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Multiple emulsions are thermodynamically stable systems that mark applications in various fields including drug delivery systems. They allow enhanced availability of drugs, greater absorption, and present reduced toxicity, among other desirable properties. In this work, we aimed to formulate a unique double emulsion (O1/W + W1/O2/W/W) with three bioactive components viz. Ocimum tenuiflorum oil, Cocos nucifera oil and crystalline Cinnamomum camphora. Three surfactants with different HLB values viz. Tween-20, Tween-80 and Triton X-100 were used for the emulsification process. The method followed was simple as compared to current methods employed for formulating multiple emulsions. Formulation was characterized using techniques of bright field microscopy, Dynamic Light Scattering (DLS), High-Resolution Transmission Electron Microscopy (HR-TEM) and Fourier-transform infrared spectroscopy (FTIR). Image processing tools were also used to characterize the formulation, which reliably cross-verified the observations from conventional characterization techniques. The potency of individual components of emulsion was compared with the prepared double emulsion model by testing the activity on two pathologically relevant bacterial strains: Fusobacterium nucleatum (FN) and Porphyromonas gingivalis (PG).

Pollen analysis is a method used for verification of the botanical and geographical honey origin. Currently, much effort is being made to introduce automated systems with the use of image analysis programs. The automatic analysis is impeded by the insufficient depth of field of objects when using a light microscope, however, this can be avoided by using image reconstruction from images obtained from different focal planes. In this method, testing was performed on the normal focus (NF) and extended-depth-of-focus (EDF) images. These two methods were compared and statistically evaluated. The number of pollen grains and selected morphometric characteristics were compared. For EDF images, a higher number of pollen grains was obtained for the analysis, and except for the length/width ratio, a statistically significant difference was observed in the characteristics of pollen grains between the compared NF and EDF methods.

LED fluorescence microscopy (MF LED) has been recommended by the World Health Organization for tuberculosis (TB) diagnosis. The aim was to validate this technique in the National Reference Laboratory for Tuberculosis, Leprosy and Mycobacteria (NRLTBLM). A validation study of the MF LED was carried out at the NRLTBLM of the "Pedro Kourí" Institute, from February-July 2018. A total of 98 slides were included: 49 smears that had a positive culture for mycobacteria and 49 slides with a negative culture. The results of the LED FM were compared with bright field microscopy (BFM) and conventional fluorescence microscopy (CFM). With the LED FM and CFM, 14 positive smears were identified that had been negative by BFM, the positivity rate being increased to 13,3%. By both fluorescent techniques, more smears (18/98) were detected with scanty acid fast bacilli than the BFM (8/98). The agreement between MF LED and BFM was considered good (k = 0,6773) and between MF LED and CFM very good (k = 0,8711). The sensitivity of the MF LED was higher (77,55%) than the BFM (48,98%). The higher sensitivity of the MF LED was confirmed, validating this technique as an alternative to Zielh Neelsen staining in the NRLTBLM. Its implementation in Cuba would be very useful in detecting and monitoring the follow up cases, as well as the management of patients with suspected TB. [ABSTRACT FROM AUTHOR]

Efficient tools for rapid antibiotic susceptibility testing (AST) are crucial for appropriate use of antibiotics, especially colistin, which is now often considered a last resort therapy with extremely drug resistant Gram-negative bacteria. Here, we developed a rapid, easy and miniaturized colistin susceptibility assay based on microfluidics, which allows for culture and high-throughput analysis of bacterial samples. Specifically, a simple microfluidic platform that can easily be operated was designed to encapsulate bacteria in nanoliter droplets and perform a fast and automated bacterial growth detection in 2 h, using standardized samples. Direct bright-field imaging of compartmentalized samples proved to be a faster and more accurate detection method as compared to fluorescence-based analysis. A deep learning powered approach was implemented for the sensitive detection of the growth of several strains in droplets. The DropDeepL AST method (Droplet and Deep learning-based method for AST) developed here allowed the determination of the colistin susceptibility profiles of 21 fast-growing Enterobacterales (E. coli and K. pneumoniae), including clinical isolates with different resistance mechanisms, showing 100 % categorical agreement with the reference broth microdilution (BMD) method performed simultaneously. Direct AST of bacteria in urine samples on chip also provided accurate results in 2 h, without the need of complex sample preparation procedures. This method can easily be implemented in clinical microbiology laboratories, and has the potential to be adapted to a variety of antibiotics, especially for last-line antibiotics to optimize treatment of patients infected with multi-drug resistant strains. [Display omitted] • Simplified workflow for bacteria encapsulation and detection in nanoliter droplets. • Automated and deep learning powered approach for droplet classification. • Digital susceptibility testing for colistin using clinical isolates in 2 hours. • Categorization of bacteria strains in complete agreement with the reference method. • Direct urine analysis on chip for rapid detection of antimicrobial resistance. [ABSTRACT FROM AUTHOR]

Pollen analysis is a method used for verification of the botanical and geographical honey origin. Currently, much effort is being made to introduce automated systems with the use of image analysis programs. The automatic analysis is impeded by the insufficient depth of field of objects when using a light microscope, however, this can be avoided by using image reconstruction from images obtained from different focal planes. In this method, testing was performed on the normal focus (NF) and extended-depth-of-focus(EDF) images. These two methods were compared and statistically evaluated. The number of pollen grains and selected morphometric characteristics were compared. For EDF images, a higher number of pollen grains was obtained for the analysis, and except for the length/width ratio, a statistically significant difference was observed in the characteristics of pollen grains between the compared NF and EDF methods. [ABSTRACT FROM AUTHOR]

Common oxide nanostructures such as silicon‐di‐oxide, magnesium‐oxide, zinc‐oxide, and copper‐oxide (CuO) having useful functional and bioactive properties have been synthesised and characterised. All these nanostructures have been found to be larvicidal towards Culex quinquefasciatus mosquito especially against lower instars in comparison with higher instars in 48 h. Only, CuO is larvicidal against late instar stages after 48 h. Moreover, CuO is larvicidal against first instar stages after 24 h (LC50 157 mg/l). However, none of these nanostructures are pupicidal. Post mortality larval morphology was found to be distorted under bright field microscopy and scanning electron microscopy images of affected larval surface appeared to be rough and uneven. Fluorescent images showed that nanostructures infiltrated inside visceral organs of larvae. Nanostructures also caused tissue oxidative stress in larvae. These results indicate that above stated oxide nanostructures are effective larvicidal agents against early instar stages of Culex larvae. [ABSTRACT FROM AUTHOR]

