Practical use of digital cameras for microphotography through the operating microscope

A practical use of personal digital cameras for taking digital photographs in the microsurgical field through an operating microscope is described. This inexpensive and practical method for acquiring microscopic images at the desired magnification combines the advantages of the digital camera and the operating microscope.

Glucocorticoid induced impairment of lung structure assessed by digital image analysis

Glucocorticoids (GC) are successfully applied in neonatology to improve lung maturation in preterm born babies. Animal studies show that GC can also impair lung development. In this investigation, we used a new approach based on digital image analysis. Microscopic images of lung parenchyma were skeletonised and the geometrical properties of the septal network characterised by analysing the 'skeletal' parameters. Inhibition of the process of alveolarisation after extensive administration of small doses of GC in newborn rats was confirmed by significant changes in the 'skeletal' parameters. The induced structural changes in the lung parenchyma were still present after 60 days in adult rats, clearly indicating a long lasting or even definitive impairment of lung development and maturation caused by GC. Conclusion: digital image analysis and skeletonisation proved to be a highly suited approach to assess structural changes in lung parenchyma.

Traumatic brain injury (TBI) produces cytoskeletal protein derangements within cortical neurons that can be attenuated by protease inhibition

TBI produces a consistent and extensive loss of neurofilament 68 (NF68) and neurofilament 200 (NF200), key intermediate cytoskeletal proteins found in neurons including axons and dendrites, in cortical samples from injured brain. The presence of low molecular weight NF68 breakdown products (BDPs) strongly suggest that calpain proteolysis at least in part contributes to neurofilament (NF) protein loss following injury. Furthermore, one and two-dimensional gel electrophoresis analyses of NF BDPs obtained from in situ and in vitro tissue also implicated the involvement of calpain 2 mediated proteolysis of neurofilaments following TBI. Immunohistochemical examination of derangements in cytoskeletal proteins following traumatic brain injury in rats indicated that preferential dendritic rather than axonal damage occurs within three hours post-TBI. Although proteolysis of cytoskeletal proteins occurred concurrently with early morphological alterations, evidence of proteolysis preceded the full expression of evolutionary histopathological changes. Furthermore, cytoskeletal immunofluorescence alterations were not restricted to the site of impact. Confocal microscopic investigations of NF68 and NF200 immunofluorescence within injured cortical neurons revealed alterations in neurofilament assembly in the absence of NF derangements detectable at the light microscopic level ($<$15 minutes post-TBI). Collectively immunohistochemistry studies suggest that derangements to neuronal processes are biochemical and evolutionary in nature, and not due solely to mechanical shearing. Importantly, a systemically administered calpain inhibitor (calpain inhibitor 2) significantly reduced NF200, NF68, and spectrin protein loss as well as providing marked preservation of NF proteins in neuronal somata, dendrites, and axons at 24 hours post-TBI. ^

Análisis y clasificación de maderas mediante técnicas no invasivas de baja energía

