal insulin+, glucagon+, somatostatin+ and PP+ cells and glucose-responsive synchronized calcium signaling as well as expression of the transcription factors Pdx-1 and NKX-6.1 in beta cells. This technical advance enabled detailed observation of sub-cellular processes in primary human and rat beta cells by super-resolution microscopy. The protocol is envisaged to have broad applicability to sophisticated analyses of pancreatic islet cells that reveal new biological insights, as demonstrated by the identification of an in vitro protocol that markedly increases proliferation of primary beta cells and is associated with a reduction in ciliated, ostensibly proliferation-suppressed beta cells.

Background Improved and affordable diagnostic or triage tests are urgently needed at the microscopy centre level. Automated digital microscopy has the potential to overcome issues related to conventional microscopy, including training time requirement and inconsistencies in results interpretation. Methods For this blinded prospective study, sputum samples were collected from adults with presumptive pulmonary tuberculosis in Lima, Peru and Ho Chi Minh City, Vietnam. TBDx performance was evaluated as a stand-alone and as a triage test against conventional microscopy and Xpert, with culture as the reference standard. Xpert was used to confirm positive cases. Findings A total of 613 subjects were enrolled between October 2014 and March 2015, with 539 included in the final analysis. The sensitivity of TBDx was 62·2% (95% CI 56·6–67·4) and specificity was 90·7% (95% CI 85·9–94·2) compared to culture. The algorithm assessing TBDx as a triage test achieved a specificity of 100% while maintaining sensitivity. Interpretation While the diagnostic performance of TBDx did not reach the levels obtained by experienced microscopists in reference laboratories, it is conceivable that it would exceed the performance of less experienced microscopists. In the absence of highly sensitive and specific molecular tests at the microscopy centre level, TBDx in a triage-testing algorithm would optimize specificity and limit overall cost without compromising the number of patients receiving up-front drug susceptibility testing for rifampicin. However, the algorithm would miss over one third of patients compared to Xpert alone.

Purpose: To investigate the effects of pterygium on corneal endothelial cell density in patients with unilateral pterygium. Methods: We performed a cross-sectional analysis of data from patients with unilateral pterygium who were selected from September 1, 2015 to July 31, 2016 at Hospital de Base do Distrito Federal to assess the corneal endothelial cell density, coefficient of variation in the cell area, hexagonality, and corneal pachymetric results. In all patients, noncontact specular microscopy was performed in both eyes and a minimum endothelial cell count of 75 cells/mm2 was required for inclusion in the study. The contralateral eye served as the control. Results: Sixty-one patients were included in the study. Twenty-nine (47.5%) patients were men and 32 (52.5%) were women (mean age, 50.84 ± 13.8). The percentage of pterygium that invaded the cornea ranged from 4.87% to 24.59% (median, 9.70% ± 4.99%). The mean corneal endothelial cell density (cells/mm) was lower in the pterygium eyes than in the controls (2451.83 ± 284.96 vs. 2549.95 ± 268.94, respectively; p=0.04). No differences in the mean coefficients of variation of cell size, hexagonality, and corneal pachymetric results were observed between the patients and controls. The Pearson correlation test showed a significant negative linear relationship between pterygium invasion and endothelial cell density [p

Contains fulltext : 173035.pdf (Publisher’s version ) (Open Access) AIMS: The quality and quantity of the infiltration of immune cells into tumour tissues have substantial impacts on patients' clinical outcomes, and are associated with response to immunotherapy. Therefore, the precise analysis of tumour-infiltrating lymphocytes (TILs) is becoming an important additional pathological biomarker. Analysis of TILs is usually performed semiquantitatively by pathologists on haematoxylin and eosin-stained or immunostained tissue sections. However, automated quantification outperforms semiquantitative approaches, and is becoming the standard. Owing to the presence of melanin pigment, this approach is seriously hampered in melanoma, because the spectrum of melanin lies close to that of commonly used immunohistochemical stains. Aim of this study is to overcome the technical issues due to the presence of melanin for an automated and accurate quantification of TILs in melanoma. METHODS AND RESULTS: Here, we successfully applied a novel multispectral imaging (MSI) technique to enumerate T cells in human primary melanomas. This microscopy technique combines imaging with spectroscopy to obtain both quantitative expression data and the tissue distributions of different cellular markers. We demonstrate that MSI allows complete and accurate analysis of TILs, successfully avoiding the blurring of images by melanin pigments, in whole tissue slide primary melanoma lesions, which could otherwise not be accurately detected by conventional digital image methodologies. CONCLUSIONS: Our study highlights the potential of MSI for accurate assessment of immune cell infiltrates, including those in notoriously difficult tissues, such as pigmented melanomas. Quantification of tumour infiltration by different immune cell types is crucial in the search for new biomarkers to predict patient responses to immunotherapies. Our findings show that this innovative microscopy technique is an important extension of the armamentarium of pathologists.