Bright ?eld imaging of biological samples stained with antibodies and/or special stains provides a rapid protocol for visualizing various macromolecules. However, this method of sample staining and imaging is rarely employed for direct quantitative analysis due to variations in sample fixations, ambiguities introduced by color composition, and the limited dynamic range of imaging instruments. We demonstrate that, through the decomposition of color signals, staining can be scored on a cell-by-cell basis. We have applied our method to Flbroblasts grown from histologically normal breast tissue biopsies obtained from two distinct populations. Initially, nuclear regions are segmented through conversion of color images into gray scale, and detection of dark elliptic features. Subsequently, the strength of staining is quanti?ed by a color decomposition model that is optimized by a graph cut algorithm. In rare cases where nuclear signal is significantly altered as a result of sample preparation, nuclear segmentation can be validated and corrected. Finally, segmented stained patterns are associated with each nuclear region following region-based tessellation. Compared to classical non-negative matrix factorization, proposed method (i) improves color decomposition, (ii) has a better noise immunity, (iii) is more invariant to initial conditions, and (iv) has a superior computing performance

Fluorescence microscopy is one of the workhorses of biomedical research and laboratory diagnosis; however, their cost, size, maintenance, and fragility has prevented their adoption in developing countries or low-resource settings. Although significant advances have decreased their size, cost and accessibility, their designs and assembly remain rather complex. Here, inspired on the simple mechanism from a nut and a bolt, we report the construction of a portable fluorescence microscope that operates in bright-field mode and in three fluorescence channels: UV, green, and red. It is assembled in under 10 min from only six 3D printed parts, basic electronic components, a microcomputer (Raspberry Pi) and a camera, all of which can be readily purchased in most locations or online for US $122. The microcomputer was programmed in Python language to capture time-lapse images and videos. Resolution and illumination conditions of the microscope were characterized, and its performance was compared with a high-end fluorescence microscope in bright-field and fluorescence mode. We demonstrate that our miniature microscope can resolve and track single cells in both modes. The instructions on how to assemble the microscope are shown in a video, and the software to control it and the design files of the 3D-printed parts are freely available online. Our portable microscope is ideal in applications where space is at a premium, such as lab-on-a-chips or space missions, and can find applications in basic and clinical research, diagnostics, telemedicine and in educational settings. [ABSTRACT FROM AUTHOR]

Single-molecule fluorescence microscopy studies of bacteria provide unique insights into the mechanisms of cellular processes and protein machineries in ways that are unrivalled by any other technique. With the cost of microscopes dropping and the availability of fully automated microscopes, the volume of microscopy data produced has increased tremendously. These developments have moved the bottleneck of throughput from image acquisition and sample preparation to data analysis. Furthermore, requirements for analysis procedures have become more stringent given the demand of various journals to make data and analysis procedures available. To address these issues we have developed a new data analysis package for analysis of fluorescence microscopy data from rod-like cells. Our software ColiCoords structures microscopy data at the single-cell level and implements a coordinate system describing each cell. This allows for the transformation of Cartesian coordinates from transmission light and fluorescence images and single-molecule localization microscopy (SMLM) data to cellular coordinates. Using this transformation, many cells can be combined to increase the statistical power of fluorescence microscopy datasets of any kind. ColiCoords is open source, implemented in the programming language Python, and is extensively documented. This allows for modifications for specific needs or to inspect and publish data analysis procedures. By providing a format that allows for easy sharing of code and associated data, we intend to promote open and reproducible research. The source code and documentation can be found via the project’s page. [ABSTRACT FROM AUTHOR]

Live cell time-lapse microscopy, a widely-used technique to study gene expression and protein dynamics in single cells, relies on segmentation and tracking of individual cells for data generation. The potential of the data that can be extracted from this technique is limited by the inability to accurately segment a large number of cells from such microscopy images and track them over long periods of time. Existing segmentation and tracking algorithms either require additional dyes or markers specific to segmentation or they are highly specific to one imaging condition and cell morphology and/or necessitate manual correction. Here we introduce a fully automated, fast and robust segmentation and tracking algorithm for budding yeast that overcomes these limitations. Full automatization is achieved through a novel automated seeding method, which first generates coarse seeds, then automatically fine-tunes cell boundaries using these seeds and automatically corrects segmentation mistakes. Our algorithm can accurately segment and track individual yeast cells without any specific dye or biomarker. Moreover, we show how existing channels devoted to a biological process of interest can be used to improve the segmentation. The algorithm is versatile in that it accurately segments not only cycling cells with smooth elliptical shapes, but also cells with arbitrary morphologies (e.g. sporulating and pheromone treated cells). In addition, the algorithm is independent of the specific imaging method (bright-field/phase) and objective used (40X/63X/100X). We validate our algorithm’s performance on 9 cases each entailing a different imaging condition, objective magnification and/or cell morphology. Taken together, our algorithm presents a powerful segmentation and tracking tool that can be adapted to numerous budding yeast single-cell studies. [ABSTRACT FROM AUTHOR]

Multiplexed bioassays, in which multiple analytes of interest are probed in parallel within a single small volume, have greatly accelerated the pace of biological discovery. Bead-based multiplexed bioassays have many technical advantages, including near solution-phase kinetics, small sample volume requirements, many within-assay replicates to reduce measurement error, and, for some bead materials, the ability to synthesize analytes directly on beads via solid-phase synthesis. To allow bead-based multiplexing, analytes can be synthesized on spectrally encoded beads with a 1:1 linkage between analyte identity and embedded codes. Bead-bound analyte libraries can then be pooled and incubated with a fluorescently-labeled macromolecule of interest, allowing downstream quantification of interactions between the macromolecule and all analytes simultaneously via imaging alone. Extracting quantitative binding data from these images poses several computational image processing challenges, requiring the ability to identify all beads in each image, quantify bound fluorescent material associated with each bead, and determine their embedded spectral code to reveal analyte identities. Here, we present a novel open-source Python software package (the mrbles analysis package) that provides the necessary tools to: (1) find encoded beads in a bright-field microscopy image; (2) quantify bound fluorescent material associated with bead perimeters; (3) identify embedded ratiometric spectral codes within beads; and (4) return data aggregated by embedded code and for each individual bead. We demonstrate the utility of this package by applying it towards analyzing data generated via multiplexed measurement of calcineurin protein binding to MRBLEs (icrospheres with atiometric arcode anthanide ncoding) containing known and mutant binding peptide motifs. We anticipate that this flexible package should be applicable to a wide variety of assays, including simple bead or droplet finding analysis, quantification of binding to non-encoded beads, and analysis of multiplexed assays that use ratiometric, spectrally encoded beads. [ABSTRACT FROM AUTHOR]

It is demonstrated that cells can be classified by pattern recognition of the subcellular structure of non-stained live cells, and the pattern recognition was performed by machine learning. Human white blood cells and five types of cancer cell lines were imaged by quantitative phase microscopy, which provides morphological information without staining quantitatively in terms of optical thickness of cells. Subcellular features were then extracted from the obtained images as training data sets for the machine learning. The built classifier successfully classified WBCs from cell lines (area under ROC curve = 0.996). This label-free, non-cytotoxic cell classification based on the subcellular structure of QPM images has the potential to serve as an automated diagnosis of single cells. [ABSTRACT FROM AUTHOR]