El estudio de materiales, especialmente biológicos, por medios no destructivos está adquiriendo una importancia creciente tanto en las aplicaciones científicas como industriales. Las ventajas económicas de los métodos no destructivos son múltiples. Existen numerosos procedimientos físicos capaces de extraer información detallada de las superficie de la madera con escaso o nulo tratamiento previo y mínima intrusión en el material. Entre los diversos métodos destacan las técnicas ópticas y las acústicas por su gran versatilidad, relativa sencillez y bajo coste. Esta tesis pretende establecer desde la aplicación de principios simples de física, de medición directa y superficial, a través del desarrollo de los algoritmos de decisión mas adecuados basados en la estadística, unas soluciones tecnológicas simples y en esencia, de coste mínimo, para su posible aplicación en la determinación de la especie y los defectos superficiales de la madera de cada muestra tratando, en la medida de lo posible, no alterar su geometría de trabajo. Los análisis desarrollados han sido los tres siguientes: El primer método óptico utiliza las propiedades de la luz dispersada por la superficie de la madera cuando es iluminada por un laser difuso. Esta dispersión produce un moteado luminoso (speckle) cuyas propiedades estadísticas permiten extraer propiedades muy precisas de la estructura tanto microscópica como macroscópica de la madera. El análisis de las propiedades espectrales de la luz laser dispersada genera ciertos patrones mas o menos regulares relacionados con la estructura anatómica, composición, procesado y textura superficial de la madera bajo estudio que ponen de manifiesto características del material o de la calidad de los procesos a los que ha sido sometido. El uso de este tipo de láseres implica también la posibilidad de realizar monitorizaciones de procesos industriales en tiempo real y a distancia sin interferir con otros sensores. La segunda técnica óptica que emplearemos hace uso del estudio estadístico y matemático de las propiedades de las imágenes digitales obtenidas de la superficie de la madera a través de un sistema de scanner de alta resolución. Después de aislar los detalles mas relevantes de las imágenes, diversos algoritmos de clasificacion automatica se encargan de generar bases de datos con las diversas especies de maderas a las que pertenecían las imágenes, junto con los márgenes de error de tales clasificaciones. Una parte fundamental de las herramientas de clasificacion se basa en el estudio preciso de las bandas de color de las diversas maderas. Finalmente, numerosas técnicas acústicas, tales como el análisis de pulsos por impacto acústico, permiten complementar y afinar los resultados obtenidos con los métodos ópticos descritos, identificando estructuras superficiales y profundas en la madera así como patologías o deformaciones, aspectos de especial utilidad en usos de la madera en estructuras. La utilidad de estas técnicas esta mas que demostrada en el campo industrial aun cuando su aplicación carece de la suficiente expansión debido a sus altos costes y falta de normalización de los procesos, lo cual hace que cada análisis no sea comparable con su teórico equivalente de mercado. En la actualidad gran parte de los esfuerzos de investigación tienden a dar por supuesto que la diferenciación entre especies es un mecanismo de reconocimiento propio del ser humano y concentran las tecnologías en la definición de parámetros físicos (módulos de elasticidad, conductividad eléctrica o acústica, etc.), utilizando aparatos muy costosos y en muchos casos complejos en su aplicación de campo. Abstract The study of materials, especially the biological ones, by non-destructive techniques is becoming increasingly important in both scientific and industrial applications. The economic advantages of non-destructive methods are multiple and clear due to the related costs and resources necessaries. There are many physical processes capable of extracting detailed information on the wood surface with little or no previous treatment and minimal intrusion into the material. Among the various methods stand out acoustic and optical techniques for their great versatility, relative simplicity and low cost. This thesis aims to establish from the application of simple principles of physics, surface direct measurement and through the development of the more appropriate decision algorithms based on statistics, a simple technological solutions with the minimum cost for possible application in determining the species and the wood surface defects of each sample. Looking for a reasonable accuracy without altering their work-location or properties is the main objetive. There are three different work lines: Empirical characterization of wood surfaces by means of iterative autocorrelation of laser speckle patterns: A simple and inexpensive method for the qualitative characterization of wood surfaces is presented. it is based on the iterative autocorrelation of laser speckle patterns produced by diffuse laser illumination of the wood surfaces. The method exploits the high spatial frequency content of speckle images. A similar approach with raw conventional photographs taken with ordinary light would be very difficult. A few iterations of the algorithm are necessary, typically three or four, in order to visualize the most important periodic features of the surface. The processed patterns help in the study of surface parameters, to design new scattering models and to classify the wood species. Fractal-based image enhancement techniques inspired by differential interference contrast microscopy: Differential interference contrast microscopy is a very powerful optical technique for microscopic imaging. Inspired by the physics of this type of microscope, we have developed a series of image processing algorithms aimed at the magnification, noise reduction, contrast enhancement and tissue analysis of biological samples. These algorithms use fractal convolution schemes which provide fast and accurate results with a performance comparable to the best present image enhancement algorithms. These techniques can be used as post processing tools for advanced microscopy or as a means to improve the performance of less expensive visualization instruments. Several examples of the use of these algorithms to visualize microscopic images of raw pine wood samples with a simple desktop scanner are provided. Wood species identification using stress-wave analysis in the audible range: Stress-wave analysis is a powerful and flexible technique to study mechanical properties of many materials. We present a simple technique to obtain information about the species of wood samples using stress-wave sounds in the audible range generated by collision with a small pendulum. Stress-wave analysis has been used for flaw detection and quality control for decades, but its use for material identification and classification is less cited in the literature. Accurate wood species identification is a time consuming task for highly trained human experts. For this reason, the development of cost effective techniques for automatic wood classification is a desirable goal. Our proposed approach is fully non-invasive and non-destructive, reducing significantly the cost and complexity of the identification and classification process.

