Semi-automated Image Analysis Research Articles

Telomere Length Measurement by Molecular Combing.

Telomeres are repetitive regions of DNA bound by specialized proteins at the termini of linear chromosomes that prevent the natural chromosome ends from being recognized as DNA double strand breaks. Telomeric DNA is gradually eroded with each round of cell division, resulting in the accumulation of critically short or dysfunctional telomeres that eventually trigger cellular senescence. Consequently, telomere length is indicative of the proliferative capacity of a cell. Multiple methods exist to measure telomere length and telomere content, but a simple and reliable technique to accurately measure individual telomere lengths is currently lacking. We have developed the Telomere length Combing Assay (TCA) to measure telomere length on stretched DNA fibers. We used TCA to measure telomere erosion in primary human fibroblasts, and to detect telomere lengthening in response to activation of telomere maintenance pathways. TCA was also used to accurately measure telomere length in healthy individuals, and to identify critically short telomeres in patients with telomere biology disorders. TCA is performed on isolated DNA, negating the need for cycling cells. TCA is amenable to semi-automated image analysis, and can be fully automated using the Genomic Vision molecular combing platform. This not only precludes sampling bias, but also provides the potential for high-throughput applications and clinical development. TCA is a simple and versatile technique to measure the distribution of individual telomere lengths in a cell population, offering improved accuracy, and more detailed biological insight for telomere length measurement applications.

18 F-FDG PET/CT brain glucose metabolism as a marker of different types of depression comorbidity in chronic heart failure patients with impaired systolic function.

Depression is an important issue in heart failure (HF). The study investigated whole-brain and regional brain glucose metabolism in HF patients and its association with depression comorbidity. Twenty-nine hospitalized patients with symptomatic systolic HF (left ventricular ejection fraction <40%), New York Heart Association (NYHA) class II-IV and mean age of 55.5 ± 12.0 years, had psychometric questionnaires before discharge and an 18 F-FDG PET/CT brain scan after discharge. Semi-automated image analysis was performed on all cases and 30 matched controls. The metabolic parameter mean standardized uptake value (SUVmean ) was calculated for the whole brain and three brain regions implicated in depression pathogenesis. A standardized SUVmean was also estimated by dividing regional brain SUVmean with whole-brain SUVmean . Cases had lower average whole-brain SUVmean (3.90 ± 1.49 vs. 5.10 ± 1.35, P=0.001) and average regional brain SUVmean (4.57 ± 2.31 vs. 9.96 ± 3.58, P < 0.001) compared to controls. Whole-brain SUVmean had a significant correlation with patient age, NYHA class, diabetes, creatinine levels, depression, and cognitive impairment. Regional brain SUVmean was correlated with whole-brain SUVmean and depression. The standardized SUVmean , in particular, was found to be a robust index that could differentiate HF patients with 'epiphenomenal' (>0.93) or 'real' (≤0.93) depression. Heart failure patients with more severe disease showed whole-brain and regional brain hypometabolism in 18 F-FDG PET/CT. Depressed HF patients (Beck Depression Inventory score >13) exhibited different metabolic patterns that could be used to differentiate between 'epiphenomenal' and 'real' depression. Namely, presence of whole-brain hypometabolism suggested 'epiphenomenal' depression, whereas absence suggested 'real' depression. Presence of significant relative regional brain hypometabolism enhanced the likelihood of 'real' depression diagnosis.

Semi-Automated Analysis of Digital Photographs for Monitoring East Antarctic Vegetation.