We present a novel intensity-gradient based algorithm specifically designed for nanometer-segmentation of cell membrane contours obtained with high-resolution optical microscopy combined with high-velocity digital imaging. The algorithm relies on the image oversampling performance and computational power of graphical processing units (GPUs). Both, synthetic and experimental data are used to quantify the sub-pixel precision of the algorithm, whose analytic performance results comparatively higher than in previous methods. Results from the synthetic data indicate that the spatial precision of the presented algorithm is only limited by the signal-to-noise ratio (SNR) of the contour image. We emphasize on the application of the new algorithm to membrane fluctuations (flickering) in eukaryotic cells, bacteria and giant vesicle models. The method shows promising applicability in several fields of cellular biology and medical imaging for nanometer-precise boundary-determination and mechanical fingerprinting of cellular membranes in optical microscopy images. Our implementation of this high-precision flicker spectroscopy contour tracking algorithm (HiPFSTA) is provided as open-source at . [ABSTRACT FROM AUTHOR]

Abstract Background Today, phase contrast microscopy (PCM) is the recommended technique for manual urinary sediment (U-sed) examination. In fact, compared to bright field microscopy (BFM), it allows a better identification of most U-sed particles. Methods The main contributions, both as original papers in medical journals and as monographs on PCM applied to U-sed examination, published in the period 1950–1982 (which was chosen because it includes the results of the most important investigations on the subject) were identified and analysed. Moreover, a brief analysis on the use of PCM in U-sed examination today was carried out. Results After the discovery of PCM by the Dutch physicist Frits Zernike in the 1930s, several contributions were published, most of which are forgotten today. All of them demonstrated the advantages of PCM over BFM in identifying the U-sed particles, especially casts, renal tubular epithelial cells, atypical urothelial cells associated with urothelial carcinoma, and erythrocytes (which for the 1st time were classified as dysmorphic - of glomerular origin - and isomorphic - of non glomerular origin). The analysis of six recent monographs on U-sed or urinalysis, written in English and with an international distribution, demonstrated that only in two of them the U-sed particles were mostly shown by PCM. Conclusion Several papers and monographs, published since the early 1950s, have demonstrated the advantages of PCM over BFM for U-sed examination. In spite of this, PCM is not as widely used as it should be. Highlights • The review of the literature of the period 1950-1982 shows that several authors have investigated the use of phase contrast microscopy in the evaluation of urinary sediment. • All the authors have described the advantages of phase contrast microscopy over traditional bright field microscopy in identifying the particles of the urinary sediment, especially epithelial cells, casts, and erythrocyte morphology. • In spite of these results, today the use of phase contrast microscopy for urinary sediment examination is not as diffused as it should be. [ABSTRACT FROM AUTHOR]

The cyanobacterium Nostoc muscorum Meg 1 was exposed to Cu2+ for seven days to assess its Cu2+ removal potential and the changes brought about in its morphology, physiology and biochemistry on Cu2+ exposure. Cu2+ binding was established by the EDX study. Identification of various functional groups on the cell surface involved in Cu2+ binding was done by FTIR analysis. AAS study showed that the organism was able to remove 96.3% Cu2+ from the medium supplemented with 3 ppm Cu2+. Cellular distribution analysis indicated internalization of 6.1% Cu2+. Another study showed that the maximum Cu2+ removal was a surface phenomenon that did not require energy. The IC50 value for Cu2+ was determined to be 9 ppm as growth was compromised by 50.5%. We used a sub-lethal dose of 3 ppm for all the experiments which are 3-fold higher than the WHO recommended value for Cu2+ (1 ppm) in drinking water. At this concentration, various photosynthetic pigments were reduced by 35.3% (chlorophyll a), 31.3% (phycocyanin), 17% (allophycocyanin), 21.3% (phycoerythrin) and 16% (carotenoids). Photosynthetic PSII activity and respiration rate were also compromised by 45% and 46.2%. Heterocyst frequency, nitrogenase and glutamine synthetase activities were declined by 18.5%, 14.8% and 16.2%. Bright field and SEM images showed distinct morphological changes, where there was filament breakage, disintegration and degradation of individual cells as well as cell elongation, distortion and shriveling. The cyanobacterium generated 436.4% more ROS compared to the control cells. [ABSTRACT FROM AUTHOR]

The design of experimental protocols that use animal models to assess the impact of a stress on a population or to determine the life span expectancy impact can be time-consuming due to the need for direct observations of dead and living animals. These experiments are usually based on the detectable activity of animals such as food intake or mobility and can sometimes produce either under- or overestimated results. The tardigrade Hypsibius exemplaris is an emerging model for the evolutionary biology of the tardigrade phylum because of its convenient laboratory breeding and the recent introduction of new molecular tools. In this report, we describe the use of a new fluorescent dye that can specifically stain dead tardigrades. Furthermore, we also monitored the absence of a toxic side effect of the death-linked fluorescent dye on tardigrade populations. Finally, we conclude that tardigrade experiments that require survival counting of the Hypsibius exemplaris species can be greatly improved by using this technique in order to limit underestimation of alive animals. [ABSTRACT FROM AUTHOR]

Organismal fitness is relevant in many contexts in biology. The most meaningful experimental measure of fitness is competitive fitness, when two or more entities (e.g., genotypes) are allowed to compete directly. In theory, competitive fitness is simple to measure: an experimental population is initiated with the different types in known proportions and allowed to evolve under experimental conditions to a predefined endpoint. In practice, there are several obstacles to obtaining robust estimates of competitive fitness in multicellular organisms, the most pervasive of which is simply the time it takes to count many individuals of different types from many replicate populations. Methods by which counting can be automated in high throughput are desirable, but for automated methods to be useful, the bias and technical variance associated with the method must be (a) known, and (b) sufficiently small relative to other sources of bias and variance to make the effort worthwhile. The nematode Caenorhabditis elegans is an important model organism, and the fitness effects of genotype and environmental conditions are often of interest. We report a comparison of three experimental methods of quantifying competitive fitness, in which wild-type strains are competed against GFP-marked competitors under standard laboratory conditions. Population samples were split into three replicates and counted (1) "by eye" from a saved image, (2) from the same image using CellProfiler image analysis software, and (3) with a large particle flow cytometer (a "worm sorter"). From 720 replicate samples, neither the frequency of wild-type worms nor the among-sample variance differed significantly between the three methods. CellProfiler and the worm sorter provide a tenfold increase in sample handling speed with little (if any) bias or increase in variance. [ABSTRACT FROM AUTHOR]

