Electron-Microscopic Evidence of the Vacuolar Nature of Phloem Exudate

We performed electron-microscopic examination of structural diurnal changes in the lumen of sieve tubes and the vacuolar system of corresponding companion cells and changes induced by the experimental blockage of assimilate export from the leaf by its cold-girdling. For these investigations, Cucurbita pepo L. and Helianthus annuus L. plants were used, that is, plant species from groups of symplastic and apoplastic plants, which differ in the type of companion cells and a mode of phloem terminal loading. The examinations showed the complete identity of changes in the electron texture of the sieve-tube lumens and companion-cell vacuoles in both plant species in the course of a day, when the level of assimilates changed, or after export blockage. Similar changes in the structure of the vacuolar labyrinths were stated in the companion cells under normal conditions and after cold-girdling, as related to the rate of sieve-tube loading with the vacuolar exudate. Vacuolar expansion and starch accumulation developing in response to changes in the assimilate level in the evening and after cold blockage of the assimilate export occurred in different types of cells, as dependent on their position in the symplast domains. However, the rate of the process similarly depended on the balance between assimilate synthesis and export. Synchronous changes in the texture of the sieve-tube lumen and companion-cell vacuoles were observed within each complex, but asynchronous changes occurred in different complexes. We suggested this phenomenon for recognizing the particular complexes, when they are grouped in a bundle. We observed no signs of cytoplasm or protein synthetic machinery in the sieve tubes. We concluded that the sieve-tube lumen and vacuoles of companion cells are common in nature. Similar electron texture of the images of the companion-cell vacuolar labyrinth and tube lumens, their connection through the lateral sieve fields, morphological modifications of the companion-cell vacuolar system as dependent on the activity of sieve tube loading—all of these facts imply the continuity of these transport compartments and fluxes in them and the similarity in the composition of the exudates from companion-cell vacuoles and phloem tubes.     

A new fluorescent staining method for callose of microspore mother cells during meiosis

To observe the dynamic behavior of callose of microspore mother cells during meiosis, we developed a convenient, rapid and efficient staining method using an improved carbol fuchsin/aniline blue solution. The stained microspore mother cells during meiosis showed yellowish green callose, red cytoplasm and dark red chromosomes when excited with blue light, which produced a contrasting image with a three-dimensional effect. When stained with only improved carbol fuchsin solution, the cells had red cytoplasm and chromosomes when excited with green light. The improved carbol fuchsin solution can be used to replace other more expensive DNA-specific dyes, such as DAPI and H33258, to reduce experimental costs.     

A Simple Method for Fixing, Dehydrating and Embedding Pollen Tubes Cultivated in Vitro for Optical and Transmission Electron Microscopy

A simple method to cultivate pollen tubes in a gelatin medium is presented. After the growth of the pollen tubes in the culture medium, they are fixed, dehydrated, and embedded in resin for ultramicrotomy. The method is easy and does not require the purchase of special materials beyond those needed for the usual techniques for studying biological specimens under transmission electron microscopy.     

一种改良的肌细胞骨架染色方法

为了观察肌细胞骨架,对传统考马斯亮蓝染色法进行改良,并与免疫荧光染色法进行了比较。培养的血管平滑肌细胞先用多聚甲醛预固定后再进行考马斯亮蓝染色,可使细胞骨架非常清晰的显色,解决了传统考马斯亮蓝染色易使肌细胞变形、脱片的问题,其效果与免疫荧光染色相近。因此,多聚甲醛预固定．考马斯亮蓝染色法是一种适于肌细胞骨架染色的简便方法。     

Basic Blue 148: A Rapid Stain for T Helper Cells

After brief exposure to an aqueous solution of the oxazine textile dye C. I. basic blue 148 following fixation in 37% formalin, 95% ethanol and glacial acetic acid, T helper cell nuclei and cytoplasm in specimens of peripheral blood displayed a deep red-violet color. No other cell in normal blood or bone marrow specimens showed intense staining of this type. The total staining time is 1 min. Basic blue 148 stain is a promising technique for hematology and immunology laboratories as a rapid screening test for T helper cells in blood specimens using a microscopic slide and ordinary incandescent illumination.     

In Situ Hybridization of Cytospin Preparations: A Rapid Nonisotopic Screening Method for Isolated Cells

A simple and rapid method for light microscopic in situ hybridization on cytospin preparations is described and demonstrated for detection of viral nucleic acid in a virus-infected cell line. Cells were fixed by acetone followed by chloroform, denatured by heat, hybridized at 37 C, and hybridized sites detected with a multiple step procedure (primary anti-biotin antibody, biotinylated second antibody, streptavidin-peroxidase). This method can be used for screening studies at the light microscope level, and offers a useful and simple way to determine optimum hybridization conditions for subsequent electron microscopic investigations.     

A novel assay based on fluorescence microscopy and image processing for determining phenotypic distributions of rod-shaped bacteria

Cell population balance (CPB) models can account for the phenotypic heterogeneity that characterizes isogenic cell populations. To utilize the predictive power of these models, however, we must determine the single-cell reaction and division rates as well as the partition probability density function of the cell population. These functions can be obtained through the Collins-Richmond inverse CPB modeling methodology, if we know the phenotypic distributions of (a) the overall cell population, (b) the dividing cell subpopulation, and (c) the newborn cell subpopulation. This study presents the development of a novel assay that combines fluorescence microscopy and image processing to determine these distributions. The method is generally applicable to rod-shaped cells dividing through the formation of a characteristic constriction. Morphological criteria were developed for the automatic identification of dividing cells and validated through direct comparison with manually obtained measurements. The newborn cell subpopulation was obtained from the corresponding dividing cell subpopulation by collecting information from the two compartments separated by the constriction. The method was applied to E. coli cells carrying the genetic toggle network with a green fluorescent marker. Our measurements for the overall cell population were in excellent agreement with the distributions obtained via flow cytometry. The new assay constitutes a powerful tool that can be used in conjunction with inverse CPB modeling to rigorously quantify single-cell behavior from data collected from highly heterogeneous cell populations.     

Acylation and cholesterol binding are not required for targeting of influenza A virus M2 protein to the hemagglutinin-defined budozone

Influenza virus assembles in the budozone, a cholesterol-/sphingolipid-enriched (“raft”) domain at the apical plasma membrane, organized by hemagglutinin (HA). The viral protein M2 localizes to the budozone edge for virus particle scission. This was proposed to depend on acylation and cholesterol binding. We show that M2–GFP without these motifs is still transported apically in polarized cells. Employing FRET, we determined that clustering between HA and M2 is reduced upon disruption of HA’s raft-association features (acylation, transmembranous VIL motif), but remains unchanged with M2 lacking acylation and/or cholesterol-binding sites. The motifs are thus irrelevant for M2 targeting in cells.     

Microfluidics and photonics for Bio‐System‐on‐a‐Chip: A review of advancements in technology towards a microfluidic flow cytometry chip

Microfluidics and photonics come together to form a field commonly referred to as ‘optofluidics’. Flow cytometry provides the field with a technology base from which both microfluidic and photonic components be developed and integrated into a useful device. This article reviews some of the more recent developments to familiarize a reader with the current state of the technologies and also highlights the requirements of the device and how researchers are working to meet these needs. (© 2008 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)