Characterization of Mayven, a Novel Actin-binding Protein Predominantly Expressed in Brain

The cytoskeleton plays an important role in neuronal morphogenesis. We have identified and characterized a novel actin-binding protein, termed Mayven, predominantly expressed in brain. Mayven contains a BTB (broad complex, tramtrack, bric-a-brac)/POZ (poxvirus, zinc finger) domain-like structure in the predicted N terminus and “kelch repeats” in the predicted C-terminal domain. Mayven shares 63% identity (77% similarity) with the Drosophila ring canal (“kelch”) protein. Somatic cell-hybrid analysis indicated that the human Mayven gene is located on chromosome 4q21.2, whereas the murine homolog gene is located on chromosome 8. The BTB/POZ domain of Mayven can self-dimerize in vitro, which might be important for its interaction with other BTB/POZ-containing proteins. Confocal microscopic studies of endogenous Mayven protein revealed a highly dynamic localization pattern of the protein. In U373-MG astrocytoma/glioblastoma cells, Mayven colocalized with actin filaments in stress fibers and in patchy cortical actin-rich regions of the cell margins. In primary rat hippocampal neurons, Mayven is highly expressed in the cell body and in neurite processes. Binding assays and far Western blotting analysis demonstrated association of Mayven with actin. This association is mediated through the “kelch repeats” within the C terminus of Mayven. Depolarization of primary hippocampal neurons with KCl enhanced the association of Mayven with actin. This increased association resulted in dynamic changes in Mayven distribution from uniform to punctate localization along neuronal processes. These results suggest that Mayven functions as an actin-binding protein that may be translocated along axonal processes and might be involved in the dynamic organization of the actin cytoskeleton in brain cells.

Assembly of the Nuclear Transcription and Processing Machinery: Cajal Bodies (Coiled Bodies) and Transcriptosomes

We have examined the distribution of RNA transcription and processing factors in the amphibian oocyte nucleus or germinal vesicle. RNA polymerase I (pol I), pol II, and pol III occur in the Cajal bodies (coiled bodies) along with various components required for transcription and processing of the three classes of nuclear transcripts: mRNA, rRNA, and pol III transcripts. Among these components are transcription factor IIF (TFIIF), TFIIS, splicing factors, the U7 small nuclear ribonucleoprotein particle, the stem–loop binding protein, SR proteins, cleavage and polyadenylation factors, small nucleolar RNAs, nucleolar proteins that are probably involved in pre-rRNA processing, and TFIIIA. Earlier studies and data presented here show that several of these components are first targeted to Cajal bodies when injected into the oocyte and only subsequently appear in the chromosomes or nucleoli, where transcription itself occurs. We suggest that pol I, pol II, and pol III transcription and processing components are preassembled in Cajal bodies before transport to the chromosomes and nucleoli. Most components of the pol II transcription and processing pathway that occur in Cajal bodies are also found in the many hundreds of B-snurposomes in the germinal vesicle. Electron microscopic images show that B-snurposomes consist primarily, if not exclusively, of 20- to 30-nm particles, which closely resemble the interchromatin granules described from sections of somatic nuclei. We suggest the name pol II transcriptosome for these particles to emphasize their content of factors involved in synthesis and processing of mRNA transcripts. We present a model in which pol I, pol II, and pol III transcriptosomes are assembled in the Cajal bodies before export to the nucleolus (pol I), to the B-snurposomes and eventually to the chromosomes (pol II), and directly to the chromosomes (pol III). The key feature of this model is the preassembly of the transcription and processing machinery into unitary particles. An analogy can be made between ribosomes and transcriptosomes, ribosomes being unitary particles involved in translation and transcriptosomes being unitary particles for transcription and processing of RNA.