Climate change is affecting Antarctica and minimally destructive long-term monitoring of its unique ecosystems is vital to detect biodiversity trends, and to understand how change is affecting these communities. The use of automated or semi-automated methods is especially valuable in harsh polar environments, as access is limited and conditions extreme. We assessed moss health and cover at six time points between 2003 and 2014 at two East Antarctic sites. Semi-automatic object-based image analysis (OBIA) was used to classify digital photographs using a set of rules based on digital red, green, blue (RGB) and hue-saturation-intensity (HSI) value thresholds, assigning vegetation to categories of healthy, stressed or moribund moss and lichens. Comparison with traditional visual estimates showed that estimates of percent cover using semi-automated OBIA classification fell within the range of variation determined by visual methods. Overall moss health, as assessed using the mean percentages of healthy, stressed and moribund mosses within quadrats, changed over the 11 years at both sites. A marked increase in stress and decline in health was observed across both sites in 2008, followed by recovery to baseline levels of health by 2014 at one site, but with significantly more stressed or moribund moss remaining within the two communities at the other site. Our results confirm that vegetation cover can be reliably estimated using semi-automated OBIA, providing similar accuracy to visual estimation by experts. The resulting vegetation cover estimates provide a sensitive measure to assess change in vegetation health over time and have informed a conceptual framework for the changing condition of Antarctic mosses. In demonstrating that this method can be used to monitor ground cover vegetation at small scales, we suggest it may also be suitable for other extreme environments where repeat monitoring via images is required.

Large-Scale 3D Optical Mapping and Quantitative Analysis of Nanoparticle Distribution in Tumor Vascular Microenvironment.

Nanoparticles (NPs) are a promising carrier for cancer therapeutics. Systemically administered NPs are transported to tumor tissues via the bloodstream, extravasated from microvessels, and delivered to cancer cells. The distribution of NPs in the tumor vascular microenvironment critically determines the therapeutic efficacy of NP-delivered drugs, but its precise assessment in 3D across a large volume remains challenging. Here, an analytical platform-termed OMNIA (for Optical Mapping of Nanoparticles and Image Analysis)-integrating tissue clearing, high-resolution optical imaging, and semiautomated image analysis is presented, which enables accurate, unbiased, and quantitative analysis of the distribution of NPs in relation to the vasculature across a large 3D volume. Application of OMNIA to tumor tissues revealed higher accumulation and more efficient extravasation of NPs in the tumor periphery than the core. Time-course analysis demonstrated that the accumulation of NPs in tumor peaked at 24 h after injection, but the relative distribution of NPs from the vasculature remained remarkably stable over time. Comparisons between 45- and 200-nm-sized NPs showed a lower accumulation of smaller NPs in tumors relative to the liver, yet better vessel permeation. Together, our results demonstrate that OMNIA facilitates precise and reliable evaluation of NP biodistribution, and mechanistic investigations on NP delivery to tumor tissues.

A Low-Cost Method for Phenotyping Wilting and Recovery of Wheat Leaves under Heat Stress Using Semi-Automated Image Analysis.

Leaf wilting is the most common symptom of dehydration stress. Methods to analyze this phenomenon are particularly relevant to evaluate crop agronomic performance, to genetically dissect out the wilting process, and for functional analysis of genetically modified plants. In this study, a low-cost, semi-automated method to quantify leaf folding of wilting plants is described that can replace visual analysis. Standardized heat-stress conditions were applied with a thermostatic drier, on plantlets or excised leaves from three wheat genotypes (Trinakria, Cappelli, and a Water-mutant of Trinakria). The best time–temperature binomial to record both the leaf wilting and recovery phases was identified using a free time-lapse application, by a smartphone camera. The quantitative description of the wilting phenomenon was obtained through the Kinovea software, which automatically tracked the leaf angle changes over time, computed various kinematic data (angular velocity, centripetal acceleration, total degrees of displacement) and constructed the graphs. The possibility of applying standardized heat-stress conditions and quantitatively describe the leaf folding kinematics means that this instrumentation and its use represents a very low cost tool for objective phenotyping of the degree of the heat-stress tolerance of wheat and of morphologically similar species.

