Biomedical subjects

Jirí Janácek

Publications and source records attributed to Jirí Janácek.

8 recordsLinked to original sources

PubMed2006

Methods for compensation of the light attenuation with depth of images captured by a confocal microscope.

A confocal laser scanning microscope (CLSM) enables us to capture images from a biological specimen in different depths and obtain a series of precisely registered fluorescent images. However, images captured from deep layers of the specimen may be darker than images from the topmost layers because of light loss distortions. This effect causes difficulties in subsequent analysis of biological objects. We propose a solution using two approaches: either an online method working already during image acquisition or an offline method assisting as a postprocessing step. In the online method, the gain value of a photomultiplier tube of a CLSM is controlled according to the difference of mean image intensities between the reference and currently acquired image. The offline method consists of two stages. In the first stage, a standard histogram maintaining relative frequencies of gray levels and improving brightness and contrast is created from all images in the series. In the second stage, individual image histograms are warped according to this standard histogram. The methods were tested on real confocal image data captured from human placenta and rat skeletal muscle specimens. It was shown that both approaches diminish the light attenuation in images captured from deep layers of the specimen.

Animals↗

PubMed2005

Nuclear distribution of actin and myosin I depends on transcriptional activity of the cell.

As previous studies suggested, nuclear myosin I (NMI) and actin have important roles in DNA transcription. In this study, we characterized the dynamics of these two proteins during transcriptional activation in phytohemagglutinin (PHA) stimulated human lymphocytes. The stimulation led to strong up-regulation of NMI both on the mRNA and protein level, while actin was relatively stably expressed. The intranuclear distribution of actin and NMI was evaluated using immunogold labeling. In nucleoli of resting cells, actin was localized predominantly to fibrillar centers (FCs), while NMI was located mainly to the dense fibrillar component (DFC). Upon stimulation, FCs remained the main site of actin localization, however, an accumulation of both actin and NMI in the DFC and in the granular component was observed. In the nucleoplasm of resting lymphocytes, both actin and NMI were localized mostly in condensed chromatin. Following stimulation, the majority of both proteins shifted towards the decondensed chromatin. In transcriptionally active cells, both actin and NMI colocalized with nucleoplasmic transcription sites. These results demonstrate that actin and NMI are compartmentalized in the nuclei where they can dynamically translocate depending on transcriptional activity of the cells.

Actins↗

PubMed2005

Three-dimensional reconstructions from non-deparaffinized tissue sections.

Three-dimensional (3-D) reconstruction from microscopic images represents a useful tool for the study of biological structures in embryology and developmental biology. However, it is usually necessary to cope with many difficulties connected with the preparation of specimens. In order to minimize mutual displacement of structures in successive sections, the applicability of non-deparaffinized tissue sections for 3-D reconstruction was tested. Chicken embryos were fixed and stained in toto with eosin and then embedded in paraffin. About 30-mum-thick non-deparaffinized serial sections were used for obtaining initial data for 3-D reconstruction of larger stacks of embryonic bodies using either fluorescence or confocal microscope. The same sections served for both collecting optical serial sections of mesonephros as source images for its 3-D reconstruction, and immunohistochemical detection of fibronectin, laminin and vimentin. It was found that sections with retained paraffin preserve the mutual spatial relationships of tissue components as well as provide an excellent differentiation of structure. It makes the process of 3-D reconstruction easier. The localization of the products of immunohistochemical reactions demonstrated the co-localization of fibronectin and laminin in basal laminas and the presence of vimentin in glomeruli and mesenchymal tissue. The use of non-deparaffinized sections represents a less time consuming and more effective alternative to thin histological sections for the purpose of 3-D reconstruction, and enables further application of material.

Animals↗

PubMed2004

Mapping of cellular compartments based on ultrastructural immunogold labeling.

Ultrastructural identification of subcellular morphologically inconspicuous compartments is based on detection of specific molecules or by a presence of specific functions. Such compartments are detected using antibodies with attached label, usually gold particles. However, the gold particles have a point pattern, while a compartment is a coherent area. In addition, some background labeling is always present that complicates identification of the labeled compartments. The aim of this study was therefore to develop a stereological method that would enable us to define cellular compartments based on delineating the borders of gold particle clusters, and to test the practical use of the method using biological experimental data. New computer program plug-ins were developed to facilitate the practical use of the stereological method. The kernel estimation method was successfully tested by detection of ribosomal rRNA over morphologically recognizable nucleoli. In a next step, we successfully detected individual chromosomal domains-nuclear compartments that cannot be distinguished in cell nuclei morphologically. The results show that the new stereological/image analysis method is well able to discriminate cellular compartments based on density of immunogold particles. The plug-ins were made available to scientific community at http://nucleus.biomed.cas.cz/gold.