Expression of γ-aminobutyric acid ρ1 and ρ1Δ450 as gene fusions with the green fluorescent protein

The functional characteristics and cellular localization of the γaminobutyric acid (GABA) ρ1 receptor and its nonfunctional isoform ρ1Δ450 were investigated by expressing them as gene fusions with the enhanced version of the green fluorescent protein (GFP). Oocytes injected with ρ1-GFP had receptors that gated chloride channels when activated by GABA. The functional characteristics of these receptors were the same as for those of wild-type ρ1 receptors. Fluorescence, because of the chimeric receptors expressed, was over the whole oocyte but was more intense near the cell surface and more abundant in the animal hemisphere. Similar to the wild type, ρ1Δ450-GFP did not lead to the expression of functional GABA receptors, and injected oocytes failed to generate currents even after exposure to high concentrations of GABA. Nonetheless, the fluorescence displayed by oocytes expressing ρ1Δ450-GFP was distributed similarly to that of ρ1-GFP. Mammalian cells transfected with the ρ1-GFP or ρ1Δ450-GFP constructs showed mostly intracellularly distributed fluorescence in confocal microscope images. A sparse localization of fluorescence was observed in the plasma membrane regardless of the cell line used. We conclude that ρ1Δ450 is expressed and transported close to, and perhaps incorporated into, the plasma membrane. Thus, ρ1- and ρ1Δ450-GFP fusions provide a powerful tool to visualize the traffic of GABA type C receptors.

The Sm domain is an ancient RNA-binding motif with oligo(U) specificity

Sm and Sm-like proteins are members of a family of small proteins that is widespread throughout eukaryotic kingdoms. These proteins form heteromers with one another and bind, as heteromeric complexes, to various RNAs, recognizing primarily short U-rich stretches. Interestingly, completion of several genome projects revealed that archaea also contain genes that may encode Sm-like proteins. Herein, we studied the properties of one Sm-like protein derived from the archaebacterium Archaeoglobus fulgidus and overexpressed in Escherichia coli. This single small protein closely reflects the properties of an Sm or Sm-like protein heteromer. It binds to RNA with a high specificity for oligo(U), and assembles onto the RNA to form a complex that exhibits, as judged by electron microscopy, a ring-like structure similar to the ones observed with the Sm core ribonucleoprotein and the like Sm (LSm) protein heteromer. Importantly, multivariate statistical analysis of negative-stain electron-microscopic images revealed a sevenfold symmetry for the observed ring structure, indicating that the proteins form a homoheptamer. These results support the structural model of the Sm proteins derived from crystallographic studies on Sm heterodimers and demonstrate that the Sm protein family evolved from a single ancestor that was present before the eukaryotic and archaeal kingdoms separated.

Human Synaptic Plasticity Gene Expression Profile and Dendritic Spine Density Changes in HIV-Infected Human CNS Cells: Role in HIV-Associated Neurocognitive Disorders (HAND)

