Immunogold Labeling of Human Leukocytes for Scanning Electron Microscopy and Light Microscopy: Quantitative Aspects of the Methodology

When cell surface antigens are labeled with the colloidal gold marker, backscattered electron images (BEI) reveal all the gold particles and, therefore, permit total counts. Secondary electron images (SEI) show only a small percentage of the gold particles and are inadequate for quantitative evaluation. For determination of the cellular labelinq index, a time-consuming method implies the screening of 100 cells by scanninq electron microscopy, at a magnification of approximately 12,000 to 15,000x, with continuous SE/BE shifts. A much more efficient method is to transfer the SEM sample or its equivalent under the light microscope and to count the total number of gold labeled cells in the epi-polarization mode. The total cell count can be evaluated under UV light, taking advantage of the autofluorescence of the glutaraldehyde fixed cells

Morphology of clean and surfactant-laden droplets in homogeneous isotropic turbulence

We perform direct numerical simulations of surfactant-laden droplets in homogeneous-isotropic turbulence with Taylor Reynolds number $Re_\lambda\approx180$. Effects of surfactant on the droplet and local flow statistics are well approximated using a lower, averaged value of surface tension, allowing us to extend the framework developed by Kolmogorov (1949) and Hinze (1955) for surfactant-free bubbles to surfactant-laden droplets. We find the Kolmogorov-Hinze scale ($d_H$) is indeed a pivotal length scale in the droplets' dynamics, separating the coalescence-dominated and the breakage-dominated regimes in the droplet size distribution. We see that droplets smaller than $d_H$ have spheroid-like shapes, whereas larger droplets have long convoluted filamentous shapes with diameters equal to $d_H$. As a result, droplets smaller than $d_H$ have areas that scale as $d^2$, while larger droplets have areas that scale as $d^3$, where $d$ is the droplet equivalent diameter. We further characterise the filamentous droplets by computing the number of handles (loops of the dispersed phase extending into the carrier phase) and voids (regions of the carrier phase enclosed by the dispersed phase) on each droplet. The number of handles per unit length of filament ($0.06d_H^{-1}$) scales inversely with surface tension, while the number of voids is independent of surface tension. Handles are indeed an unstable feature of the interface and are destroyed by the restoring effect of surface tension, whereas voids can move freely inside the droplets.Comment: 31 pages, 13 figure

Immuno-Scanning Electron Microscopy of Normal and Leukemic Leukocytes Labeled with Colloidal Gold

The immunogold method, utilizing 40 nm colloidal gold particles which can be selectively visualized with the scanning electron microscope (SEM) in the backscattered electron imaging mode was used for the study of blood cells incubated with various monoclonal antibodies. Numerous antileukocyte monoclonal antibodies still recognize lightly glutaraldehyde prefixed antigens and can be used to identify various blood cell types and even to recognize their different maturation stages. Clearcut differences in surface morphology exist among peripheral blood normal leukocytes and even among the principal lymphocyte subclasses. Marked heterogeneity in surface morphology is, on the other hand, evident when studying precursors or leukemic cells. Immature cells show, nevertheless, relatively smooth surfaces while some distinct surface features appear on cells already committed toward a specific differentiation lineage. Hairy cells can also be precisely identified, especially when in small number in heterogeneous populations, combining their typical surface morphology with their positivity for B1 and Leu M5 monoclonal antibodies

Immuno-Cytochemistry with Backscattered Electrons

Some cytochemical reaction products are visible inside the cytoplasm of cells observed with the scanning electron microscope (SEM) using the backscattered electron imaging (BEI) mode. Methods can be utilized whenever they result in the deposition of heavy metal, like silver, lead or osmium at the sites of the enzymatic reaction. More recently the BEI mode of the SEM has been demonstrated to improve the detection of immunogold labeled cell surface antigens. Colloidal gold particles, 40 to 15 nm in diameter can be efficiently used for immuno-specific labeling. Moreover, cytochemical reactions can be applied to previously immunogold labeled cells, therefore combining the results of enzyme cytochemistry and of surface labeling at the level of each individual cell. The choice of fixative, incubation media, dehydration and drying methods should be guided by considerations on the sample characteristics for optimal electron scattering. Cytochemical as well as immuno-labeling reactions are not used per se but in combination with the study of cell surface morphology which needs, therefore, to be sufficiently well preserved. Coating should provide good conductivity and secondary electron emission, while emitting a minimal number of backscattered electrons. The application of these methods considerably enhances our capacity to characterize with the SEM the surface morphology of precisely identified subpopulations of many cell types

