Visualization of BrdU-labelled DNA in cyanobacterial cells by Hilbert differential contrast transmission electron microscopy

We have attempted to observe the native shape of DNA in rapidly frozen whole cyanobacterial cells through 5-bromo-2-deoxyuridine (BrdU) incorporation and visualization with a Hilbert differential contrast transmission electron microscopy (HDC TEM). The incorporation of BrdU into the DNA of Synechococcus elongatus PCC 7942 was confirmed with fluorescently labelled anti-BrdU antibodies and through EDX analysis of ultra-thin sections. HDC TEM observed cells that had incorporated BrdU into their DNA exhibited electron dense areas at the location corresponding to fluorescently labelled BrdU. Since various strings and strands were observed in high contrast with the HDC TEM, we conclude that the method promises to allow us to identify and understand bulk structural changes of the in vivo DNA and the nucleoid through observation at high resolution.     

Comparative study of DNA synthesis and nucleolar organizer regions of sinusoid littoral cells in mouse regenerating liver.

Variations in DNA synthesis (DNAs) and Nucleolar Organizer Regions (NORs) were studied in the littoral cell population from regenerating liver of C3HS inbred mice standardized for periodicity analysis. Immunohistochemical detection of Bromodeoxyuridine (BrdU) with a monoclonal antibody and silver staining of NORs (AgNORs) were assessed by means of a digital image analysis system in histological sections. Tissue samples were obtained every four hours from the 30th to the 54th hours after a partial hepatectomy. The results showed, in both parameters, a gradual increment of the values during the period studied, with highest values (DNAs 107.1 +/- 16.1 SE; AgNORs 77.3 +/- 3.4 SE) located at 16:00/54 Time of Day / Hours Post-Hepatectomy (TD/HPH), which were significantly different (p <0.001) from the values of the first sample (DNAs 38.1 +/- 9.5 SE; AgNORs 27.3 +/- 1.0 SE) taken at 16:00/30 TD/HPH. The results of our experiment demonstrate the existence of a strong correlation of DNA synthesis measured by BrdU immunohistochemistry and AgNORs numbers in sinusoid littoral cells from mouse regenerating liver.     

Visualization of the native shape of bodipy‐labeled DNA in Escherichia coli by correlative microscopy

The native shape and intracellular distribution of newly synthesized DNA was visualized by correlative (light and electron) microscopy in ice embedded whole cells of Escherichia coli. For that purpose, the commercially available modified nucleoside triphosphate named BODIPY® FL‐14‐dUTP was enzymatically incorporated in vivo into the genome of E. coli mutant K12 strain, which cannot synthesize thymine. The successful incorporation of this thymidine analogue was confirmed first by fluorescence microscope, where the cells were stained in the typical for bodipy green color. Later the preselected labeled E. coli were observed by Hilbert Differential Transmission Electron Microscope (HDC TEM) and the distribution of elemental boron (contained in bodipy) was visualized at high‐resolution by an electron spectroscopic imaging (ESI) technique. The practical detection limit of boron was found to be around 5 ～ 10 mmol/kg in area of 0.1 μm², which demonstrated that ESI is a suitable approach to study the cytochemistry and location of labeled nucleic fragments within the cytoplasmic chromosomal area. In addition, the fine cellular fibrous and chromosomal ultrastructures were revealed in situ by combing of phase‐plate HDC TEM and ESI. The obtained results conclude that the correlation between fluorescent microscopy with phase‐plate HDC TEM and ESI is a powerful approach to explore the structural and conformation dynamics of DNA replication machinery in frozen cells close to the living state.     

Immunolocalization indicates that both original and regenerated lizard tail tissues contain populations of long retaining cells,putative stem/progenitor cells