The role of imaging in early detection of patients at risk for neuropathy during primary chemotherapy for ovarian carcinoma.

e18071 Background: Neuropathy is a common adverse event requiring dose reduction or regimen change in patients receiving platinum-taxane chemotherapy for ovarian cancer. Our aim was to determine whether specific biomarkers, such as skeletal muscle, visceral and subcutaneous fat cross-sectional areas, measured using computerized tomography (CT), were associated with the development of neuropathy. Analytic Morphomics, a semi-automated image analysis method, has been used to predict oncologic outcomes with immunotherapy and to identify patients at higher risk for postoperative complications from major cancer surgeries. We hypothesized that patients who required dose reductions or regimen changes would have CT imaging findings that distinguished them from their counterparts who did not require dose reduction or regimen change. Methods: All patients at our institution diagnosed with high-grade ovarian carcinoma between January 1, 2014 and December 31, 2018 were identified retrospectively. Analytic Morphomics was used to extract measures of body composition from CT images at the level of the third lumbar vertebra. Demographic and clinical characteristics were abstracted from the electronic medical record. We analyzed several binary outcomes (neuropathy, delay in chemotherapy, or dose reduction by cycle 1, 3 or 6) and used unpaired, two-tailed t-tests to compare means between each outcome group at each timepoint. Results: Of 156 patients screened for the study, 138 patients were included and had pre-treatment CT scans available and completed six cycles of platinum-taxane chemotherapy. Patients who developed neuropathy in any of the six cycles, or had a change in their chemotherapy regimen by cycle six, had higher mean visceral fat area than those who did not develop neuropathy. This finding was statistically significant throughout three cycles (p = 0.023) and six cycles (p = 0.030). Conclusions: Increased visceral fat on CT imaging was significantly associated with a higher incidence of neuropathy and change in chemotherapy regimen in patients undergoing primary platinum-taxane chemotherapy for ovarian carcinoma. Weight and BMI did not have a significant association with development of neuropathy, dose reduction, or treatment delay. Further study is needed to determine if dose reductions or regimen changes due to neuropathy can be better predicted using morphomics.

Primary Human Osteoblasts Cultured in a 3D Microenvironment Create a Unique Representative Model of Their Differentiation Into Osteocytes.

Microengineered systems provide an in vitro strategy to explore the variability of individual patient response to tissue engineering products, since they prefer the use of primary cell sources representing the phenotype variability. Traditional in vitro systems already showed that primary human osteoblasts embedded in a 3D fibrous collagen matrix differentiate into osteocytes under specific conditions. Here, we hypothesized that translating this environment to the organ-on-a-chip scale creates a minimal functional unit to recapitulate osteoblast maturation toward osteocytes and matrix mineralization. Primary human osteoblasts were seeded in a type I collagen hydrogel, to establish the role of lower (2.5 × 105 cells/ml) and higher (1 × 106 cells/ml) cell density on their differentiation into osteocytes. A custom semi-automatic image analysis software was used to extract quantitative data on cellular morphology from brightfield images. The results are showing that cells cultured at a high density increase dendrite length over time, stop proliferating, exhibit dendritic morphology, upregulate alkaline phosphatase (ALP) activity, and express the osteocyte marker dental matrix protein 1 (DMP1). On the contrary, cells cultured at lower density proliferate over time, do not upregulate ALP and express the osteoblast marker bone sialoprotein 2 (BSP2) at all timepoints. Our work reveals that microengineered systems create unique conditions to capture the major aspects of osteoblast differentiation into osteocytes with a limited number of cells. We propose that the microengineered approach is a functional strategy to create a patient-specific bone tissue model and investigate the individual osteogenic potential of the patient bone cells.

HyLength: a semi-automated digital image analysis tool for measuring the length of roots and fungal hyphae of dense mycelia.