High-fat diet (HFD)-induced leaky gut syndrome combined with low-grade inflammation increase reactive oxygen species (ROS) in the intestine and may contribute to dysbiosis and metabolic syndrome (MetS). Poorly bioavailable and only partially metabolizable dietary polyphenols, such as proanthocyanidins (PACs), may exert their beneficial effects on metabolic health by scavenging intestinal ROS. To test this hypothesis, we developed and validated a novel, noninvasive, in situ method for visualizing intestinal ROS using orally administered ROS-sensitive indocyanine green (ICG) dye. C57BL/6J mice fed HFD for 10 weeks accumulated high levels of intestinal ROS compared to mice fed low-fat diet (LFD). Oral administration of poorly bioavailable grape polyphenol extract (GPE) and β-carotene decreased HFD-induced ROS in the gut to levels comparable to LFD-fed mice, while administration of more bioavailable dietary antioxidants (α-lipoic acid, vitamin C, vitamin E) did not. Forty percent of administered GPE antioxidant activity was measured in feces collected over 24 h, confirming poor bioavailability and persistence in the gut. The bloom of beneficial anaerobic gut bacteria, such as Akkermansia muciniphila, associated with improved metabolic status in rodents and humans may be directly linked to protective antioxidant activity of some dietary components. These findings suggest a possible mechanistic explanation for the beneficial effects of poorly bioavailable polyphenols on metabolic health. [ABSTRACT FROM AUTHOR]

Sequential labeling and imaging in fluorescence microscopy allows the imaging of multiple structures in the same cell using a single fluorophore species. In super-resolution applications, the optimal dye suited to the method can be chosen, the optical setup can be simpler and there are no chromatic aberrations between images of different structures. We describe a method based on DNA strand displacement that can be used to quickly and easily perform the labeling and removal of the fluorophores during each sequence. Site-specific tags are conjugated with unique and orthogonal single stranded DNA. Labeling for a particular structure is achieved by hybridization of antibody-bound DNA with a complimentary dye-labeled strand. After imaging, the dye is removed using toehold-mediated strand displacement, in which an invader strand competes off the dye-labeled strand than can be subsequently washed away. Labeling and removal of each DNA-species requires only a few minutes. We demonstrate the concept using sequential dSTORM super-resolution for multiplex imaging of subcellular structures. [ABSTRACT FROM AUTHOR]

Studies on monolayer cultures and whole-animal models for the prediction of the response of native human tissue are associated with limitations. Therefore, more and more laboratories are tending towards multicellular spheroids grown in vitro as a model of native tissues. In addition, they are increasingly used in a wide range of biofabrication methodologies. These 3D microspheroids are generated through a self-assembly process that is still poorly characterised, called cellular aggregation. Here, a system is proposed for the automated, non-invasive and high throughput monitoring of the morphological changes during cell aggregation. Microwell patterned inserts were used for spheroid formation while an automated microscope with 4x bright-field objective captured the morphological changes during this process. Subsequently, the acquired time-lapse images were automatically segmented and several morphological features such as minor axis length, major axis length, roundness, area, perimeter and circularity were extracted for each spheroid. The method was quantitatively validated with respect to manual segmentation on four sets of ± 60 spheroids. The average sensitivities and precisions of the proposed segmentation method ranged from 96.67–97.84% and 96.77–97.73%, respectively. In addition, the different morphological features were validated, obtaining average relative errors between 0.78–4.50%. On average, a spheroid was processed 73 times faster than a human operator. As opposed to existing algorithms, our methodology was not only able to automatically monitor compact spheroids but also the aggregation process of individual spheroids, and this in an accurate and high-throughput manner. In total, the aggregation behaviour of more than 700 individual spheroids was monitored over a duration of 16 hours with a time interval of 5 minutes, and this could be increased up to 48,000 for the described culture format. In conclusion, the proposed system has the potential to be used for unravelling the mechanisms involved in spheroid formation and monitoring their formation during large-scale manufacturing protocols. [ABSTRACT FROM AUTHOR]

Establishing the tissue source of epithelial cells within a biological sample is an important capability for forensic laboratories. In this study we used Imaging Flow Cytometry (IFC) to analyze individual cells recovered from buccal, epidermal, and vaginal samples that had been dried between 24 hours and more than eight weeks. Measurements capturing the size, shape, and fluorescent properties of cells were collected in an automated manner and then used to build a multivariate statistical framework for differentiating cells based on tissue type. Results showed that epidermal cells could be distinguished from vaginal and buccal cells using a discriminant function analysis of IFC measurements with an average classification accuracy of ~94%. Ultimately, cellular measurements such as these, which can be obtained non-destructively, may provide probative information for many types of biological samples and complement results from standard genetic profiling techniques. [ABSTRACT FROM AUTHOR]

In this paper we report the development of a cost-effective, modular, open source, and fully automated slide-scanning microscope, composed entirely of easily available off-the-shelf parts, and capable of bright field and fluorescence modes. The automated X-Y stage is composed of two low-cost micrometer stages coupled to stepper motors operated in open-loop mode. The microscope is composed of a low-cost CMOS sensor and low-cost board lenses placed in a 4f configuration. The system has approximately 1 micron resolution, limited by the f/# of available board lenses. The microscope is compact, measuring just 25×25×30 cm, and has an absolute positioning accuracy of ±1 μm in the X and Y directions. A Z-stage enables autofocusing and imaging over large fields of view even on non-planar samples, and custom software enables automatic determination of sample boundaries and image mosaicking. We demonstrate the utility of our device through imaging of fluorescent- and transmission-dye stained blood and fecal smears containing human and animal parasites, as well as several prepared tissue samples. These results demonstrate image quality comparable to high-end commercial microscopes at a cost of less than US$400 for a bright-field system, with an extra US$100 needed for the fluorescence module. [ABSTRACT FROM AUTHOR]

Cellphones equipped with high-quality cameras and powerful CPUs as well as GPUs are widespread. This opens new prospects to use such existing computational and imaging resources to perform medical diagnosis in developing countries at a very low cost. Many relevant samples, like biological cells or waterborn parasites, are almost fully transparent. As they do not exhibit absorption, but alter the light’s phase only, they are almost invisible in brightfield microscopy. Expensive equipment and procedures for microscopic contrasting or sample staining often are not available. Dedicated illumination approaches, tailored to the sample under investigation help to boost the contrast. This is achieved by a programmable illumination source, which also allows to measure the phase gradient using the differential phase contrast (DPC) [, ] or even the quantitative phase using the derived qDPC approach []. By applying machine-learning techniques, such as a convolutional neural network (CNN), it is possible to learn a relationship between samples to be examined and its optimal light source shapes, in order to increase e.g. phase contrast, from a given dataset to enable real-time applications. For the experimental setup, we developed a 3D-printed smartphone microscope for less than 100 $ using off-the-shelf components only such as a low-cost video projector. The fully automated system assures true Koehler illumination with an LCD as the condenser aperture and a reversed smartphone lens as the microscope objective. We show that the effect of a varied light source shape, using the pre-trained CNN, does not only improve the phase contrast, but also the impression of an improvement in optical resolution without adding any special optics, as demonstrated by measurements. [ABSTRACT FROM AUTHOR]