Airborne precautions are required at hospital admission for patients with suspected pulmonary tuberculosis. The isolation is maintained until 3 serially collected sputum smears are acid-fast bacilli negative, a time- and labor-intensive method with limited sensitivity and specificity, which has a great impact on patient flow management. We evaluated the possibility of replacing the result of microscopy by the semiquantitative result of the molecular point-of-care test Xpert MTB/RIF to assess patients' transmission risk to quickly guide airborne isolation decisions in low-endemic countries. The performance of the Xpert MTB/RIF, used as a first-line test, was compared to the results of microscopy for specimens (n=242) collected from May 2010 to December 2014 in Lausanne, Switzerland. The sensitivity and specificity of Xpert MTB/RIF were 91.5% (65/71) and 99.6% (170/171), respectively, vs. 64.8% (46/71) and 94.2% (161/171) for microscopy. Samples with negative Xpert MTB/RIF were all smear negative for Mycobacterium tuberculosis (negative predictive value, 100%). The semiquantitative results of Xpert MTB/RIF—high, medium, low or very low—were found to correlate with acid-fast bacilli detection: positive predictive value of 100% (6/6), 96.5% (27/28), 52.2% (12/23) and 11.1% (1/9) respectively. Finally, when including clinical criteria, we identified 11 smear-negative but Xpert MTB/RIF–positive patients with a significant transmission potential. In conclusion, our data support the introduction of an Xpert MTB/RIF–based strategy as a replacement of smear microscopy for a faster and more accurate management of tuberculosis patients' transmission risk in a low-prevalence country.

BACKGROUND: Macrophages are present before the onset of blood flow, but very little is known about their function in vascular development. We have developed a technique to concurrently label both endothelial cells and macrophages for time-lapse microscopy using co-injection of fluorescently conjugated acetylated low-density lipoprotein (AcLDL) and phagocytic dye PKH26-PCL.RESULTS: We characterize double-labeled cells to confirm specific labeling of macrophages. Double-labeled cells circulate, roll along the endothelium, and extravasate from vessels. Most observed macrophages are integrated into the vessel wall, showing an endothelial-like morphology. We used transgenic quail that express a fluorescent protein driven by the endothelial-specific promoter Tie1 in conjugation with the phagocytic dye to analyze these cells. Circulating PKH26-PCL-labeled cells are mostly Tie1-, but those which have integrated into the vessel wall are largely Tie1+. The endothelial-like phagocytic cells were generally stationary during normal vascular development. We, therefore, induced vascular remodeling and found that these cells could be recruited to sites of remodeling.CONCLUSIONS: The active interaction of endothelial cells and macrophages support the hypothesis that these cells are involved in vascular remodeling. The presence of phagocytic endothelial-like cells suggests either a myeloid-origin to certain endothelial cells or that circulating endothelial cells/hematopoietic stem cells have phagocytic capacity.

PURPOSE: To compare morphometric features between fellow acute primary angle-closure (APAC) eyes and glaucomatous or suspect eyes with narrow angle (NA). METHODS: Fellow eyes of 30 patients with unilateral APAC and 30 with NA were evaluated by ultrasound biomicroscopy (UBM) under light and dark conditions. UBM parameters such as anterior chamber depth (ACD), angle opening distance at 250 µm/500 µm from the scleral spur (AOD250/AOD500), trabecular ciliary process distance (TCPD) and iris-lens contact distance (ILCD) were measured in the superior (SQ) and inferior (IQ) quadrants. RESULTS: Significant differences between APAC fellow and NA eyes were found in ACD, P