In plant-fungus phenotyping, determining fungal hyphal and plant root lengths by digital image analysis can reduce labour and increase data reproducibility. However, the degree of software sophistication is often prohibitive and manual measuring is still used, despite being very time-consuming. We developed the HyLength tool for measuring the lengths of hyphae and roots in in vivo and in vitro systems. The HyLength was successfully validated against manual measures of roots and fungal hyphae obtained from all systems. Compared with manual methods, the HyLength underestimated Medicago sativa roots in the in vivo system and Rhizophagus irregularis hyphae in the in vitro system by about 12cm per m and allowed to save about 1h for a single experimental unit. As regards hyphae of R. irregularis in the in vivo system, the HyLength overestimated the length by about 21cm per m compared with manual measures, but time saving was up to 20.5h per single experimental unit. Finally, with hyphae of Aspergillus oryzae, the underestimation was about 8cm per m with a time saving of about 10min for a single germinating spore. By benchmarking the HyLength against the AnaMorf plugin of the ImageJ/Fiji, we found that the HyLength performed better for dense fungal hyphae, also strongly reducing the measuring time. The HyLength can allow measuring the length over a whole experimental unit, eliminating the error due to sub-area selection by the user and allowing processing a high number of samples. Therefore, we propose the HyLength as a useful freeware tool for measuring fungal hyphae of dense mycelia.

Influence of observer-dependency on left ventricular hypertrabeculation mass measurement and its relationship with left ventricular volume and ejection fraction - comparison between manual and semiautomatic CMR image analysis methods.

Recent studies concerning left ventricular noncompaction (LVNC) suggest that the extent of left ventricular (LV) hypertrabeculation has no impact on prognosis. The variety of methods of LV noncompacted myocardial mass (NCM) assessment may influence the results. Hence, we compared two methods of NCM estimation: largely observer-independent Hautvast's(H) computed algorithm-based approach and commonly used Jacquier's(J) method, and their associations with LV end-diastolic volume (EDV) and ejection fraction (EF). Cardiac magnetic resonance images of 77 persons (45±17yo) - 42 LVNC, 15 non-ischemic dilative cardiomyopathy, 20 control group were analyzed. LVNC patients were divided into the subgroup with normal (LVNCN) and high EDV (LVNCDCM). NCM and total left ventricular mass (LVM) were estimated by Hautvast's [excluding intertrabecular blood (ITB) and including papillary muscles (PMs) into NCM] and Jacquier's approach (including ITB and PMs, if unclearly distinguished, into NCM). The cut-off value of NCM for LVNC diagnosis was 22% (AUC 0.933) for NCMH/LVMH and 26% (AUC 0.883) for NCMJ/LVMJ. Inter- and intra-observer variability (estimated by coefficient of variation [CoV] and intraclass correlation coefficient [ICC]) of NCMH/LVMH appeared better than of NCMJ/LVMJ (CoV 4.3%, ICC 0.981 and CoV 4.9%, ICC 0.978; respectively for NCMH/LVMH, while for NCMJ/LVMJ: CoV 19.7%, ICC 0.15 and CoV 12.9%, ICC 0.504). In LVNCN subgroup, the correlation between EDV and NCMH was stronger than NCMJ (r = 0.677, p<0.001 vs. r = 0.480, p = 0.038; respectively). In LVNC the EDV correlated with NCMH/LVMH (r = 0.391, p<0.01), but not with NCMJ/LVMJ. In the overall group a relationship was present between EF and NCMH/LVMH (r = -0.449, p<0.001), but not NCMJ/LVMJ. Only NCMH/LVMH explained the variability of EDV (b 0.434, p<0.001). Choosing a method of NCM assessment that is less observer-dependent might increase the reliability of results. The impact of method selection on the LV parameters and cut-off values for hypertrabeculation should be further investigated.

Routine Optical Clearing of 3D-Cell Cultures: Simplicity Forward.