Background: Conventional gold standard characterization of chigger mites involves chemical preparation procedures (i.e. specimen clearing) for visualization of morphological features, which however contributes to destruction of the arthropod host DNA and any endosymbiont or pathogen DNA harbored within the specimen. Methodology/Principal findings: In this study, a novel work flow based on autofluorescence microscopy was developed to enable identification of trombiculid mites to the species level on the basis of morphological traits without any special preparation, while preserving the mite DNA for subsequent genotyping. A panel of 16 specifically selected fluorescence microscopy images of mite features from available identification keys served for complete chigger morphological identification to the species level, and was paired with corresponding genotype data. We evaluated and validated this method for paired chigger morphological and genotypic ID using the mitochondrial cytochrome c oxidase subunit I gene (coi) in 113 chigger specimens representing 12 species and 7 genera (Leptotrombidium, Ascoschoengastia, Gahrliepia, Walchia, Blankaartia, Schoengastia and Schoutedenichia) from the Lao People’s Democratic Republic (Lao PDR) to the species level (complete characterization), and 153 chiggers from 5 genera (Leptotrombidium, Ascoschoengastia, Helenicula, Schoengastiella and Walchia) from Thailand, Cambodia and Lao PDR to the genus level. A phylogenetic tree constructed from 77 coi gene sequences (approximately 640 bp length, n = 52 new coi sequences and n = 25 downloaded from GenBank), demonstrated clear grouping of assigned morphotypes at the genus levels, although evidence of both genetic polymorphism and morphological plasticity was found. Conclusions/Significance: With this new methodology, we provided the largest collection of characterized coi gene sequences for trombiculid mites to date, and almost doubled the number of available characterized coi gene sequences with a single study. The ability to provide paired phenotypic-genotypic data is of central importance for future characterization of mites and dissecting the molecular epidemiology of mites transmitting diseases like scrub typhus. [ABSTRACT FROM AUTHOR]

The Sch9 kinase of Saccharomyces cerevisiae is one of the major TOR pathway effectors and regulates diverse processes in the cell. Sch9 belongs to the AGC kinase family. In human, amplification of AGC kinase genes is connected with cancer. However, not much is known about the effects of Sch9 overproduction in yeast cells. To fill this gap, we developed a model system to monitor subcellular location and aggregation state of overproduced Sch9 or its regions fused to a fluorescent protein. With this system, we showed that Sch9-YFP forms detergent-resistant aggregates, and multiple protein regions are responsible for this. This finding corroborated the fact that Sch9-YFP is visualized as various fluorescent foci. In addition, we found that Sch9 overproduction caused cell elongation, and this effect was determined by its C-terminal region containing kinase domains. The constructs we present can be exploited to create superior yeast-based model systems to study processes behind kinase overproduction in cancers. [ABSTRACT FROM AUTHOR]

Background: Phase contrast microscopy is the recommended technique for urine sediment examination. Bright field microscopy does not differentiate objects with slight changes in the refractive index and hence phase contrast is a superior alternative. Methods: In this article, we describe a novel method to improve contrast in bright field microscopy. A strategically placed disc of specific dimensions enhances the diffraction of rays by Fresnel principle causing a shift in wavelength in the rays which are perceived as differences in contrast by the eye due to constructive and destructive interference. Results: Epithelial cells, red blood cells (RBCs), dysmorphic red blood cells, casts, bacteria and crystals are easily seen and differentiated by this technique. Conclusions: The images obtained are similar to those obtained by phase contrast microscopy. [ABSTRACT FROM AUTHOR]

Objectives: Myocardial infarction (MI) causes significant loss of cardiomyocytes, myocardial tissue damage, and impairment of myocardial function. The inability of cardiomyocytes to proliferate prevents the heart from self-regeneration. The treatment for advanced heart failure following an MI is heart transplantation despite the limited availability of the organs. Thus, stem-cell-based cardiac therapies could ultimately prevent heart failure by repairing injured myocardium that reverses cardiomyocyte loss. However, stem-cell-based therapies lack understanding of the mechanisms behind a successful therapy, including difficulty tracking stem cells to provide information on cell migration, proliferation and differentiation. In this study, we have investigated the interaction between different types of stem and inflammatory cells and cell-targeted imaging molecules, 18F-FDG and 6-NBDG, to identify uptake patterns and pharmacokinetics in vitro. Methods: Macrophages (both M1 and M2), human induced pluripotent stem cells (hiPSCs), and human amniotic mesenchymal stem cells (hAMSCs) were incubated with either 18F-FDG or 6-NBDG. Excess radiotracer and fluorescence were removed and a 100 μm-thin CdWO4 scintillator plate was placed on top of the cells for radioluminescence microscopy imaging of 18F-FDG uptake, while no scintillator was needed for fluorescence imaging of 6-NBDG uptake. Light produced following beta decay was imaged with a highly sensitive inverted microscope (LV200, Olympus) and an Electron Multiplying Charge-Couple Device (EM-CCD) camera. Custom-written software was developed in MATLAB for image processing. Results: The average cellular activity of 18F-FDG in a single cell of hAMSCs (0.670±0.028 fCi/μm2, P = 0.001) was 20% and 36% higher compared to uptake in hiPSCs (0.540±0.026 fCi/μm2, P = 0.003) and macrophages (0.430±0.023 fCi/μm2, P = 0.002), respectively. hAMSCs exhibited the slowest influx (0.210 min-1) but the fastest efflux (0.327 min-1) rate compared to the other tested cell lines for 18F-FDG. This cell line also has the highest phosphorylation but exhibited the lowest rate of de-phosphorylation. The uptake pattern for 6-NBDG was very different in these three cell lines. The average cellular activity of 6-NBDG in a single cell of macrophages (0.570±0.230 fM/μm2, P = 0.004) was 38% and 14% higher compared to hiPSCs (0.350±0.160 fM/μm2, P = 0.001) and hAMSCs (0.490±0.028 fM/μm2, P = 0.006), respectively. The influx (0.276 min-1), efflux (0.612 min-1), phosphorylation (0.269 min-1), and de-phosphorylation (0.049 min-1) rates were also highest for macrophages compared to the other two tested cell lines. Conclusion: hAMSCs were found to be 2–3× more sensitive to 18F-FDG molecule compared to hiPSCs/macrophages. However, macrophages exhibited the most sensitivity towards 6-NBDG. Based on this result, hAMSCs targeted with 18F-FDG could be more suitable for understanding the mechanisms behind successful therapy for treating MI patients by gathering information on cell migration, proliferation and differentiation. [ABSTRACT FROM AUTHOR]