Cell Compartmentation↗

PubMed2004

Dynamics of DNA replication: an ultrastructural study.

DNA replication in cells takes place in domains scattered throughout the nucleoplasm. We have characterized the dynamics of DNA synthesis in synchronized mid-S-phase HeLa cells. Saponin-permeabilized cells were allowed to elongate nascent DNA chains in presence of biotin-dUTP for 5, 15, and 30 min (a pulse experiment), or for 5 min followed by an incubation with unlabeled precursors for 10 or 25 min (a pulse-and-chase experiment). The replication foci were then identified in ultrathin sections using immunogold labeling of the incorporated biotin. Total number of particles per nucleus, total scanned area of the nucleus, size, shape, and gold particle number of each labeled cluster, and the density of clusters per nucleus were evaluated. We have demonstrated that as replication proceeds, the labeled sites increase in size up to 240 nm (30 min incorporation) while maintaining a broadly round shape. In pulse-and-chase experiments the labeled DNA was shown to spread to occupy DNA foci of approximately 400 nm in diameter. These results demonstrate that DNA replication is compartmentalized within cell nuclei at the level of DNA foci and support the view that the synthetic centers are spatially constrained while the chromatin loops are dynamic during DNA synthesis.

Binding Sites↗

PubMed2003

Three-dimensional computer reconstruction of large tissue volumes based on composing series of high-resolution confocal images by GlueMRC and LinkMRC software.

Computer-based visualization of large tissue volumes with high resolution based on composing series of high-resolution confocal images is presented. GlueMRC and LinkMRC programs are introduced, implementing composition of overlapping series of optical sections captured by a confocal microscope, registration and subsequent composition of successive confocal stacks. Both programs are using an interactive approach in combination with automatic algorithms for image registration. Further, the method for obtaining surface renderings of microscopical structure under study is described. For this purpose, structure contours visible in the sections are interactively digitized using a Colon plug-in module running in Ellipse environment. Then the coordinates of the contours are processed by special modules in the graphic programming environment IRIS Explorer and the structure surface is rendered. The method is shown on the 3-D reconstruction of the capillary bed of human placental villi and chick embryonic gut and its vascular bed.

Animals↗

PubMed2003

Stereology techniques in radiation biology.

Clinicians involved in conventional radiation therapy are very concerned about the dose-response relationships of normal tissues. Before proceeding to new clinical protocols, radiation biologists involved with conformal proton therapy believe it is necessary to quantify the dose response and tolerance of the organs and tissues that will be irradiated. An important focus is on the vasculature. This presentation reviews the methodology and format of using confocal microscopy and stereological methods to quantify tissue parameters, cell number, tissue volume and surface area, and vessel length using the microvasculature as a model tissue. Stereological methods and their concepts are illustrated using an ongoing study of the dose response of the microvessels in proton-irradiated hemibrain. Methods for estimating the volume of the brain and the brain cortex, the total number of endothelial cells in cortical microvessels, the length of cortical microvessels, and the total surface area of cortical microvessel walls are presented step by step in a way understandable for readers with little mathematical background. It is shown that stereological techniques, based on a sound theoretical basis, are powerful and reliable and have been used successfully.

Animals↗

PubMedDate not supplied

Topological properties and spatial organization of villous capillaries in normal and diabetic placentas.

Spatial arrangement and complexity of the capillary bed of placental terminal villi were analyzed in 9 normal and 11 diabetic placentas. Specimens were taken by systematic random sampling, fixed and stained in toto, and embedded in paraffin. Fifteen fields of view were sampled systematically from 120-microm-thick sections of specimens and examined using a confocal laser scanning microscope. Series of thin optical sections of terminal villi and their developmental forms were recorded by the confocal microscope and used as initial data for three-dimensional visualization of the spatial arrangement of villous capillaries. Vascular topology and branching were studied by focusing through the villus, making a schematic drawing of the villous capillary bed and counting redundant capillary connections. It was found that the basic arrangement of villous capillaries is similar in both normal and diabetic placentas. Nevertheless, the proportion of simple forms of the capillary bed without redundant connections is significantly higher in normal placentas and the mean number of redundant connections per villus is significantly higher in diabetic placentas. It is concluded that both the longitudinal growth and branching of capillaries contribute to the increase in the placental capillary bed in late gestation and that the capillary bed of diabetic villi is more complicated due to more intense capillary branching.

Adult↗