This review aims at presenting state-of-the-art knowledge on the composition and functions of the endothelial glycocalyx. The endothelial glycocalyx is a network of membrane-bound proteoglycans and glycoproteins, covering the endothelium luminally. Both endothelium- and plasma-derived soluble molecules integrate into this mesh. Over the past decade, insight has been gained into the role of the glycocalyx in vascular physiology and pathology, including mechanotransduction, hemostasis, signaling, and blood cell-vessel wall interactions. The contribution of the glycocalyx to diabetes, ischemia/reperfusion, and atherosclerosis is also reviewed. Experimental data from the micro- and macrocirculation alludes at a vasculoprotective role for the glycocalyx. Assessing this possible role of the endothelial glycocalyx requires reliable visualization of this delicate layer, which is a great challenge. An overview is given of the various ways in which the endothelial glycocalyx has been visualized up to now, including first data from two-photon microscopic imaging.

Background: Tuberculosis (TB) is the most common opportunistic infection and the leading cause of death in HIV-positive people worldwide. Diagnosing TB is difficult, and is more challenging in resource-scarce settings where culture-based diagnostic methods rely on poorly sensitive smear microscopy by Ziehl-Neelsen stain (ZN). Methods: We performed a cross-sectional study examining the diagnostic utility of Microscopic Observation Drug Susceptibility liquid culture (MODS) versus traditional Ziehl-Neelsen staining (ZN) and Lowenstein Jensen culture (LJ) of pulmonary tuberculosis (TB) and multidrug-resistant tuberculosis (MDRTB) in HIV-infected patients in Bolivia. For sputum scarce individuals we assessed the value of the string test and induced sputum for TB diagnosis. The presence of Mycobacterium tuberculosis (Mtb) in the sputum of 107 HIV-positive patients was evaluated by ZN, LJ, and MODS. Gastric secretion samples obtained by the string test were evaluated by MODS in 102 patients. Results: The TB-HIV co-infection rate of HIV patients with respiratory symptoms by sputum sample was 45 % (48/107); 46/48 (96 %) were positive by MODS, 38/48 (79 %) by LJ, and 30/48 (63 %) by ZN. The rate of MDRTB was 9 % (4/48). Median time to positive culture was 10 days by MODS versus 34 days by LJ (p

The etiologic agents of acute gastroenteritis are diverse. The diagnosis of bacterial pathogens is particularly challenging given the large amount of vastly diverse indigenous gastrointestinal organisms present in stool. Multiple methods must be used by the clinical microbiology laboratories to diagnose the cause of acute gastroenteritis, including bacterial cultures, ELISA, and microscopy. Due to the limitations of conventional methods, there is still room for improvement in the detection of pathogens by using the molecular methods. This paper discusses these different diagnostic approaches and limitations.

In this study, 132 patients with lymphadenopathy were investigated. Fifty-two (39.4%) were diagnosed with tuberculosis (TB). The microscopic observation drug susceptibility (MODS) assay provided rapid (13 days), accurate diagnosis (sensitivity, 65.4%) and reliable drug susceptibility testing (DST). Despite its lower sensitivity than that of other methods, its faster results and simultaneous DST are advantageous in resource-poor settings, supporting the incorporation of MODS into diagnostic algorithms for extrapulmonary TB.