The regeneration of the tail in lizards is likely sustained by stem/progenitor cells located in the stump after amputation of the tail. This microscopic and ultrastructural study shows the localization of 5‐bromo‐deoxy‐uridine (5BrdU)‐long retaining labeled cells in different tissues of the tail stump. These putative stem/progenitor cells are sparsely detected in the epidermis of scales, adipose tissue, intermuscle connective septa, myosatellite cells, and perichondrion of the vertebrae. Most of 5BrdU‐labeled cells are present in the bone marrow of vertebrae as hemocytoblasts and reticulate cells, whereas more numerous myelocytes and polychromatophilic erythroblasts show a variable level of nuclear labeling. 5BrdU and tritiated‐thymidine labeled and unlabeled hemopoietic cells are seen in circulating vessels and in the blastema where their maturation is completed. This observation indicates that the entire differentiation span of both white and red blood cells, at least during tail regeneration, lasts longer than 4 weeks. Labeled polychromatophilic erythroblasts and heterophilic and basophilic myelocytes are present in the synusoidal vessels of the regenerating tail. This study indicates that extravasating blood cells involved in immunity make large part of the forming blastema cell population, but are replaced by mesenchymal cells of different origin. The presence of long retaining labeled cells in tissues of the tail stump is likely connected to the production of blastema mesenchymal cells. Although no direct cell‐lineage study has been done, histological, immunocytochemical, and autoradiographic studies have indicated that it is from these tissues that proliferating cells appear mainly localized after tail amputation and blastema formation. Microsc. Res. Tech. 78:1032–1045, 2015. © 2015 Wiley Periodicals, Inc.     

Enhanced immunological detection of epigenetic modifications of DNA in healthy and cancerous cells by fluorescence microscopy

Epigenetic modifications of DNA, including methylation, hydroxymethylation, formylation, and carboxylation of cytosines, are proposed to function in gene regulation during reproduction and development. Changes in cytosine methylation are associated with a range of diseases, such as cancer. Immunofluorescence uses specific antibodies to quantitatively detect the global amount of cytosine modifications by fluorescence microscopy. The most critical stage of immunofluorescence is the antigen retrieval to remove the protein content around the DNA, allowing specific antibodies to bind to DNA epitopes. Acid treatments have commonly been used for antigen retrieval. Previously, trypsin was added after acid in the protocol, which increased the amount of detectable DNA methylation. In this study, the protocol was further enhanced by the addition of pepsin, which is able to target charged hydrophobic amino acids in proteins, unlike trypsin, which breaks positive hydrophilic amino acids. The global levels of cytosine modifications in CF‐1, HeLa, and AR42J cells were compared using this protocol. In all cells, the sequential treatment of trypsin and pepsin increased the specificity of the staining. With the synergistic effect of the two enzymes, it is possible to target different protein groups packaging DNA molecules and removing them effectively. The findings suggest that this revised protocol can be conveniently used for each cytosine modification in the cells examined, and should be optimized for other cells. These new antigen retrieval conditions may more accurately detect the changes in cytosine modifications during development and in diseases.     

Nucleotide and protein distribution in BrdU-labelled polytene chromosomes revealed by ion probe mass spectrometry

Detailed chemical maps of BrdU-labelled polytene chromosomes of Drosophila melanogaster, obtained by imaging secondary ion mass spectrometry, reveal separately the distribution of DNA and proteins in the chromosomes. The thymidine-analogue BrdU within the chromosomal DNA is localized by detecting the Br^? secondary ion signal, while both nucleic acid and protein content are mapped through the abundantly emitted CN^? signal. This novel approach supercedes, and helps explain the origin of, the banding patterns that are observed by conventional staining techniques. The high spatial resolution and chemical and isotopic sensitivity of this technique should enhance the localization of specific genes by in situ hybridization in mitotic chromosomes.     

Cell cycle behavior and MyoD expression in response to T3 differ in normal and mdx dystrophic primary muscle cell cultures

