Two-color immunoperoxidase staining: visualization of anatomic relationships between immunoreactive neural elements

A method for differentially staining for two antigens in single sections is described. Paraffin-embedded or Vibratome sections are incubated in two sequences of immunoperoxidase (PAP) reagents using a diaminobenzidine (DAB)-nickel ammonium sulfate solution to localize the first antigen, and DAB alone to localize the second antigen. With these chromagens, black and amber-colored reaction products are generated at the locations of the first and second antigens, respectively. The reaction products are stable and provide excellent color contrast. With this technique, the anatomical relationships between two sets of immunoreactive elements can be studied in individual sections. Intimate spatial associations that would probably not be detected by an examination of adjacent sections stained for each antigen can be visualized with this two-color immunoperoxidase method.     

Structural response of relaxed and constricted arterioles

Analysis of viscoelastic properties of blood vessel walls presents difficult analytical problems in view of their non-homogeneity, anisotropy, and non-linear viscoelastic characteristics. The analytical technique used in this study is the numerical method of direct stiffness which has been successfully applied in preliminary studies. The direct stiffness method constitutes a finite element analysis, where the wall structure in an electron micrograph is represented by an assembly of triangular elements. Different material properties can be assigned to each triangular element; the method is thus ideally suited for computer analysis of non-homogeneous biological structures. Anatomical components considered in the analysis were two types of collagen fibers and smooth muscle cells. Typical circumferential stress-time and radius-time histories for selected points have been obtained in relaxed and constricted arterioles under static pressure loads. Plots of the stress distribution in cross sections of the vessel wall have been obtained in relaxed and constricted arterioles.     

The use of secondary ion mass spectrometry (SIMS) to evaluate internal contamination.

Secondary ion mass spectrometry (SIMS) permits the detection of stable and radioactive elements in microvolume. Based on the ablation of specimens by ion bombardment, this mass spectrometry method allows a rapid assessment of trace elements in biological samples and enables accurate isotopic ratio determination. In this work, an application of SIMS in studies involving element microdistribution is illustrated on the basis of analyses of duodenal tissue sections from rats contaminated with either cerium or thorium. For this purpose, tests are performed with SIMS to analyze tissue sections obtained 12, 24 and 48 hr after contamination. In this report, strengths and limitations of SIMS are pointed out as an important tool in biological research.     

A hypothesis on the role of transposons

Genomic transposable elements, or transposons, are sequences of DNA that can move to different positions in the genome; in the process, they can cause chromosomal rearrengements and changes in gene expression. Despite their prevalence in the genomes of many species, their function is largely unknown: for this reason, they have been labelled “junk” DNA. “Epigenetic Tracking” is a model of development that, combined with a standard evolutionary algorithm, become an evo-devo method able to generate arbitrary shapes of any kind and complexity (in terms of number of cells, number of colours, etc.). The model of development has been also shown to be able to produce the artificial version of key biological phenomena such as the phenomenon of ageing, and the process of carcinogenesis. In this paper the evo-devo core of the method is explored and the result is a novel hypothesis on the biological role of transposons, according to which transposition in somatic cells during development drives cellular differentiation and transposition in germ cells is an indispensable tool to boost evolution. Thus, transposable elements, far from being “junk”, have one of the most important roles in multicellular biology.     

植物组织石蜡切片的扫描电镜观察方法研究

石蜡切片的扫描电镜观察法有其独到之处:集光镜和扫捕电镜特长于一体,在大量的石蜡切片光镜观察的基础上,挑选具有研究线索的切片,采用此法转移到扫描电镜下作高分辩研究,既可普查切片全貌,又可处得切片中亚微结构的三维图像,这对结构的准确分辩十分有利,且便于作连续切片观察。本文简要介绍这一实验技术。     

A critique of the contributions of immunoperoxidase cytochemistry to our understanding of pituitary cell function, as illustrated by our current studies of gonadotropes, corticotropes and endogenous pituitary GnRH and TRH

Summary The peroxidase-antiperoxidase technique has been used to study sites of pituitary hormone storage and binding. Some recent findings from our laboratory show that the technique can make intriguing contributions to our understanding of pituitary cell function. In serial ulbra-thin sections, one can identify two or three hormones in a given cell. During pre-pubertal development, gonadotropes may contain adrenocorticotropin immunoreactivity. Brain releasing hormones may be stored or sequestered in granules of cells they stimulate. This report includes a discussion andcritique of our recent findings and interpretations which must be considered before one draws any conclusions about their biological significance.     