Tuberculosis viability microscopy predicted, within 1 hour, quantitative culture results that became available weeks later. Viability microscopy provides promising results for informing decisions concerning drug susceptibility testing, treatment changes, and infection control measures in resource-constrained settings where most tuberculosis occurs., Background. It is difficult to determine whether early tuberculosis treatment is effective in reducing the infectiousness of patients' sputum, because culture takes weeks and conventional acid-fast sputum microscopy and molecular tests cannot differentiate live from dead tuberculosis. Methods. To assess treatment response, sputum samples (n = 124) from unselected patients (n = 35) with sputum microscopy–positive tuberculosis were tested pretreatment and after 3, 6, and 9 days of empiric first-line therapy. Tuberculosis quantitative viability microscopy with fluorescein diacetate, quantitative culture, and acid-fast auramine microscopy were all performed in triplicate. Results. Tuberculosis quantitative viability microscopy predicted quantitative culture results such that 76% of results agreed within ±1 logarithm (rS = 0.85; P < .0001). In 31 patients with non-multidrug-resistant (MDR) tuberculosis, viability and quantitative culture results approximately halved (both 0.27 log reduction, P < .001) daily. For patients with non-MDR tuberculosis and available data, by treatment day 9 there was a >10-fold reduction in viability in 100% (24/24) of cases and quantitative culture in 95% (19/20) of cases. Four other patients subsequently found to have MDR tuberculosis had no significant changes in viability (P = .4) or quantitative culture (P = .6) results during early treatment. The change in viability and quantitative culture results during early treatment differed significantly between patients with non-MDR tuberculosis and those with MDR tuberculosis (both P < .001). Acid-fast microscopy results changed little during early treatment, and this change was similar for non-MDR tuberculosis vs MDR tuberculosis (P = .6). Conclusions. Tuberculosis quantitative viability microscopy is a simple test that within 1 hour predicted quantitative culture results that became available weeks later, rapidly indicating whether patients were responding to tuberculosis therapy.

Aquaporin-3 (AQP3) and aquaporin-4 (AQP4) are homologous proteins expressed in the basolateral plasma membrane of kidney collecting duct principal cells, where they mediate the exit pathway for apically reabsorbed water. Although both proteins are localized to the same plasma membrane domain, it is unknown if they are sorted together in the Golgi, or arrive in the same or different vesicles at the plasma membrane. We addressed these questions using high resolution deconvolution imaging, spinning disk and laser scanning confocal microscopy of cells expressing AQP3 and AQP4. AQP3 and AQP4 were observed mostly in separate post-Golgi carriers, and spinning disk microscopy showed that most of AQP3 and AQP4 were delivered to the plasma membrane in separate vesicles. In contrast, VSV-G and LDL-R, two well-charcterized basolateral proteins, co-localized to a high degree in the same post-Golgi carriers, indicating that the differential sorting of AQP3 and AQP4 is specific and regulated. Significantly, a chimeric AQP3 containing the AQP4 cytoplasmic tails co-localized with AQP4 in post-Golgi vesicles. These results indicate that AQP3 and AQP4 are separated into different post-Golgi carriers based on different cytoplasmic domain sorting signals, and are then delivered separately to the plasma membrane. Aquaporin-3 (AQP3) and aquaporin-4 (AQP4) are homologous proteins expressed in the basolateral plasma membrane of kidney collecting duct principal cells, where they mediate the exit pathway for apically reabsorbed water. Although both proteins are localized to the same plasma membrane domain, it is unknown if they are sorted together in the Golgi, or arrive in the same or different vesicles at the plasma membrane. We addressed these questions using high resolution deconvolution imaging, spinning disk and laser scanning confocal microscopy of cells expressing AQP3 and AQP4. AQP3 and AQP4 were observed mostly in separate post-Golgi carriers, and spinning disk microscopy showed that most of AQP3 and AQP4 were delivered to the plasma membrane in separate vesicles. In contrast, VSV-G and LDL-R, two well-charcterized basolateral proteins, co-localized to a high degree in the same post-Golgi carriers, indicating that the differential sorting of AQP3 and AQP4 is specific and regulated. Significantly, a chimeric AQP3 containing the AQP4 cytoplasmic tails co-localized with AQP4 in post-Golgi vesicles. These results indicate that AQP3 and AQP4 are separated into different post-Golgi carriers based on different cytoplasmic domain sorting signals, and are then delivered separately to the plasma membrane.