Since mdx limb muscle regeneration in vivo is accompanied by rapid myoblast proliferation and differentiation compared to normal, we tested the possibility that proliferation and differentiation were differentially regulated in normal and mdx dystrophic muscle cells. Cell cycle behavior, MyoD expression, and the effects of thyroid hormone (T3) treatment were examined in primary cultures. Using a 4-hour pulse time for bromodeoxyuridine (BrdU) incorporation during S-phase, the phases of the cell cycle (early S, late S, G(2)/M, and G(0)/G(1)) were separated by 2-colour fluorescence (BrdU/PI) analysis using flow cytometry. The G(0)/G(1)-early S and the late S-G(2)/M transitions were examined under the influence of T3 in cycling normal and mdx muscle cell cultures over a 20-hour time period. Myogenesis and differentiation were assessed morphologically and by immunostaining for MyoD protein. Mdx cultures had fewer cells in G(0)/G(1) at 20 hours and more cells in early and late S-phase compared to normal cultures. T3 significantly increased the proportion of normal cells in early S-phase by 20 hours, and reduced the proportions in G(2)/M phase. Over the same time interval in parallel cultures, the proportion of MyoD+ normal cells decreased significantly. In the absence of T3, mdx cell cultures showed greater proportions of cells in S-phase than normal cultures, and similar increases in S-phase and loss of MyoD expression over time. However, mdx cultures had no change in the proportion that were MyoD+ during T3 treatment. The results confirm that T3 in primary cultures increased proliferation and prevented the de-differentiation of mdx cells to a greater degree than was typical of normal cells. The different susceptibilities to T3-related shifts between proliferation and differentiation observed in vitro support the idea that committed mdx myoblasts may be more activated and proliferative than normal myoblasts during regeneration in vivo.     

Carotid body chemoreceptors in dissociated cell culture

Carotid body (CB) glomus or type 1 cells act as peripheral chemoreceptors which detect changes in arterial PO(2), PCO(2), and pH and help maintain homeostasis via the reflex control of ventilation. Over the last approximately 12 years significant progress has been made towards understanding chemotransduction mechanisms using freshly isolated or cultured type 1 cells. The latter preparation allows several powerful experimental manipulations (e.g., co-culture with sensory neurons) resulting in significant advances in our understanding of CB chemoreception. Here, we review several properties of type 1 cells after several days to weeks in culture. Typically, cultured type 1 cells grow in monolayer clusters enveloped by glial-like, type II, or sustentacular cells, which are immunopositive for the glial marker, glial fibrillary acid protein (GFAP). These cells can undergo DNA synthesis, evidenced by uptake of bromodeoxyuridine (BrdU), and show a limited capacity for cell division. Mitosis and survival of type 1 cells can be regulated by oxygen tension and/or growth factors (e.g., bFGF, insulin). In the rat, type 1 cells are immunopositive for several monoaminergic markers, including tyrosine hydroxylase (TH), dopamine transporter (DAT), and 5-HT. They also express cholinergic markers (e.g., vesicular acetylcholine transporter; VAChT), the highly conserved synaptic vesicle protein (SV2), and gap junctional proteins including Connexin 32 (Cx32). Moreover, in long-term culture ( approximately 2 weeks) they retain expression of O(2)-sensitive, TASK-1-like, and Ca(2+)-dependent (BK), K(+) channels as revealed by immunocytochemistry or RT-PCR analysis of mRNA extracted from type 1 clusters after removal from the culture surface.     

From egg to pole cells: ultrastructural aspects of early cleavage and germ cell determination in insects.

Insect eggs are giant and very complex cells covered by an extremely resistant shell. Both the egg cell and surrounding eggshell express anteroposterior and ventrodorsal polarity. The molecular and cytoplasmic organization of both axes originates during oogenesis and leads to the production of an ooplasmic system which consists of euplasm and deutoplasm (yolk) and contains a nucleus as well as extranuclear determinants of maternal origin. Both are part of the store of information for early embryogenesis. In addition, the deutoplasm serves as raw material and early nutrient supply for building the embryo. The insect egg cell, which is arrested in the first maturation division when released from the ovary during oviposition, will be activated by different stimuli among different species to complete meiosis and start embryogenesis. The zygote nucleus undergoes a number of synchronous mitotic divisions leading to cleavage energids which initially form a syncytial blastoderm and subsequently the cellular blastoderm. In many insects, prior to blastoderm formation, polar granules (or oosome material) are incorporated in a single cell or a small number of cells which bud off at the posterior pole. These so called pole cells give rise to the primordial germ cells. Therefore, polar granules or the oosome material mark the germ line, and while structural counterparts of determinants of body pattern formation have so far not been found, the polar granules or oosome serve as an autonomous ooplasmic determinant for the pole or germ cells. Anteroposterior body polarity can arise independent of the germ plasm.     