HIV-associated neurocognitive disorders (HAND) is characterized by development of cognitive, behavioral and motor abnormalities, and occur in approximately 50% of HIV infected individuals. Our current understanding of HAND emanates mainly from HIV-1 subtype B (clade B), which is prevalent in USA and Western countries. However very little information is available on neuropathogenesis of HIV-1 subtype C (clade C) that exists in Sub-Saharan Africa and Asia. Therefore, studies to identify specific neuropathogenic mechanisms associated with HAND are worth pursuing to dissect the mechanisms underlying this modulation and to prevent HAND particularly in clade B infection. In this study, we have investigated 84 key human synaptic plasticity genes differential expression profile in clade B and clade C infected primary human astrocytes by using RT2 Profile PCR Array human Synaptic Plasticity kit. Among these, 31 and 21 synaptic genes were significantly (≥3 fold) down-regulated and 5 genes were significantly (≥3 fold) up-regulated in clade B and clade C infected cells, respectively compared to the uninfected control astrocytes. In flow-cytometry analysis, down-regulation of postsynaptic density and dendrite spine morphology regulatory proteins (ARC, NMDAR1 and GRM1) was confirmed in both clade B and C infected primary human astrocytes and SK-N-MC neuroblastoma cells. Further, spine density and dendrite morphology changes by confocal microscopic analysis indicates significantly decreased spine density, loss of spines and decreased dendrite diameter, total dendrite and spine area in clade B infected SK-N-MC neuroblastoma cells compared to uninfected and clade C infected cells. We have also observed that, in clade B infected astrocytes, induction of apoptosis was significantly higher than in the clade C infected astrocytes. In conclusion, this study suggests that down-regulation of synaptic plasticity genes, decreased dendritic spine density and induction of apoptosis in astrocytes may contribute to the severe neuropathogenesis in clade B infection.

Técnica para segmentação automática de imagens microscópicas de componentes sanguíneos e classificação diferencial de leucócitos baseada em lógica fuzzy

Automatic detection of blood components is an important topic in the field of hematology. The segmentation is an important stage because it allows components to be grouped into common areas and processed separately and leukocyte differential classification enables them to be analyzed separately. With the auto-segmentation and differential classification, this work is contributing to the analysis process of blood components by providing tools that reduce the manual labor and increasing its accuracy and efficiency. Using techniques of digital image processing associated with a generic and automatic fuzzy approach, this work proposes two Fuzzy Inference Systems, defined as I and II, for autosegmentation of blood components and leukocyte differential classification, respectively, in microscopic images smears. Using the Fuzzy Inference System I, the proposed technique performs the segmentation of the image in four regions: the leukocyte’s nucleus and cytoplasm, erythrocyte and plasma area and using the Fuzzy Inference System II and the segmented leukocyte (nucleus and cytoplasm) classify them differentially in five types: basophils, eosinophils, lymphocytes, monocytes and neutrophils. Were used for testing 530 images containing microscopic samples of blood smears with different methods. The images were processed and its accuracy indices and Gold Standards were calculated and compared with the manual results and other results found at literature for the same problems. Regarding segmentation, a technique developed showed percentages of accuracy of 97.31% for leukocytes, 95.39% to erythrocytes and 95.06% for blood plasma. As for the differential classification, the percentage varied between 92.98% and 98.39% for the different leukocyte types. In addition to promoting auto-segmentation and differential classification, the proposed technique also contributes to the definition of new descriptors and the construction of an image database using various processes hematological staining

Sistema de contagem com morfologia matemática fuzzy

Mathematical Morphology presents a systematic approach to extract geometric features of binary images, using morphological operators that transform the original image into another by means of a third image called structuring element and came out in 1960 by researchers Jean Serra and George Matheron. Fuzzy mathematical morphology extends the operators towards grayscale and color images and was initially proposed by Goetherian using fuzzy logic. Using this approach it is possible to make a study of fuzzy connectives, which allows some scope for analysis for the construction of morphological operators and their applicability in image processing. In this paper, we propose the development of morphological operators fuzzy using the R-implications for aid and improve image processing, and then to build a system with these operators to count the spores mycorrhizal fungi and red blood cells. It was used as the hypothetical-deductive methodologies for the part formal and incremental-iterative for the experimental part. These operators were applied in digital and microscopic images. The conjunctions and implications of fuzzy morphology mathematical reasoning will be used in order to choose the best adjunction to be applied depending on the problem being approached, i.e., we will use automorphisms on the implications and observe their influence on segmenting images and then on their processing. In order to validate the developed system, it was applied to counting problems in microscopic images, extending to pathological images. It was noted that for the computation of spores the best operator was the erosion of Gödel. It developed three groups of morphological operators fuzzy, Lukasiewicz, And Godel Goguen that can have a variety applications