International audience; BACKGROUND: Second Harmonic Generation (SHG) microscopy recently appeared as an efficient optical imaging technique to probe unstained collagen-rich tissues like cornea. Moreover, corneal remodeling occurs in many diseases and precise characterization requires overcoming the limitations of conventional techniques. In this work, we focus on diabetes, which affects hundreds of million people worldwide and most often leads to diabetic retinopathy, with no early diagnostic tool. This study then aims to establish the potential of SHG microscopy for in situ detection and characterization of hyperglycemia-induced abnormalities in the Descemet's membrane, in the posterior cornea. METHODOLOGY/PRINCIPAL FINDINGS: We studied corneas from age-matched control and Goto-Kakizaki rats, a spontaneous model of type 2 diabetes, and corneas from human donors with type 2 diabetes and without any diabetes. SHG imaging was compared to confocal microscopy, to histology characterization using conventional staining and transmitted light microscopy and to transmission electron microscopy. SHG imaging revealed collagen deposits in the Descemet's membrane of unstained corneas in a unique way compared to these gold standard techniques in ophthalmology. It provided background-free images of the three-dimensional interwoven distribution of the collagen deposits, with improved contrast compared to confocal microscopy. It also provided structural capability in intact corneas because of its high specificity to fibrillar collagen, with substantially larger field of view than transmission electron microscopy. Moreover, in vivo SHG imaging was demonstrated in Goto-Kakizaki rats. CONCLUSIONS/SIGNIFICANCE: Our study shows unambiguously the high potential of SHG microscopy for three-dimensional characterization of structural abnormalities in unstained corneas. Furthermore, our demonstration of in vivo SHG imaging opens the way to long-term dynamical studies. This method should be easily generalized to other structural remodeling of the cornea and SHG microscopy should prove to be invaluable for in vivo corneal pathological studies.

PURPOSE: To study the mechanisms of pupillary block in eyes with occludable angle by ultrasound biomicroscopy. METHODS: Initially, a pilot study of 13 eyes with acute primary angle-closure without medication was executed. Ultrasound biomicroscopy measurements of the angle, posterior chamber depth and iris thickness were performed in the temporal quadrant under light and dark conditions. Afterwards, ultrasound biomicroscopy measurements of iris-lens contact distance and iris-lens angle in the temporal quadrant and central anterior chamber depht were made in 32 eyes with acute primary angle-closure or intermittent primary angle-closure without medication, under light and dark conditions before and after laser peripheral iridectomy. RESULTS: In the pilot study, a significant decrease in the angle as well as a significant increase in the iris thickness occurred when comparing light to dark conditions. Before and after laser peripheral iridectomy (second study), significant differences were found in iris-lens contact distance (P

Recent findings have attested the protective effects of erythropoietin (EPO) in ischemically challenged organs. We therefore aimed at elaborating the underlying mechanism of EPO-mediated protection in musculocutaneous tissue undergoing persistent ischemia after acute injury. Mice were assigned to five experimental groups equipped with a randomly perfused flap fixed in a dorsal skinfold chamber, whereas the sixth group did not undergo flap preparation: EPO, L-Name, EPO and L-Name, EPO and bevacizumab, untreated flap, and nonischemic chamber (control). Intravital fluorescence microscopic analysis of microhemodynamics, apoptotic cell death, macromolecular leakage and angiogenesis was carried out over a 10-day period. Further, immunohistochemical analysis was used to study the protein expression of endothelial nitric oxide synthase (eNOS) and vascular endothelial growth factor (VEGF). Increased expression of eNOS in EPO-administered mice correlated with significant arteriolar dilation and thus increased blood flow resulting in a maintained functional capillary density (FCD) at day 10. In addition, EPO induced a VEGF upregulation, which was associated with newly formed capillaries. In addition, EPO was able to reduce ischemia-induced apoptotic cell death and finally to significantly reduce flap necrosis. In contrast, coadministration of L-Name abolished EPO-mediated tissue protection by abrogating the dilatory effect resulting in reduced FCD and tissue survival, without counteracting angiogenesis and apoptotic cell death, whereas additional administration of bevacizumab did not influence the beneficial effect of EPO on flap survival despite abrogating angiogenesis. Macromolecular leakage was found to be increased in all treatment groups. This study shows that EPO administration prevents musculocutaneous tissue from ischemic necrosis as a consequence of an eNOS-dependent arteriolar hyperperfusion maintaining capillary perfusion, thus representing a promising approach to pharmacologically protect ischemically challenged tissue.