The interaction between actin filaments and myosin molecular motors is a power source of a variety of cellular functions including cell division, cell motility, and muscular contraction. In vitro motility assay examines actin filaments interacting with myosin molecules that are adhered to a substrate (e.g., glass surface). This assay has been the standard method of studying the molecular mechanisms of contraction under an optical microscope. While the force generation has been measured through an optically trapped bead to which an actin filament is attached, a force vector vertical to the glass surface has been largely ignored with the in vitro motility assay. The vertical vector is created by the gap (distance) between the trapped bead and the glass surface. In this report, we propose a method to estimate the angle between the actin filament and the glass surface by optically determining the gap size. This determination requires a motorized stage in a standard epi-fluorescence microscope equipped with optical tweezers. This facile method is applied to force measurements using both pure actin filaments, and thin filaments reconstituted from actin, tropomyosin and troponin. We find that the angle-corrected force per unit filament length in the active condition (pCa = 5.0) decreases as the angle between the filament and the glass surface increases; i.e. as the force in the vertical direction increases. At the same time, we demonstrate that the force on reconstituted thin filaments is approximately 1.5 times larger than that on pure actin filaments. The range of angles we tested was between 11° and 36° with the estimated measurement error less than 6°. These results suggest the ability of cytoplasmic tropomyosin isoforms maintaining actomyosin active force to stabilize cytoskeletal architecture. [ABSTRACT FROM AUTHOR]

The ubiquitous presence of SPFH (tomatin, rohibitin, lotillin, flK/HflC) proteins in all domains of life suggests that their function would be conserved. However, SPFH functions are diverse with organism-specific attributes. SPFH proteins play critical roles in physiological processes such as mechanosensation and respiration. Here, we characterize the stomatin ORF19.7296/SLP3 in the opportunistic human pathogen Candida albicans. Consistent with the localization of stomatin proteins, a Slp3p-Yfp fusion protein formed visible puncta along the plasma membrane. We also visualized Slp3p within the vacuolar lumen. Slp3p primary sequence analyses identified four putative S-palmitoylation sites, which may facilitate membrane localization and are conserved features of stomatins. Plasma membrane insertion sequences are present in mammalian and nematode SPFH proteins, but are absent in Slp3p. Strikingly, Slp3p was present in yeast cells, but was absent in hyphal cells, thus categorizing it as a yeast-phase specific protein. Slp3p membrane fluorescence significantly increased in response to cellular stress caused by plasma membrane, cell wall, oxidative, or osmotic perturbants, implicating SLP3 as a general stress-response gene. A slp3Δ/Δ homozygous null mutant had no detected phenotype when slp3Δ/Δ mutants were grown in the presence of a variety of stress agents. Also, we did not observe a defect in ion accumulation, filamentation, endocytosis, vacuolar structure and function, cell wall structure, or cytoskeletal structure. However, SLP3 over-expression triggered apoptotic-like death following prolonged exposure to oxidative stress or when cells were induced to form hyphae. Our findings reveal the cellular localization of Slp3p, and for the first time associate Slp3p function with the oxidative stress response. [ABSTRACT FROM AUTHOR]

Background: In countries with high tuberculosis (TB) burden, there is urgent need for rapid, large-scale screening to detect smear-positive patients. We developed a computer-aided whole smear screening system that focuses in real-time, captures images and provides diagnostic grading, for both bright-field and fluorescence microscopy for detection of acid-fast-bacilli (AFB) from respiratory specimens. Objectives: To evaluate the performance of dual-mode screening system in AFB diagnostic algorithms on concentrated smears with auramine O (AO) staining, as well as direct smears with AO and Ziehl-Neelsen (ZN) staining, using mycobacterial culture results as gold standard. Methods: Adult patient sputum samples requesting for M. tuberculosis cultures were divided into three batches for staining: direct AO-stained, direct ZN-stained and concentrated smears AO-stained. All slides were graded by an experienced microscopist, in parallel with the automated whole smear screening system. Sensitivity and specificity of a TB diagnostic algorithm in using the screening system alone, and in combination with a microscopist, were evaluated. Results: Of 488 direct AO-stained smears, 228 were culture positive. These yielded a sensitivity of 81.6% and specificity of 74.2%. Of 334 direct smears with ZN staining, 142 were culture positive, which gave a sensitivity of 70.4% and specificity of 76.6%. Of 505 concentrated smears with AO staining, 250 were culture positive, giving a sensitivity of 86.4% and specificity of 71.0%. To further improve performance, machine grading was confirmed by manual smear grading when the number of AFBs detected fell within an uncertainty range. These combined results gave significant improvement in specificity (AO-direct:85.4%; ZN-direct:85.4%; AO-concentrated:92.5%) and slight improvement in sensitivity while requiring only limited manual workload. Conclusion: Our system achieved high sensitivity without substantially compromising specificity when compared to culture results. Significant improvement in specificity was obtained when uncertain results were confirmed by manual smear grading. This approach had potential to substantially reduce workload of microscopists in high burden countries. [ABSTRACT FROM AUTHOR]

Prostate specific membrane antigen (PSMA) targeted microbubbles (MBs) were developed using bioorthogonal chemistry. Streptavidin-labeled MBs were treated with a biotinylated tetrazine (MBTz) and targeted to PSMA expressing cells using trans-cyclooctene (TCO)-functionalized anti-PSMA antibodies (TCO-anti-PSMA). The extent of MB binding to PSMA positive cells for two different targeting strategies was determined using an in vitro flow chamber. The initial approach involved pretargeting, where TCO-anti-PSMA was first incubated with PSMA expressing cells and followed by MBTz, which subsequently showed a 2.8 fold increase in the number of bound MBs compared to experiments performed in the absence of TCO-anti-PSMA. Using direct targeting, where TCO-anti-PSMA was linked to MBTz prior to initiation of the assay, a 5-fold increase in binding compared to controls was observed. The direct targeting approach was subsequently evaluated in vivo using a human xenograft tumor model and two different PSMA-targeting antibodies. The US signal enhancements observed were 1.6- and 5.9-fold greater than that for non-targeted MBs. The lead construct was also evaluated in a head-to-head study using mice bearing both PSMA positive or negative tumors in separate limbs. The human PSMA expressing tumors exhibited a 2-fold higher US signal compared to those tumors deficient in human PSMA. The results demonstrate both the feasibility of preparing PSMA-targeted MBs and the benefits of using bioorthogonal chemistry to create targeted US probes. [ABSTRACT FROM AUTHOR]