Ultrastructural autometallography: a method for silver amplification of catalytic metals

The autometallographic technique involves application of a silver bromide-containing emulsion on the surface of ultrathin sections placed on grids that are subsequently exposed to a photographic developer. In tissue sections from animals treated intravitally with gold, silver, or mercury compounds, accumulations of the metals are visualized by autometallography and can be used for quantitative studies. After amplification, sections can be stained with lead citrate and uranyl acetate. Using autometallography, particles of colloidal gold dispersed in a film of gelatin showed a time-dependent growth and were gradually amplified up to 3.5-fold after 15 min of development. Hence the method may prove useful tracing colloidal gold particles in sections with low particle density, and be a powerful tool for revealing metals in biological tissues.     

X-ray microanalysis of biological specimens by high voltage electron microscopy

For the purpose of analyzing and imaging chemical components of cells and tissues at the electron microscopic level, 3 fundamental methods are available, chemical, physical and biological. Among the physical methods, two methods qualifying and quantifying the elements in the structural components are very often employed. The first method is radioautography which can demonstrate the localization of radiolabeled compounds which were incorporated into cells and tissues after the administration of radiolabeled compounds. The second method is X-ray microanalysis which can qualitatively analyze and quantify the total amounts of elements present in cells and tissues. We have developed the two methodologies in combination with intermediate high or high voltage transmission electron microscopy (200–400 kV) and applied them to various kinds of organic and inorganic compounds present in biological materials. As for the first method, radioautography, I had already contributed a chapter to PHC (37/2). To the contrary, this review deals with another method, X-ray microanalysis, using semi-thin sections and intermediate high voltage electron microscopy developed in our laboratory.

X-ray microanalysis is a useful method to qualify and quantify basic elements in biological specimens. We first quantified the end-products of histochemical reactions such as Ag in radioautographs, Ce in phosphatase reaction and Au in colloidal gold immunostaining using semithin sections and quantified the reaction products observing by intermediate high voltage transmission electron microscopy at accelerating voltages from 100 to 400 kV. The P/B ratios of all the end products Ag, Ce and Au increased with the increase of the accelerating voltages from 100 to 400 kV. Then we analyzed various trace elements such as Zn, Ca, S and Cl which originally existed in cytoplasmic matrix or cell organelles of various cells, or such elements as Al which was absorbed into cells and tissues after oral administration, using both conventional chemical fixation and cryo-fixation followed by cryo-sectioning and freeze-drying, or freeze-substitution and dry-sectioning, or freeze-drying and dry-sectioning producing semithin sections similarly to radioautography. As the results, some trace elements which originally existed in cytoplasmic matrix or cell organelles of various cells in different organs such as Zn, Ca, S and Cl, were effectively detected. Zn was demonstrated in Paneth cell granules of mouse intestines and its P/B ratios showed a peak at 300 kV. Ca was found in human ligaments and rat mast cells with a maximum of P/B ratios at 350 kV. S and Cl were detected in mouse colonic goblet cells with maxima of P/B ratios at 300 kV. On the other hand, some elements which were absorbed by experimental administration into various cells and tissues in various organs, such as Al in lysosomes of hepatocytes and uriniferous tubule cells in mice was detected with a maximum of P/B ratios at 300 kV.

From the results, it was shown that X-ray microanalysis using semi-thin sections observed by intermediate high voltage transmission electron microscopy at 300–400 kV was very useful resulting in high P/B ratios for quantifying some trace elements in biological specimens. These methodologies should be utilized in microanalysis of various compounds and elements in various cells and tissues in various organs.     

A novel and rapid method for isolating sequences adjacent to rare cutting sites and their use in physical mapping.

We describe a simple PCR based technique which can be used to isolate sequences adjacent to rare cutter sites and can subsequently be employed for the construction of long range physical maps. The method involves the ligation of an adaptor to rare cutter sequences and its use as a target for forward priming in PCR. Primers to Alu repeat elements initiate synthesis of the reverse strand. Using this technique any rare cutter site which has a repeat element within amplification range can be cloned. We have isolated six unique sequences around NotI sites from an irradiation reduced hybrid containing a fragment of human chromosome 22 and are using these for physical mapping around the Ewing's sarcoma translocation breakpoint on chromosome 22.     