OBJETIVO: Avaliar o endotélio corneano na presença de precipitados ceráticos em pacientes portadores de uveítes infecciosas e não-infecciosas com emprego da microscopia especular de não-contato. MÉTODOS: Prospectivamente foram incluídos 25 pacientes com média de idade de 40,5 (±14,2 anos). Os pacientes foram divididos em dois grupos de acordo com a etiologia da uveíte. O microscópio especular Topcon SP-2000P foi usado para capturar imagens endoteliais. As variáveis densidade endotelial, área celular média e coeficiente de variação das células foram analisados estatisticamente. RESULTADOS: Dos 25 pacientes incluídos no estudo, 16 (44%) olhos apresentavam uveítes infecciosas, 19(53%) uveítes não-infecciosas e 1 (3%) olho foi excluído devido à impossibilidade de obtenção da reflexão especular. A densidade endotelial média foi 2.628 ± 204 cel/mm² no grupo infeccioso e 2.622 ± 357 cel/mm² no não-infeccioso. As áreas celulares média nos grupos infeccioso e não-infeccioso foram respectivamente 385 ± 31 µm² e 390 ± 60 µm². Os coeficientes de variação das áreas celulares adjacentes aos precipitados ceráticos foram 26,36 ± 3,44 no infeccioso e 27,69 ± 4,61 no não-infeccioso. As diferenças encontradas entre os grupos em todas as variáveis não foram estatisticamente significantes (p

Case presentation: : A 46-year-old man suffering from progressive deafness since childhood received a Clarion 90 K cochlear implant with the HiRes® preformed electrode in his left ear in October 2006. A persistent Staphylococcus aureus infection failed to be treated with corticoids, amoxicillin/ clavulanate, ciprofloxaxin, and rifampin. The processor was removed on July 2007. Interventions: : The removed cochlear implant processor was treated with different reagents, with the aim of detecting a S. aureus and S. aureus biofilm: (1) fluorescein-coupled Fc of anti-human serum, (2) polyclonal anti-polysaccharide intercellular adhesion antibodies coupled to Alexa Fluor 568 goat anti-rabbit immunoglobulin (Ig)G, (3) crystal violet, (4) methylene blue, (5) acridine orange, (6) Gram stain, and (7) live/dead fluorescent stain. Results: : S. aureus and the major constituent of the S. aureus biofilm, the polysaccharide intercellular adhesion, were detected on the surface of the implant. S. aureus was isolated after a simple contact between the implant and a solid growth medium. The ability of the isolated S. aureus strain to produce biofilm in vitro was confirmed. Interpretation: : S. aureus biofilm was documented on the implant. Initial bacterial colonization could be related to the pocket of the removable magnet. Colonies of S. aureus without biofilm were found attached to the electrode wire. Conclusion: : We report one case of a S. aureus biofilm infection documented on a cochlear implant, as assessed by immuno-microscopy. The biofilm was likely responsible for the persistent infection which manifested for many months after the implant surgery and could explain the unusual bacterial phenotypic resistance against administered antimicrobial agents

OBJECTIVE: To describe the cytological aspect of peritoneal washings in benign multicystic peritoneal mesothelioma (BMPM). METHODS: Three peritoneal washing specimens stained by standard cytological and histological procedures and analysed by light microscopy. RESULTS: The specimens showed an abundance of monomorphous mesothelial cells devoid of atypia or mitoses. The mesothelial cells were calretinin positive. They also showed numerous squamous metaplastic cells arranged in flat sheets or isolated cells. The background contained some inflammatory cells. CONCLUSION: The combination of cytology of the peritoneal washing, histology (cell block and surgical specimen) and clinical history allow differentiation of BMPM from other cystic lesions (cystic lymphangioma and malignant mesothelioma).

Background: Acquisition of lineage-specific cell cycle duration is a central feature of metazoan development. The mechanisms by which this is achieved during early embryogenesis are poorly understood. In the nematode Caenorhabditis elegans , differential cell cycle duration is apparent starting at the two-cell stage, when the larger anterior blastomere AB divides before the smaller posterior blastomere P 1 . How anterior-posterior (A-P) polarity cues control this asynchrony remains to be elucidated. Results : We establish that early C. elegans embryos possess a hitherto unrecognized DNA replication checkpoint that relies on the PI-3-like kinase atl-1 and the kinase chk-1 . We demonstrate that preferential activation of this checkpoint in the P 1 blastomere contributes to asynchrony of cell division in two-cell-stage wild-type embryos. Furthermore, we show that preferential checkpoint activation is largely abrogated in embryos that undergo equal first cleavage following inactivation of Gα signaling. Conclusion: Our findings establish that differential checkpoint activation contributes to acquisition of distinct cell cycle duration in two-cell-stage C. elegans embryos and suggest a novel mechanism coupling asymmetric division to acquisition of distinct cell cycle duration during development.