Three-dimensional cell cultures, such as spheroids and organoids, serve as increasingly important models in fundamental and applied research and start to be used for drug screening purposes. Optical tissue clearing procedures are employed to enhance visualization of fluorescence-stained organs, tissues, and three-dimensional cell cultures. To get a more systematic overview about the effects and applicability of optical tissue clearing on three-dimensional cell cultures, we compared six different clearing/embedding protocols on seven types of spheroid- and chip-based three-dimensional cell cultures of approximately 300 μm in size that were stained with nuclear dyes, immunofluorescence, cell trackers, and cyan fluorescent protein. Subsequent whole mount confocal microscopy and semi-automated image analysis were performed to quantify the effects. Quantitative analysis included fluorescence signal intensity and signal-to-noise ratio as a function of z-depth as well as segmentation and counting of nuclei and immunopositive cells. In general, these analyses revealed five key points, which largely confirmed current knowledge and were quantified in this study. First, there was a massive variability of effects of different clearing protocols on sample transparency and shrinkage as well as on dye quenching. Second, all tested clearing protocols worked more efficiently on samples prepared with one cell type than on co-cultures. Third, z-compensation was imperative to minimize variations in signal-to-noise ratio. Fourth, a combination of sample-inherent cell density, sample shrinkage, uniformity of signal-to-noise ratio, and image resolution had a strong impact on data segmentation, cell counts, and relative numbers of immunofluorescence-positive cells. Finally, considering all mentioned aspects and including a wish for simplicity and speed of protocols – in particular, for screening purposes – clearing with 88% Glycerol appeared to be the most promising option amongst the ones tested.

Choroidal Vascularity Index: An In-Depth Analysis of This Novel Optical Coherence Tomography Parameter.

Remarkable improvements in optical coherence tomography (OCT) technology have resulted in highly sophisticated, noninvasive machines allowing detailed and advanced morphological evaluation of all retinal and choroidal layers. Postproduction semiautomated imaging analysis with dedicated public-domain software allows precise quantitative analysis of binarized OCT images. In this regard, the choroidal vascularity index (CVI) is emerging as a new imaging tool for the measurement and analysis of the choroidal vascular system by quantifying both luminal and stromal choroidal components. Numerous reports have been published so far regarding CVI and its potential applications in healthy eyes as well as in the evaluation and management of several chorioretinal diseases. Current literature suggests that CVI has a lesser variability and is influenced by fewer physiologic factors as compared to choroidal thickness. It can be considered a relatively stable parameter for evaluating the changes in the choroidal vasculature. In this review, the principles and the applications of this advanced imaging modality for studying and understanding the contributing role of choroid in retinal and optic nerve diseases are discussed. Potential advances that may allow the widespread adoption of this tool in the routine clinical practice are also presented.

Tumor proportion in colon cancer: results from a semiautomatic image analysis approach

The tumor stroma ratio (TSR) is a promising prognostic biomarker in colon cancer, which could provide additional risk stratification for therapy adaption. The objective of our study was the investigation of the prognostic significance of TSR at different tumor sites in a simple semiautomatic approach with the open-source program ImageJ. We investigated 206 pT3 and pT4 adenocarcinomas of no special type. According to our established thresholds, 31 tumors (15%) were classified as low tumor proportion (TP) (≤ 15% TP), 42 tumors (20%) were classified as high TP (≥ 54% TP), and 133 tumors (65%) were classified as medium TP. High and low TP were associated with an adverse overall survival in comparison to medium TP (p = 0.001 and p = 0.03). Furthermore, the TP was an independent risk factor of occurrence of distant metastasis next to T status, microsatellite status, and tumor budding. The 5-year survival rate was 49% in patients with high TP, 48% in patients with low TP, and 68% in patients with medium TP (p = 0.042, n = 160). Patients with a high TP had less often tumor budding (p = 0.012), lymphovascular invasion (p = 0.049), and less harvested lymph nodes (p = 0.042) in comparison to low TP tumors. The results provide first evidence that a high tumor proportion/low stroma proportion is also associated with an adverse prognosis and that this subgroup might be difficult to identify with other classical histopathologic characteristics that are linked to an adverse prognosis.