A p-type finite element solution for the simulation of O2 transport in articular cartilage tissue: heterogeneous and porous media

The partial pressure of oxygen (pO2) is suggested to have a regulatory effect on chondrocyte biosynthetic activities, and its effect during expansion is unknown. The authors hypothesize that oxygen tension due to mechanical deformation or swelling could be as important as direct mechanical effects on cell biosynthetic activities. While there are plenty of studies on measuring and/or modelling pO2 in articular cartilage (AC) for static (rest) conditions, to the best of the authors' knowledge there are very few such studies on pO2 in AC for dynamic conditions such as swelling or tissue deformation. In this study, it is attempted to develop a model to study the dynamics of oxygen transport in AC. A high-precision hybrid element is designed using the p-type finite element method, by which diffusion and convection are incorporated as a single element. A domain decomposition method is used that allows the use of a different type of discretization with independent discretization variables in non-overlapping sub-domains, for a generic three-dimensional approach to elliptic boundary value problems of order 2 or higher. The formulation developed in this study might be used in determining the necessary flow conditions to cultivate tissue constructs in tissue repair and tissue engineering.     

Reliability estimation for cutting tools based on logistic regression model using vibration signals

As an important part of CNC machine, the reliability of cutting tools influences the whole manufacturing effectiveness and stability of equipment. The present study proposes a novel reliability estimation approach to the cutting tools based on logistic regression model by using vibration signals. The operation condition information of the CNC machine is incorporated into reliability analysis to reflect the product time-varying characteristics. The proposed approach is superior to other degradation estimation methods in that it does not necessitate any assumption about degradation paths and probability density functions of condition parameters. The three steps of new reliability estimation approach for cutting tools are as follows. First, on-line vibration signals of cutting tools are measured during the manufacturing process. Second, wavelet packet (WP) transform is employed to decompose the original signals and correlation analysis is employed to find out the feature frequency bands which indicate tool wear. Third, correlation analysis is also used to select the salient feature parameters which are composed of feature band energy, energy entropy and time-domain features. Finally, reliability estimation is carried out based on logistic regression model. The approach has been validated on a NC lathe. Under different failure threshold, the reliability and failure time of the cutting tools are all estimated accurately. The positive results show the plausibility and effectiveness of the proposed approach, which can facilitate machine performance and reliability estimation.     

Fixed structure compensator design using a constrained hybrid evolutionary optimization approach

This paper presents an efficient technique for designing a fixed order compensator for compensating current mode control architecture of DC−DC converters. The compensator design is formulated as an optimization problem, which seeks to attain a set of frequency domain specifications. The highly nonlinear nature of the optimization problem demands the use of an initial parameterization independent global search technique. In this regard, the optimization problem is solved using a hybrid evolutionary optimization approach, because of its simple structure, faster execution time and greater probability in achieving the global solution. The proposed algorithm involves the combination of a population search based optimization approach i.e. Particle Swarm Optimization (PSO) and local search based method. The op-amp dynamics have been incorporated during the design process. Considering the limitations of fixed structure compensator in achieving loop bandwidth higher than a certain threshold, the proposed approach also determines the op-amp bandwidth, which would be able to achieve the same. The effectiveness of the proposed approach in meeting the desired frequency domain specifications is experimentally tested on a peak current mode control dc−dc buck converter.     

Metabolic labeling of Escherichia coli genomic DNA with erythrosine‐11‐dUTP for functional imaging via correlative microscopy

The fluorescent metabolic labeling of microorganisms genome is an advanced imaging technique to observe and study the native shapes, structural changes, functions, and tracking of nucleic acids in single cells or tissues. We have attempted to visualize the newly synthesized DNA within the intact nucleoid of ice‐embedded proliferating cells of Escherichia coli K‐12 (thymidine‐requiring mutant, strain N4316) via correlative light‐electron microscopy. For that purpose, erythrosine‐11‐dUTP was synthesized and used as a modified analog of the exogenous thymidine substrate for metabolic incorporation into the bacterial chromosome. The formed fluorescent genomic DNA during in cellulo polymerase reaction caused a minimal cellular arrest and cytotoxicity of E. coli at certain controlled conditions. The stained cells were visualized in typical red emission color via an epifluorescence microscope. They were further ice‐embedded and examined with a Hilbert differential contrast transmission electron microscopy. At high‐resolution, the ultrastructure of tagged nucleoid appeared with significantly higher electron dense in comparison to the unlabeled one. The enhanced contrast areas in the chromosome were ascribed to the presence of iodine contents from erythrosine dye. The presented labeling approach might be a powerful strategy to reveal the structural and dynamic changes in natural DNA replication including the relationship between newly synthesized in vivo nucleic acid and the physiological state of the cell.     