Scanning Electron Microscope Cytochemistry of Blood Cells

The backscattered electron imaging (BEI) mode of scanning electron microscopy (SEM) has been applied to study various histo-cytochemical reactions in biological specimens since the early seventies. Due to numerous, recent technical improvements the BEI mode of SEM now belongs to the routine of many SEM laboratories. For cytochemistry, BEI has been mainly used to: visualize intracellular structures and organelles; recognize the different cell types in heterogeneous populations or tissues; study the correlations between enzymatic activities and cell surface features. We have evaluated the most relevant results obtained in the study of blood cells and the possible future applications of these techniques

Cell Surface Changes of Hemopoietic Cells During Normal and Leukemic Differentiation: An Immuno-Scanning Electron Microscopy Study

Hemopoietic cells display a wide range of cell surface antigens which are either lineage specific or acquired during differentiation. Monoclonal antibodies can be used, in conjunction with colloidal gold markers, to identify under the scanning electron microscopy (SEM) at the single cell level, specific lineage or maturation stages in the hemopoietic bone marrow. Normal bone marrow cells, either gradient separated or purified by immuno-magnetic methods and leukemic cell samples, which can be considered as frozen stages of hemopoietic differentiation, have been studied with this method. Typical cell surface morphologies, which characterize immature progenitor cells and cells committed or differentiated towards the lymphoid, myeloid, erythroid and megakaryocytic lineage have been identified. Correlations between cell surface features and some hemopoietic cells functions have been attempted on the basis of these findings

Ocorrência de giberela nos genótipos de trigo do ensaio de VCU da Embrapa - ano 2012.

Editores técnicos: Joseani Mesquita Antunes, Ana Lídia Variani Bonato, Márcia Barrocas Moreira Pimentel

Germinação e vigor de sementes de trigo inoculadas com Azospirillum brasilense.

Editores técnicos: Joseani Mesquita Antunes, Ana Lídia Variani Bonato, Márcia Barrocas Moreira Pimentel

Caracterização fenológica da coleção brasileira de trigo em Passo Fundo - RS.

Editores técnicos: Joseani Mesquita Antunes, Ana Lídia Variani Bonato, Márcia Barrocas Moreira Pimentel

Distribution in the brain and possible neuroprotective effects of intranasally delivered multi-walled carbon nanotubes

Carbon nanotubes (CNTs) are currently under active investigation for their use in several biomedical applications, especially in neurological diseases and nervous system injury due to their electrochemical properties. Nowadays, no CNT-based therapeutic products for internal use appear to be close to the market, due to the still limited knowledge on their fate after delivery to living organisms and, in particular, on their toxicological profile. The purpose of the present work was to address the distribution in the brain parenchyma of two intranasally delivered MWCNTs (MWCNTs 1 and a-MWCNTs 2), different from each other, the first being non electroconductive while the second results in being electroconductive. After intranasal delivery, the presence of CNTs was investigated in several brain areas, discriminating the specific cell types involved in the CNT uptake. We also aimed to verify the neuroprotective potential of the two types of CNTs, delivering them in rats affected by early diabetic encephalopathy and analysing the modulation of nerve growth factor metabolism and the effects of CNTs on the neuronal and glial phenotypes. Our findings showed that both CNT types, when intranasally delivered, reached numerous brain areas and, in particular, the limbic area that plays a crucial role in the development and progression of major neurodegenerative diseases. Furthermore, we demonstrated that electroconductive MWCNTs were able to exert neuroprotective effects through the modulation of a key neurotrophic factor and probably the improvement of neurodegeneration-related gliosis