Airway tapering: an objective image biomarker for bronchiectasis

PurposeTo estimate airway tapering in control subjects and to assess the usability of tapering as a bronchiectasis biomarker in paediatric populations.MethodsAirway tapering values were semi-automatically quantified in 156 children with control CTs collected in the Normal Chest CT Study Group. Airway tapering as a biomarker for bronchiectasis was assessed on spirometer-guided inspiratory CTs from 12 patients with bronchiectasis and 12 age- and sex-matched controls. Semi-automatic image analysis software was used to quantify intra-branch tapering (reduction in airway diameter along the branch), inter-branch tapering (reduction in airway diameter before and after bifurcation) and airway-artery ratios on chest CTs. Biomarkers were further stratified in small, medium and large airways based on three equal groups of the accompanying vessel size.ResultsControl subjects showed intra-branch tapering of 1% and inter-branch tapering of 24–39%. Subjects with bronchiectasis showed significantly reduced intra-branch of 0.8% and inter-branch tapering of 19–32% and increased airway–artery ratios compared with controls (p < 0.01). Tapering measurements were significantly different between diseased and controls across all airway sizes. Difference in airway–artery ratio was only significant in small airways.ConclusionPaediatric normal values for airway tapering were established in control subjects. Tapering showed to be a promising biomarker for bronchiectasis as subjects with bronchiectasis show significantly less airway tapering across all airway sizes compared with controls. Detecting less tapering in larger airways could potentially lead to earlier diagnosis of bronchiectasis. Additionally, compared with the conventional airway–artery ratio, this novel biomarker has the advantage that it does not require pairing with pulmonary arteries.Key Points• Tapering is a promising objective image biomarker for bronchiectasis that can be extracted semi-automatically and has good correlation with validated visual scoring methods.• Less airway tapering was observed in patients with bronchiectasis and can be observed sensitively throughout the bronchial tree, even in the more central airways.• Tapering values seemed to be less influenced by variety in scanning protocols and lung volume making it a more robust biomarker for bronchiectasis detection.

BioVision Tracker: A semi-automated image analysis software for spatiotemporal gene expression tracking in Arabidopsis thaliana.

Fluorescence microscopy can produce large quantities of data that reveal the spatiotemporal behavior of gene expression at the cellular level in plants. Automated or semi-automated image analysis methods are required to extract data from these images. These data are helpful in revealing spatial and/or temporal-dependent processes that influence development in the meristematic region of plant roots. Tracking spatiotemporal gene expression in the meristem requires the processing of multiple microscopy imaging channels (one channel used to image root geometry which serves as a reference for relating locations within the root, and one or more channels used to image fluorescent gene expression signals). Many automated image analysis methods rely on the staining of cell walls with fluorescent dyes to capture cellular geometry and overall root geometry. However, in long time-course imaging experiments, dyes may fade which hinders spatial assessment in image analysis. Here, we describe a procedure for analyzing 3D microscopy images to track spatiotemporal gene expression signals using the MATLAB-based BioVision Tracker software. This software requires either a fluorescence image or a brightfield image to analyze root geometry and a fluorescence image to capture and track temporal changes in gene expression.

Cell Tracking Profiler - a user-driven analysis framework for evaluating 4D live-cell imaging data.

Accurate measurements of cell morphology and behaviour are fundamentally important for understanding how disease, molecules and drugs affect cell function in vivo Here, by using muscle stem cell (muSC) responses to injury in zebrafish as our biological paradigm, we established a 'ground truth' for muSC behaviour. This revealed that segmentation and tracking algorithms from commonly used programs are error-prone, leading us to develop a fast semi-automated image analysis pipeline that allows user-defined parameters for segmentation and correction of cell tracking. Cell Tracking Profiler (CTP) is a package that runs two existing programs, HK Means and Phagosight within the Icy image analysis suite, to enable user-managed cell tracking from 3D time-lapse datasets to provide measures of cell shape and movement. We demonstrate how CTP can be used to reveal changes to cell behaviour of muSCs in response to manipulation of the cell cytoskeleton by small-molecule inhibitors. CTP and the associated tools we have developed for analysis of outputs thus provide a powerful framework for analysing complex cell behaviour in vivo from 4D datasets that are not amenable to straightforward analysis.