Apoptotic DNA fragmentation can be revealed in situ: An ultrastructural approach

A common pattern of apoptotic death is DNA cleavage, initially producing large fragments (50 kbp), followed by the production of nucleosomic/oligonucleosomic fragments. Nevertheless, apoptosis without DNA fragmentation, at least of the nucleosomic type, has been reported. To investigate the spatial relationship between DNA cleavage and chromatin condensation, we applied the TUNEL technique to the ultrastructural analysis of apoptotic cells. A modified method, utilizing a gold‐conjugated antidigoxigenin antibody, was carried out on U937 versus Molt‐4 cells, both exposed to UVB radiation or staurosporine treatment. Gold particle density in the different domains of apoptotic cells was evaluated by a four‐way ANOVA test. Gold labelling was more strongly localised in condensed chromatin than in the diffuse chromatin. U937 cells, which evidenced in vitro oligonucleosomic fragmentation after both UVB and staurosporine treatments, revealed a significantly higher gold particle density, when compared with Molt‐4, which did not show, on the other hand, oligonucleosomic cleavage even in the presence of ≤50 kbp cleavage. Thus, a correlation between DNA fragment sizes and gold particle density appears. TUNEL applied to electron microscopy is an effective approach to study the relationship between apoptotic chromatin condensation and DNA cleavage. Both these events indeed appear in the apoptotic nucleus, but their reciprocal correlation is still greatly unknown. Microsc. Res. Tech. 2009. © 2009 Wiley‐Liss, Inc.     

AFM imaging in solution of protein-DNA complexes formed on DNA anchored to a gold surface

A chemical procedure for anchoring DNA molecules to gold surfaces was used to facilitate the imaging of DNA and DNA-protein complexes in buffer solution by tapping mode atomic force microscopy (TMAFM). For preparing flat gold surfaces, a novel approach was employed by evaporating small amounts of gold onto freshly cleaved mica to give flat films that were stable under aqueous buffer conditions. The thickness of the investigated films ranged from 1 to 10 nm. For typical films of 4-6 nm, which were stable under aqueous buffer conditions, the root mean square (RMS) roughness ranged between 0.25 and 0.5 nm, as measured by atomic force microscopy (AFM). This roughness is comparable to that of obtained by the template stripped gold (TSG) technique, which is widely used in scanning probe microscopy but involves more preparation steps. In order to visualize DNA and DNA-protein complexes by TMAFM, the DNA was chemisorbed to the gold surface through a linker carrying a terminal thiol group at the 5'-end of each of the DNA strands. The modified DNA fragments were bound to the gold films and imaged in buffer solution, while unmodified DNA could not be visualized. Since the DNA was not dried during the process, it can be assumed that its native conformation was retained. This mode of anchoring did not prevent interaction with proteins, as confirmed by the observation that the topology of a complex formed by adding the protein to a surface-anchored DNA was the same as that obtained by anchoring a pre-formed complex to the gold surface. We attribute this observation to the fact that the DNA is anchored to the gold surfaces only through its ends, therefore the DNA-support interaction is minimized but imaging is still possible.     

Probing DNA structure and function with a multiwavelength fluorescence confocal laser microscope

Three levels of organization in DNA structure in the interphase cell nucleus are assessed by confocal laser scanning microscopy: (i) the conformational state of the double helix; (ii) the distribution of eu- and heterochromatin; and (iii) the localization of replication complexes throughout S phase. Multi-parameter measurements were carried out in each optical section using two laser sources and combined stereoscopic reconstructions were used to assess the co-localization of nuclear components. DNA is highly polymorphic and can adopt a variety of different helical conformations as well as unusual structures (curved, cruciform, multi-stranded). We have assessed by laser scanning microscopy the presence of left-handed Z-DNA in polytene chromosomes of Diptera as well as the spatio-temporal distribution of Z-DNA binding proteins in whole-mount Drosophila embryos and ovaries. We have determined the 3-D distribution of replication sites relative to heterochromatin regions, nucleoli and nuclear membrane by using short pulses of BrdU incorporation in synchronized mouse and human fibroblasts. Replication sites were visualized with a monoclonal anti-BrdU antibody combined with DNA fluorescent staining and antibody labelling of nuclear lamin. The implications of dynamic DNA movement and structural rearrangement to the organization of the nucleus in domains are discussed.     