Protein encapsulation and release from PEO-b-polyphosphoester templated calcium carbonate particles

International audience

Láser Q-Switched en el manejo de ocronosis exógena: revisión sistemática de la literatura

Antecedentes: La ocronosis Exógena (OE) es una enfermedad subdiagnosticada y de difícil manejo (1). El láser Q-Switched (QS) surge como una alternativa para el tratamiento de esta (2). Objetivo: Describir las características de los pacientes, del láser QS y los desenlaces en el tratamiento de OE. Métodos: Se realizó una búsqueda de la literatura en las bases PubMed, Embase, PMC, Scielo, Elselvier, BMJ Case Reports, Journal of Medical Case Reports, Cases Journal e International Medical Case Reports Journal, desde enero del 2000 a marzo del 2016, pacientes con ocronosis exógena, 18 a 70 años, tratados con láser QS. Los artículos fueron evaluados mediante la herramienta de evaluación de validez y valor educativo de reportes de caso descrito por Pierson (3). Resultados: Se encontraron 256 artículos, 63 fueron seleccionados: 28 repetidos y 31 no cumplieron criterios de inclusión. Se escogieron 4 artículos que reportan 12 casos de pacientes con ocronosis exógena diagnosticada mediante estudio histopatológico y tratada con láser QS. Discusión: Hay poca experiencia con el láser QS en OE. En la práctica clínica se usa para tatuajes y patologías pigmentarias dérmicas con resultados satisfactorios. El pigmento dérmico en OE y la corta duración de pulso de láser QS, podrían ser el pilar de tratamiento para OE. Conclusión: El láser QS puede ser útil para el tratamiento en OE, con nivel de evidencia 3 y grado de recomendación D. Se sugiere realizar estudios clínicos con mayor grado de evidencia.

Convex Total Variation Denoising of Poisson Fluorescence Confocal Images With Anisotropic Filtering

Fluorescence confocal microscopy (FCM) is now one of the most important tools in biomedicine research. In fact, it makes it possible to accurately study the dynamic processes occurring inside the cell and its nucleus by following the motion of fluorescent molecules over time. Due to the small amount of acquired radiation and the huge optical and electronics amplification, the FCM images are usually corrupted by a severe type of Poisson noise. This noise may be even more damaging when very low intensity incident radiation is used to avoid phototoxicity. In this paper, a Bayesian algorithm is proposed to remove the Poisson intensity dependent noise corrupting the FCM image sequences. The observations are organized in a 3-D tensor where each plane is one of the images acquired along the time of a cell nucleus using the fluorescence loss in photobleaching (FLIP) technique. The method removes simultaneously the noise by considering different spatial and temporal correlations. This is accomplished by using an anisotropic 3-D filter that may be separately tuned in space and in time dimensions. Tests using synthetic and real data are described and presented to illustrate the application of the algorithm. A comparison with several state-of-the-art algorithms is also presented.

A protocol for preparing GFP-labeled neurons previously imaged in vivo and in slice preparations for light and electron microscopic analysis.

In vivo imaging of green fluorescent protein (GFP)-labeled neurons in the intact brain is being used increasingly to study neuronal plasticity. However, interpreting the observed changes as modifications in neuronal connectivity needs information about synapses. We show here that axons and dendrites of GFP-labeled neurons imaged previously in the live mouse or in slice preparations using 2-photon laser microscopy can be analyzed using light and electron microscopy, allowing morphological reconstruction of the synapses both on the imaged neurons, as well as those in the surrounding neuropil. We describe how, over a 2-day period, the imaged tissue is fixed, sliced and immuno-labeled to localize the neurons of interest. Once embedded in epoxy resin, the entire neuron can then be drawn in three dimensions (3D) for detailed morphological analysis using light microscopy. Specific dendrites and axons can be further serially thin sectioned, imaged in the electron microscope (EM) and then the ultrastructure analyzed on the serial images.