Real-time imaging of multivesicular body-plasma membrane fusion to quantify exosome release from single cells.

Exosomes are small extracellular vesicles with a diameter of 40-150 nm, and are implicated in cellular homeostasis and cell-cell communication. They can be secreted in bulk in response to cell-extrinsic and cell-intrinsic signals that cause multivesicular body (MVB) fusion with the plasma membrane (PM). However, research on the regulation of exosome release is hampered by the failure of current methods to capture the dynamics of exosome release. Here we describe how live imaging with tetraspanin-based pH-sensitive fluorescent reporters can quantify the MVB-PM fusion rate of single cells. Our approach enables identification of exogenous stimuli, signaling pathways, and fusion complexes, and can map subcellular sites of fusion events. In addition, dual-color imaging can be used to assess simultaneous release of different cargo by MVB exocytosis. This protocol describes the complete imaging experiment, consisting of transient expression of tetraspanin reporters (2 d), live-cell (dual-color) total internal reflection fluorescence microscopy (30-60 min per condition), and semiautomatic image analysis by using a newly developed ImageJ macro (±30 min per condition).

Determination of Size Distribution of Precipitation Aggregates Using Non-Invasive Microscopy and Semiautomated Image Processing and Analysis

Particle size distribution (PSD) determination is a typical practice for the characterization of the slurries generated in a precipitation plant. Furthermore, the precipitates generated in these processes form colloidal or aggregated suspensions. Nevertheless, the conventional methods used to estimate PSD (e.g., laser diffraction and/or a cyclosizer) have not been designed to measure particles that tend to aggregate or disaggregate, since they include external forces (e.g., centrifugal, agitation, pumping and sonication). These forces affect the true size of the aggregates formed in a unit operation, thereby losing representativeness in terms of aggregates particle size. This study presents an alternative method of measuring the size distribution of particles with aggregation behavior, particularly, by using non-invasive microscopy and image processing and analysis. The samples used were obtained from an experimental precipitation process by applying sulfidization to treat the cyanide-copper complexes contained in a cyanidation solution. This method has been validated with statistical tools and compared with a conventional analysis based on laser diffraction (Mastersizer). The PSD results obtained with optical microscopy show a bi-modal behavior of the precipitates. However this behavior could be not determined when using the laser diffraction technique. The PSD obtained for the sample tested by microscopy had a mean of 119.7 μm, a median of 147 μm and a 90% distribution reached a particle size of 312.5 μm. These values differ with those obtained by the laser diffraction technique. Our results show significant differences between the methods analyzed, demonstrating that the image processing and analysis obtained by optical microscopy is an excellent and non-invasive alternative to obtain size distributions of aggregates in precipitation processes.

Alterations of retinal vessels in patients with sepsis

To compare the retinal vasculature of septic patients with age-matched healthy volunteers. This is a single-centre prospective observational study from January 2018 to May 2019 in a third-level ICU. We performed a single fundus imaging using a hand-held digital fundus camera in patients with sepsis or septic shock (n = 40) during the first 24h after ICU admission and compared these data with age-matched healthy controls (n = 20). Semi-automated image analysis was performed. The average retinal arteriolar and venular caliber were calculated and summarized as the central retinal arteriolar equivalent (CRAE) and central retinal venular equivalent (CRVE). Arteriole:venular ratio (AVR) was defined as the ratio of CRAE:CRVE. The vascular length density of segmented retinal vessels was = defined as the skeletonized vessel area/total area × 100%. Median CRAE of septic patients was significantly higher in comparison to healthy controls (165[149-187] vs. 146[142-158]µm, p = 0.002). However, median CRVE and AVR of septic patients did not differ with healthy controls (247[223-282] vs. 244[215-272], p = 0.396 and 0.64[0.58-0.74] vs. 0.61[0.55-0.68], p = 0.145) respectively. Patients with sepsis showed a significant decrease in retinal vascular length density compared with healthy subjects (p < 0.001). Retinal observation using a hand-held fundus imaging device showed signs of arteriolar vasodilation with decreased vascular density in septic patients in comparison to healthy controls.