Highly sensitive immunodetection of DNA on sections with exogenous terminal deoxynucleotidyl transferase and non-isotopic nucleotide analogues.

A new method is described for locating DNA on ultra-thin sections. Sections of aldehyde-fixed, plastic-embedded cells were incubated in a medium containing terminal deoxynucleotidyl transferase (TdT) and various non-isotopic nucleotide analogues. The labeled nucleotides bound to the surface of ultra-thin sections were then visualized by an indirect immunogold labeling technique. The resulting labeling pattern was strongly dependent on the divalent cation used in the TdT medium. The method revealed with great precision the specific DNA-containing structures within Ehrlich tumor cells, even where DNA was present in very low amounts. The method is compatible with all usual fixation and embedding procedures and can be combined with cytochemical methods. The in situ TdT method provides a very useful tool for pinpointing the precise location of DNA within biological material at the ultrastructural level.     

Histological Methods for Assessing Myelin Sheaths and Axons in Human Nerve Trunks

Although there are many histological techniques for assessing myelin sheaths and axons in paraffin embedded or frozen sections of the peripheral nervous system, modern approaches usually use plastic embedded material. Although plastic embedding is superior for small cutaneous branches, this method has limited value for histological assessment of nerve trunks. We report three methods which together yield a comprehensive approach for thorough and detailed investigation of human nerve trunks. The rapid osmication method permitted assessment of myelinated nerve fibers from frozen sections at operation, thus providing the surgeon with guidance on the extent of nerve resection. The modification presented here resulted in permanent slides, allowing comparison of results with those of the other two procedures. The new osmium-hematoxylin technique could be performed on paraffin embedded nerves. Paraffin, unlike plastic, permitted the study of the whole cross sectional area of the nerve in single sections. Moreover, the sharp image of the myelin permitted computerized morphometry. The significantly modified axonal silver impregnation technique was performed on frozen sections mounted on glass slides, as opposed to the time-consuming impregnation of free-floating sections. The latter technique had a high success rate and permitted semiquantitative assessment of axons in nerve trunks. These methods can be performed in any routine histology laboratory and resulted in greater accuracy compared to conventional methods.     

High-resolution whole-mount imaging of three-dimensional tissue organization and gene expression enables the study of Phloem development and structure in Arabidopsis

Currently, examination of the cellular structure of plant organs and the gene expression therein largely relies on the production of tissue sections. Here, we present a staining technique that can be used to image entire plant organs using confocal laser scanning microscopy. This technique produces high-resolution images that allow three-dimensional reconstruction of the cellular organization of plant organs. Importantly, three-dimensional domains of gene expression can be analyzed with single-cell precision. We used this technique for a detailed examination of phloem cells in the wild type and mutants. We were also able to recognize phloem sieve elements and their differentiation state in any tissue type and visualize the structure of sieve plates. We show that in the altered phloem development mutant, a hybrid cell type with phloem and xylem characteristics develops from initially normally differentiated protophloem cells. The simplicity of sieve element data collection allows for the statistical analysis of structural parameters of sieve plates, essential for the calculation of phloem conductivity. Taken together, this technique significantly improves the speed and accuracy of the investigation of plant growth and development.     

江豚耳蜗切片的计算机三维重建

在自己组装的微型机图象系统上,实现了对生物组织连续切片的三维重建。重建结果以灰度阴影方式在彩色显示器上显示,并可在打印机上以多灰度等级方式打印输出。结合一例江豚内耳的连续切片,对其耳蜗骨迷路部份进行了三维重建。并将重建结果以动态形式在显示器上转动,以便观察其各个侧面的情况。     

Finite element simulation of skeletal muscular structures obtained from images of histological serial sections

In this study, we present a method for the three-dimensional reconstruction of objects obtained from histological serial sections (exemplified by those of a pennate striated skeletal muscle) and its application to the finite element method. A hyperelastic material model is used for modeling biological soft tissue. The reconstruction process relies on the direct construction of a volumetric mesh using an octree approach which leads to a stable finite element method. Stability can be expressed in the spectral matrix condition number. To visualize stress patterns within the underlying anatomy the simulation results are projected onto images of the histological scenario.     