Development of artificial cell models requires encapsulation of biomolecules within membrane-bound compartments. There have been limited studies of using mammalian cell-free expression (CFE) system as the ‘cytosol’ of artificial cells. We exploit glass capillary droplet microfluidics for the encapsulation of mammalian CFE within double emulsion templated vesicles. The complexity of the physicochemical properties of HeLa cell-free lysate poses a challenge compared with encapsulating simple buffer solutions. In particular, we discovered the formation of aggregates in double emulsion templated vesicles encapsulating mammalian HeLa CFE, but not with bacterial CFE. The aggregates did not arise from insolubility of the proteins made from CFE nor due to the interaction of mammalian CFE with the organic solvents in the middle phase of the double emulsions. We found that aggregation is dependent on the concentration of poly(vinyl) alcohol (PVA) surfactant, a critical double emulsion-stabilizing surfactant, and the lysate concentration in mammalian CFE. Despite vesicle instability and reduced protein expression, we demonstrate protein expression by encapsulating mammalian CFE system. Using mass spectrometry and Western blot, we identified and verified that actin is one of the proteins inside the mammalian CFE that aggregated with PVA surfactant. Our work establishes a baseline description of mammalian CFE system encapsulated in double emulsion templated vesicles as a platform for building artificial cells. [ABSTRACT FROM AUTHOR]

Kodo millet is an underutilized crop that could be used as a new starch source. Raw starch has many flaws that must be modified through treatment. Cold plasma is a well-adopted nonthermal technology for starch modification. In the current study, millet starch was treated in a plasma reactor at different voltages of 10 kV, 20 kV, and 30 kV for a duration of 10 min, 20 min, and 30 min. The effects of treatments on their hydration, structural, and physicochemical properties were explored. Water absorption and oil absorption capacities increased from 1.43 to 1.55 g/g and 1.01–1.34 g/g respectively. On the other hand, amylose content and pH of starch declined from 31.06 to 25.94 g/hg and 7.5 to 6.9 respectively. Whiteness index value increased from 82.71 to 84.45. IR spectra exhibited functional groups present in the starch. X-ray diffractogram showed changes in crystallinity along with treatment. Relative crystallinity decreased from 23.06 to 21.63%. The significance of the results was assessed at a 95% confidence level (α) at p ≤ 0.05. Scanning electron microscope (SEM) and Bright field microscopy (BFM) showed microstructural changes in the starch. Plasma improved the hydration and structural properties of kodo millet starch, and it can be useful for preparing various food products with good quality characteristics. [Display omitted] • Cold plasma treatment altered the hydration properties of Kodo millet starch. • Color properties were enhanced in treated starch samples. • Amylose content, pH, and crystallinity of starch were reduced with exposure to plasma. • SEM & BFM micrographs have shown structural changes in starch granules. [ABSTRACT FROM AUTHOR]

Neutral lipids—involved in many cellular processes—are stored as lipid droplets (LD), those mainly cytosolic (cLD) along with a small nuclear population (nLD). nLD could be involved in nuclear-lipid homeostasis serving as an endonuclear buffering system that would provide or incorporate lipids and proteins involved in signalling pathways as transcription factors and as enzymes of lipid metabolism and nuclear processes. Our aim was to determine if nLD constituted a dynamic domain. Oleic-acid (OA) added to rat hepatocytes or HepG2 cells in culture produced cellular-phenotypic LD modifications: increases in TAG, CE, C, and PL content and in cLD and nLD numbers and sizes. LD increments were reversed on exclusion of OA and were prevented by inhibition of acyl-CoA synthetase (with Triacsin C) and thus lipid biosynthesis. Under all conditions, nLD corresponded to a small population (2–10%) of total cellular LD. The anabolism triggered by OA, involving morphologic and size changes within the cLD and nLD populations, was reversed by a net balance of catabolism, upon eliminating OA. These catabolic processes included lipolysis and the mobilization of hydrolyzed FA from the LD to cytosolic-oxidation sites. These results would imply that nLD are actively involved in nuclear processes that include lipids. In conclusion, nLD are a dynamic nuclear domain since they are modified by OA through a reversible mechanism in combination with cLD; this process involves acyl-CoA-synthetase activity; ongoing TAG, CE, and PL biosynthesis. Thus, liver nLD and cLD are both dynamic cellular organelles. [ABSTRACT FROM AUTHOR]

Background: The necessity of extracellular Ca2+ for fertilization and early embryonic development in the African clawed frog, Xenopus laevis, is controversial. Ca2+ entry into X. laevis sperm is reportedly required for the acrosome reaction, yet fertilization and embryonic development have been documented to occur in high concentrations of the Ca2+ chelator BAPTA. Here we sought to resolve this controversy. Methodology/principal finding: Using the appearance of cleavage furrows as an indicator of embryonic development, we found that X. laevis eggs inseminated in a solution lacking added divalent cations developed normally. By contrast, eggs inseminated in millimolar concentrations of BAPTA or EGTA failed to develop. Transferring embryos to varying solutions after sperm addition, we found that extracellular Ca2+ is specifically required for events occurring within the first 30 minutes after sperm addition, but not after. We found that the fluorescently stained sperm were not able to penetrate the envelope of eggs inseminated in high BAPTA, whereas several had penetrated the vitelline envelope of eggs inseminated without a Ca2+ chelator, or with BAPTA and saturating CaCl2. Together these results indicate that fertilization does not occur in high concentrations of Ca2+ chelators. Finally, we found that the jelly coat includes >5 mM of readily diffusible Ca2+. Conclusions/Significance: Taken together, these data are consistent with requirement of extracellular Ca2+ for fertilization. Based on our findings, we hypothesize that the jelly coat surrounding the egg acts as a reserve of readily available Ca2+ ions to foster fertilization in changing extracellular milieu. [ABSTRACT FROM AUTHOR]

Whole slide imaging (WSI) is a useful tool for multi-modal imaging, and in our work, we have often combined WSI with darkfield microscopy. However, traditional darkfield microscopy cannot use a single condenser to support high- and low-numerical-aperture objectives, which limits the modality of WSI. To overcome this limitation, we previously developed a darkfield internal reflection illumination (DIRI) microscope using white light-emitting diodes (LEDs). Although the developed DIRI is useful for biological applications, substantial problems remain to be resolved. In this study, we propose a novel illumination technique called color DIRI. The use of three-color LEDs dramatically improves the capability of the system, such that color DIRI (1) enables optimization of the illumination color; (2) can be combined with an oil objective lens; (3) can produce fluorescence excitation illumination; (4) can adjust the wavelength of light to avoid cell damage or reactions; and (5) can be used as a photostimulator. These results clearly illustrate that the proposed color DIRI can significantly extend WSI modalities for biological applications. [ABSTRACT FROM AUTHOR]