Novel Genetic Locus Influencing Retinal Venular Tortuosity Is Also Associated With Risk of Coronary Artery Disease.

The retina may provide readily accessible imaging biomarkers of global cardiovascular health. Increasing evidence suggests variation in retinal vascular traits is highly heritable. This study aimed to identify the genetic determinants of retinal vascular traits. Approach and Results: We conducted a meta-analysis of genome-wide association studies for quantitative retinal vascular traits derived using semi-automatic image analysis of digital retinal photographs from the GoDARTS (Genetics of Diabetes Audit and Research in Tayside; N=1736) and ORCADES (Orkney Complex Disease Study; N=1358) cohorts. We identified a novel genome-wide significant locus at 19q13 (ACTN4/CAPN12) for retinal venular tortuosity (TortV), and one at 13q34 (COL4A2) for retinal arteriolar tortuosity (TortA); these 2 loci were subsequently confirmed in 3 independent cohorts (Ntotal=1413). In the combined analysis of discovery and replication cohorts, the lead single-nucleotide polymorphism in ACTN4/CAPN12 was rs1808382 (βs.d.=-0.109; SE=0.015; P=2.39×10-13) and in COL4A2 was rs7991229 (βs.d.=0.103; SE=0.015; P=4.66×10-12). Notably, the ACTN4/CAPN12 locus associated with TortV is also associated with coronary artery disease, heart rate, and atrial fibrillation. Genetic determinants of retinal vascular tortuosity are also linked to cardiovascular health. These findings provide a molecular pathophysiological foundation for the use of retinal vascular traits as biomarkers for cardiovascular diseases.

Histological differences in cancer cells, stroma, and luminal spaces strongly correlate with in vivo MRI-detectability of prostate cancer

The current study aimed to investigate the plausible histopathological factors that affect the detectability of prostate cancers on multiparametric magnetic resonance imaging (MP-MRI). This retrospective study included 59 consecutive patients who had undergone MP-MRI and subsequent radical prostatectomy. The cases were standardized according to the tumor size ranging from 10 to 20 mm on the final pathological diagnosis. Histopathological review and semi-automated imaging analysis were performed to evaluate the relative area fractions of the histological components, including cancer cells, stroma, and luminal spaces. Among the 59 prostatectomy specimens, no case showed two or more foci of cancer that matched the size criteria. Of the 59 lesions, 35 were MRI-detectable [Prostate Imaging Reporting and Data System (PIRADS) score of 3 or greater] and 24 were MRI-undetectable (PIRADS score of 2 or less). No significant differences were observed in Gleason Grade Group, percentage of Gleason pattern 4, and predominant subtype of Gleason pattern 4 between MRI-detectable and MRI-undetectable cancers. On the other hand, significantly higher mean area fraction of cancer cells (60.9% vs. 42.7%, P < 0.0001) and lower mean area fractions of stroma (33.8% vs. 45.1%, P = 0.00089) and luminal spaces (5.2% vs. 12.2%, P < 0.0001) were observed in MRI-detectable cancers than in MRI-undetectable cancers. In a multivariable analysis performed upon exclusion of area fraction of stroma due to its multicollinearity with that of cancer cells, area fractions of cancer cells (P = 0.0031) and luminal space (P = 0.0035) demonstrated strong positive and negative correlation with MRI-detectability, respectively. Changes in cancer cells, stroma, and luminal spaces, rather than conventional histological parameters, could be considered one of the best predictors to clinical, in vivo MRI-detectability of prostate cancer.