Use of immunohistochemistry to diagnose chytridiomycosis in dyeing poison dart frogs (Dendrobates tinctorius)

Chytridiomycosis, caused by Batrachochytrium dendrobatidis, is an emerging disease of both wild and captive amphibians, posing a threat to their survival in many parts of the world. As the disease can be difficult to diagnose on routine pathologic sections, the purpose of this study was to develop an additional method for visualization. To accomplish this, immunohistochemical staining was applied to histologic skin sections from four experimentally infected Dyeing poison dart frogs (Dendrobates tinctorius). Staining of the positive tissue sections was distinct and readily visualized, making this technique a valuable ancillary diagnostic test for this important disease.     

A novel technique for immunohistoperoxidase staining of unfixed whole joints of small animals

Summary A method has been developed to cut unfixed and undecalcified sections of rat paws from animals with adjuvant arthritis and to stain them by a biotin-avidin immunoperoxidase technique. Good tissue integrity and morphology throughout the immunohistochemical procedure were retained if the sections were first mounted on transparent sellotape. The method is illustrated with two monoclonal antibodies (mAb) and is generally applicable with any mAb or polyclonal antibody and with joints from other small animals. For rats with adjuvant arthritis it was found important to block endogenous peroxidase before immunostaining. Complete inhibition of this enzyme without loss of antigenicity was best achieved after application of the mAb and biotinylated anti-IgG conjugate to the unfixed tissue sections.     

Predicting bacterial community assemblages using an artificial neural network approach

Understanding the interactions between the Earth's microbiome and the physical, chemical and biological environment is a fundamental goal of microbial ecology. We describe a bioclimatic modeling approach that leverages artificial neural networks to predict microbial community structure as a function of environmental parameters and microbial interactions. This method was better at predicting observed community structure than were any of several single-species models that do not incorporate biotic interactions. The model was used to interpolate and extrapolate community structure over time with an average Bray-Curtis similarity of 89.7. Additionally, community structure was extrapolated geographically to create the first microbial map derived from single-point observations. This method can be generalized to the many microbial ecosystems for which detailed taxonomic data are currently being generated, providing an observation-based modeling technique for predicting microbial taxonomic structure in ecological studies.     

Reducing data acquisition for light‐sheet microscopy by extrapolation between imaged planes

Light‐sheet fluorescence microscopy (LSFM) is a powerful technique that can provide high‐resolution images of biological samples. Therefore, this technique offers significant improvement for three‐dimensional (3D) imaging of living cells. However, producing high‐resolution 3D images of a single cell or biological tissues, normally requires high acquisition rate of focal planes, which means a large amount of sample sections. Consequently, it consumes a vast amount of processing time and memory, especially when studying real‐time processes inside living cells. We describe an approach to minimize data acquisition by interpolation between planes using a phase retrieval algorithm. We demonstrate this approach on LSFM data sets and show reconstruction of intermediate sections of the sparse samples. Since this method diminishes the required amount of acquisition focal planes, it also reduces acquisition time of samples as well. Our suggested method has proven to reconstruct unacquired intermediate planes from diluted data sets up to 10× fold. The reconstructed planes were found correlated to the original preacquired samples (control group) with correlation coefficient of up to 90%. Given the findings, this procedure appears to be a powerful method for inquiring and analyzing biological samples.     

Laser-guidance based cell detection for identifying malignant cancerous cells without any fluorescent markers

Laser guidance technique employs the optical forces generated from a focused Gaussian laser beam incident on a biological cell to trap and guide the cell along the laser propagation direction. The optical force, which determines the guidance speed, is dependent on the cellular characteristics of the cell being guided, such as size, shape, composition and morphology. Different cell populations or subpopulations can be detected without any fluorescent markers by measuring their guidance speeds. We found that cell guidance speeds were sensitive enough to monitor the subtle changes during the progression of mouse fibroblast cells from normal to cancerous phenotype. The results also demonstrated that this technique can effectively distinguish mouse mammary cancerous cells with different metastatic competence. Laser guidance technique can be used as a label-free cell detection method for basic cell biological investigation and cancer diagnosis.