Signal standardization of the secondary ion mass spectrometry (SIMS) microscope for quantification of halogens and calcium in biological applications

The secondary ion mass spectrometry (SIMS) microscope is able to map chemical elements in tissue sections. Although absolute quantification of an element remains difficult, a relative quantitative approach is possible for soft tissue by using carbon (¹²C) as an internal reference present at large homogeneous and constant concentration in specimen and embedding resin. In this study, this approach is used to standardize the signal of an SIMS microscope for the quantification of halogens (¹⁹F^—, ³⁵Cl^— and ⁷⁹Br^—) and calcium (⁴⁰Ca⁺). Standard preparation was determined based on homogeneity and stability criteria by molecular incorporation (halogens) or mixing (calcium) in methacrylate resin. Standard measurements were performed by depth analysis on areas of 8 μm (halogens) and 150 μm (calcium) in diameter for 10–30 min, under Cs⁺ (halogens) or O₂⁺ (calcium) bombardment. Results obtained from 100–120 measurements for each standard dilution show that the relationship between the signal intensity measured and the elemental concentration (μg/mg of wet tissue or mm ) is linear in the range of biological concentrations. This quantitative approach was applied firstly to bromine of the 5-bromo-2′-deoxyuridine (BrdU) used as nuclear marker of rat hepatocytes in proliferation. The second model concerns depletion of calcium concentration in cortical compartment in Paramecium tetraurelia during exocytosis. Then signal standardization in SIMS microscopy allows us to correlate quantitative results with those obtained from other methods.     

Application of photoconversion technique for correlated confocal and ultrastructural studies in organotypic slice cultures

Photoconversion of fluorescent staining into stable diaminobenzidine (DAB) precipitate is widely used for neuroanatomical and developmental studies. An important advantage of the approach is to make correlations between light and electron microscopy analyses possible, the DAB reaction product formed during photoconversion being electron dense. By combining a photoconversion approach with biolistic transfection of neurons in organotypic hippocampal slice cultures, we describe here a methodology that allowed us to study at the electron microscopy level the fine details of cells expressing specific genes of interest. The same approach has also been used to analyze the ultrastructural characteristics of specific cells such as neurons recorded with patch clamp techniques. This approach revealed particularly useful for studies of dendritic arborisation, dendritic spines, and axon varicosities of identified cells, as precise morphometric parameters of these structures can only be obtained by electron microscopy. The techniques used for fluorescent staining and photoconversion of these different cell structures and the results obtained by electron microscopic analyses are described.     

Cloud‐based decision support system for the detection and classification of malignant cells in breast cancer using breast cytology images

The advancement of computer‐ and internet‐based technologies has transformed the nature of services in healthcare by using mobile devices in conjunction with cloud computing. The classical phenomenon of patient–doctor diagnostics is extended to a more robust advanced concept of E‐health, where remote online/offline treatment and diagnostics can be performed. In this article, we propose a framework which incorporates a cloud‐based decision support system for the detection and classification of malignant cells in breast cancer, while using breast cytology images. In the proposed approach, shape‐based features are used for the detection of tumor cells. Furthermore, these features are used for the classification of cells into malignant and benign categories using Naive Bayesian and Artificial Neural Network. Moreover, an important phase addressed in the proposed framework is the grading of the affected cells, which could help in grade level necessary medical procedures for patients during the diagnostic process. For demonstrating the e effectiveness of the proposed approach, experiments are performed on real data sets comprising of patients data, which has been collected from the pathology department of Lady Reading Hospital of Pakistan. Moreover, a cross‐validation technique has been performed for the evaluation of the classification accuracy, which shows performance accuracy of 98% as compared to physical methods used by a pathologist for the detection and classification of the malignant cell. Experimental results show that the proposed approach has significantly improved the detection and classification of the malignant cells in breast cytology images.