Large benthic foraminifera are unicellular calcifying reef organisms that can form symbiotic relationships with a range of different microalgae. However, the cellular functions, such as symbiosis and calcification, and other aspects of cellular physiology in large benthic foraminifera are not fully understood. Amphisorus kudakajimensis was used as a model to determine the detailed cellular characteristics of large benthic foraminifera. We used calcein acetoxymethyl ester (calcein AM) as a fluorescent indicator for live confocal imaging. We demonstrated that calcein AM is a useful fluorescent indicator to stain the fine network of reticulopodia and the cytoplasm in living A. kudakajimensis. We showed that at least two types of reticulopodia exist in A. kudakajimensis: the straight bundle of reticulopodia that spreads from the aperture and the fine reticulopodia along the surface of the aperture and chamber walls. The cytoplasm in outer chambers was highly branched and contained a few dinoflagellates. In contrast, the inner chamberlets contained condensed cytoplasm and many dinoflagellates, suggesting that the cytoplasm of A. kudakajimensis performs different functions based on its location within the large test. Our confocal detailed image analysis provides real-time cellular morphology and cell physiology of living foraminifera. [ABSTRACT FROM AUTHOR]

Schistosomiasis is a parasitic disease affecting over 200 million people worldwide. This study reports the design and development of a microfiltration device for isolating schistosome eggs in urine for rapid diagnostics of urogenital schistosomiasis. The design of the device comprises a linear array of microfluidic traps to immobilize and separate schistosome eggs. Sequential loading of individual eggs is achieved autonomously by flow resistance, which facilitates observation and enumeration of samples with low-abundance targets. Computational fluid dynamics modeling and experimental characterization are performed to optimize the trapping performance. By optimizing the capture strategy, the trapping efficiency could be achieved at 100% with 300 μl/min and 83% with 3000 μl/min, and the filtration procedure could be finished within 10 min. The trapped eggs can be either recovered for downstream analysis or preserved in situ for whole-mount staining. On-chip phenotyping using confocal laser fluorescence microscopy identifies the microstructure of the trapped schistosome eggs. The device provides a novel microfluidic approach for trapping, counting and on-chip fluorescence characterization of urinal Schistosoma haematobium eggs for clinical and investigative application. [ABSTRACT FROM AUTHOR]

The polarity of proliferative and differentiated cellular compartments of colonic crypts is believed to be specified by gradients of key mitogens and morphogens. Indirect evidence demonstrates a tight correlation between Wnt- pathway activity and the basal-luminal patterning; however, to date there has been no direct experimental manipulation demonstrating that a chemical gradient of signaling factors can produce similar patterning under controlled conditions. In the current work, colonic organoids (colonoids) derived from cultured, multicellular organoid fragments or single stem cells were exposed in culture to steep linear gradients of two Wnt-signaling ligands, Wnt-3a and R-spondin1. The use of a genetically engineered Sox9-Sox9EGFP:CAGDsRED reporter gene mouse model and EdU-based labeling enabled crypt patterning to be quantified in the developing colonoids. Colonoids derived from multicellular fragments cultured for 5 days under a Wnt-3a or a combined Wnt-3a and R-spondin1 gradient were highly polarized with proliferative cells localizing to the region of the higher morphogen concentration. In a Wnt-3a gradient, Sox9EGFP polarization was 7.3 times greater than that of colonoids cultured in the absence of a gradient; and the extent of EdU polarization was 2.2 times greater than that in the absence of a gradient. Under a Wnt-3a/R-spondin1 gradient, Sox9EGFP polarization was 8.2 times greater than that of colonoids cultured in the absence of a gradient while the extent of EdU polarization was 10 times greater than that in the absence of a gradient. Colonoids derived from single stem cells cultured in Wnt-3a/R-spondin1 gradients were most highly polarized demonstrated by a Sox9EGFP polarization 20 times that of colonoids grown in the absence of a gradient. This data provides direct evidence that a linear gradient of Wnt signaling factors applied to colonic stem cells is sufficient to direct patterning of the colonoid unit in culture. [ABSTRACT FROM AUTHOR]

In this paper, we present an objective method for localization of proteins in blood brain barrier (BBB) vasculature using standard immunohistochemistry (IHC) techniques and bright-field microscopy. Images from the hippocampal region at the BBB are acquired using bright-field microscopy and subjected to our segmentation pipeline which is designed to automatically identify and segment microvessels containing the protein glucose transporter 1 (GLUT1). Gabor filtering and k-means clustering are employed to isolate potential vascular structures within cryosectioned slabs of the hippocampus, which are subsequently subjected to feature extraction followed by classification via decision forest. The false positive rate (FPR) of microvessel classification is characterized using synthetic and non-synthetic IHC image data for image entropies ranging between 3 and 8 bits. The average FPR for synthetic and non-synthetic IHC image data was found to be 5.48% and 5.04%, respectively. [ABSTRACT FROM AUTHOR]

Yeasts can form multicellular patterns as they expand on agar plates, a phenotype that requires a functional copy of the FLO11 gene. Although the biochemical and molecular requirements for such patterns have been examined, the mechanisms underlying their formation are not entirely clear. Here we develop quantitative methods to accurately characterize the size, shape, and surface patterns of yeast colonies for various combinations of agar and sugar concentrations. We combine these measurements with mathematical and physical models and find that FLO11 gene constrains cells to grow near the agar surface, causing the formation of larger and more irregular colonies that undergo hierarchical wrinkling. Head-to-head competition assays on agar plates indicate that two-dimensional constraint on the expansion of FLO11 wild type (FLO11) cells confers a fitness advantage over FLO11 knockout (flo11Δ) cells on the agar surface. [ABSTRACT FROM AUTHOR]

Lymphatic Filariasis, a Neglected Tropical Disease, is caused by thread-like parasitic worms, including B. malayi, which migrate to the human lymphatic system following transmission. The parasites reside in collecting lymphatic vessels and lymph nodes for years, often resulting in lymphedema, elephantiasis or hydrocele. The mechanisms driving worm migration and retention within the lymphatics are currently unknown. We have developed an integrated in vitro imaging platform capable of quantifying B. malayi migration and behavior in a multicellular microenvironment relevant to the initial site of worm injection by incorporating the worm in a Polydimethylsiloxane (PDMS) microchannel in the presence of human dermal lymphatic endothelial cells (LECs) and human dermal fibroblasts (HDFs). The platform utilizes a motorized controllable microscope with CO2 and temperature regulation to allow for worm tracking experiments with high resolution over large length and time scales. Using post-acquisition algorithms, we quantified four parameters: 1) speed, 2) thrashing intensity, 3) percentage of time spent in a given cell region and 4) persistence ratio. We demonstrated the utility of our system by quantifying these parameters for L3 B. malayi in the presence of LECs and HDFs. Speed and thrashing increased in the presence of both cell types and were altered within minutes upon exposure to the anthelmintic drug, tetramisole. The worms displayed no targeted migration towards either cell type for the time course of this study (3 hours). When cells were not present in the chamber, worm thrashing correlated directly with worm speed. However, this correlation was lost in the presence of cells. The described platform provides the ability to further study B. malayi migration and behavior. [ABSTRACT FROM AUTHOR]