For didactic and documentation purposes the dermatoscopic ABCD rule and the dermatoscopic risk stratification have been proposed. The aim of this investigation was to compare the ability of the 2 methods to separate patients with cutaneous malignant melanoma from individuals with other pigmented skin lesions. Three dermatologists, experienced users of dermatoscopy, assessed macroscopic clinical and dermatoscopic slides from 258 patients referred to the skin cancer outpatient clinic by the ABCD rule and risk stratification methods. Diagnostic performance of the 2 methods was compared by receiver operating characteristics curve analysis. When all pigmented skin lesions were compared, there was a trend for the observers to perform better using risk stratification. When only lesions with a well-defined pigment network were included, the diagnostic performance of the risk stratification method was superior to the dermatoscopic ABCD rule (areas under the receiver operating characteristics curve median 0.93 vs. 0.80, p

Background Laboratory capacity to confirm malaria cases in Tanzania is low and presumptive treatment of malaria is being practiced widely. In malaria endemic areas WHO now recommends systematic laboratory testing when suspecting malaria. Currently, the use of Rapid Diagnostic Tests (RDTs) is recommended for the diagnosis of malaria in lower level peripheral facilities, but not in health centres and hospitals. In this study, the following parameters were evaluated: (1) the quality of routine microscopy, and (2) the effects of RDT implementation on the positivity rate of malaria test results at three levels of the health system in Dar es Salaam, Tanzania. Methods During a baseline cross-sectional survey, routine blood slides were randomly picked from 12 urban public health facilities in Dar es Salaam, Tanzania. Sensitivity and specificity of routine slides were assessed against expert microscopy. In March 2007, following training of health workers, RDTs were introduced in nine public health facilities (three hospitals, three health centres and three dispensaries) in a near-to-programmatic way, while three control health facilities continued using microscopy. The monthly malaria positivity rates (PR) recorded in health statistics registers were collected before (routine microscopy) and after (routine RDTs) the intervention in all facilities. Results At baseline, 53% of blood slides were reported as positive by the routine laboratories, whereas only 2% were positive by expert microscopy. Sensitivity of routine microscopy was 71.4% and specificity was 47.3%. Positive and negative predictive values were 2.8% and 98.7%, respectively. Median parasitaemia was only three parasites per 200 white blood cells (WBC) by routine microscopy compared to 1226 parasites per 200 WBC by expert microscopy. Before RDT implementation, the mean test positivity rates using routine microscopy were 43% in hospitals, 62% in health centres and 58% in dispensaries. After RDT implementation, mean positivity rates using routine RDTs were 6%, 7% and 8%, respectively. The sensitivity and specificity of RDTs using expert microscopy as reference were 97.0% and 96.8%. The positivity rate of routine microscopy remained the same in the three control facilities: 71% before versus 72% after. Two cross-sectional health facility surveys confirmed that the parasite rate in febrile patients was low in Dar es Salaam during both the rainy season (13.6%) and the dry season (3.3%). Conclusions The quality of routine microscopy was poor in all health facilities, regardless of their level. Over-diagnosis was massive, with many false positive results reported as very low parasitaemia (1 to 5 parasites per 200 WBC). RDTs should replace microscopy as first-line diagnostic tool for malaria in all settings, especially in hospitals where the potential for saving lives is greatest.

Purpose: To compare central corneal thickness (CCT) measurements of healthy individuals obtained with ultrasonic pachymetry (UP) and non-contact specular microscopy (NCSM). Method: In total, 148 eyes of 74 subjects with no ocular or systemic diseases were included in the study. Central corneal thickness measurements of all patients performed with UP and NCCM were compared. Results: A total of 74 subjects (38 females) were included in this study. The mean age was 45.2 ± 18.4 (range 12-85) years. The mean central corneal thickness of all 148 eyes was 546.9 ± 40 μm with UP and 510.8 ± 42 μm with NCSM. The mean central corneal thickness measured with NCSM was 35 μm thinner than that measured with UP (p