The identification of pollen grains using cathodoluminescence in the scanning electron microscope

A scanning electron microscope fitted with a sensitive cathodoluminescence detector system has been used for the identification of pollen grains stained with acridine orange or unstained. This approach provides a means of identifying and studying the morphological details of pollens. Cathodoluminescence detection can, in principle, extend the limits of resolution beyond those obtainable with the fluorescence microscope.     

Study of rice pollen grains by multispectral imaging microscopy

Cellular images obtained by light microscopy have been analyzed qualitatively, but there is still a lack of quantitative information about the variations in cellular metabolism of selective substances. A new approach using the multispectral imaging microscope (MIM) to observe rice (Oryza sativa L.) pollen grains is reported. A liquid crystal tunable filter device was used for wavelength selection from 400 to 720 nm and a cooled two-dimensional monochrome charge coupled device for image detection. Rice pollen were stained respectively by acetocarmine, Coomassie blue, or iodine potassium-iodine, and then imaged by MIM. The images were processed by the WuDa Image Analysis System 2003 (computer software), and the transmittance spectra for pollen grain images were obtained. The statistical analysis of the transmittance data showed that the macromolecular amount (nucleic acid, protein, starch) of male-sterile line (MSL) pollen grains was less than those of the fertility-maintaining line (FML). For instance there was a significantly lower nucleic acid content in the MSL than in the FML pollen. The results revealed that pollen abortion was directly related to the diminution of intracellular substances for metabolism. Consequently, we have established a quantitative criterion to determine pollen sterility. Comparing the spectra features of the FML with the MSL, we found that certain spectra features can be used to identify various types of abortion pollens and the deficient cytoplasm of male-sterile rice. Our experimental results offer the first quantitative understanding for evaluating cell morphological structure correlated with cellular physiological status.     

Cryo‐scanning electron microscopy and light microscopy for the study of fungi interactions

The application of the cryo‐scanning electron microscopy and light microscopy for the study of the interactions at different environmental conditions between Penicillium oxalicum and Fusarium verticillioides is described. A dual microculture was developed for the light microscopy analysis of the interaction. The microscope and macroscopic examinations were compared. Analysis of Petri plates revealed that F. verticillioides was a competitor for space and nutrients while P. oxalicum was a mycoparasite under the microscopic observations. Microsc. Res. Tech., 2010. © 2010 Wiley‐Liss, Inc.     

Detection of pollen grains in multifocal optical microscopy images of air samples

Pollen is a major cause of allergy and monitoring pollen in the air is relevant for diagnostic purposes, development of pollen forecasts, and for biomedical and biological researches. Since counting airborne pollen is a time-consuming task and requires specialized personnel, an automated pollen counting system is desirable. In this article, we present a method for detecting pollen in multifocal optical microscopy images of air samples collected by a Burkard pollen sampler, as a first step in an automated pollen counting procedure. Both color and shape information was used to discriminate pollen grains from other airborne material in the images, such as fungal spores and dirt. A training set of 44 images from successive focal planes (stacks) was used to train the system in recognizing pollen color and for optimization. The performance of the system has been evaluated using a separate set of 17 image stacks containing 65 pollen grains, of which 86% was detected. The obtained precision of 61% can still be increased in the next step of classifying the different pollen in such a counting system. These results show that the detection of pollen is feasible in images from a pollen sampler collecting ambient air. This first step in automated pollen detection may form a reliable basis for an automated pollen counting system. Microsc. Res. Tech., 2009. © 2009 Wiley-Liss, Inc.     

Taxonomic study of Triblemma by using light and scanning electron microscopy

Triblemma Ching is a genus proposed by Ching Ren‐Chang in 1978, and it is composed of two species, Triblemma lancea (Thunb.) Ching and Triblemma zeylanica (Hook.) Ching. There has been much debate on the systematic position of Triblemma, and this genus has always been included in Diplazium. Here, we have described new features of tracheary elements, epidermis, spore, scale, and rachis of Triblemma revealed using light and scanning electron microscopy and proposed that Triblemma is closely related to Athyriopsis, which conflicts with the traditional viewpoint.     

Comparative light microscopy,scanning electron microscopy and transmission electron microscopy of selected organic walled microfossils

Two simple techniques are described and illustrated. The first is for the study of one specimen by both light microscopy (LM) and scanning electron microscopy (SEM). The second is for the study of one selected specimen by LM, SEM and in ultrathin section by transmission electron microscopy (TEM). Although these techniques were developed for the comparative study of Precambrian organic walled microfossils (OWMs), they could be used for a wide range of other specimens.     

Discrimination of heavy and light elements in a specimen by use of scanning transmission electron microscopy

Heavier elements have a larger scattering cross-section for elastically scattered electrons than lighter ones. Furthermore, the maximum number of scattered electrons is at higher scattering angles for heavier atoms. These differences can be used, in principle, to distinguish heavy and light elements from each other in dark field Scanning Transmission Electron Microscopy (STEM). We have achieved such discrimination in practice by collecting the electrons in a STEM experiment at two different angles. The information about the elemental composition that these two images together contain is visualized by forming linear combinations of the images which are specific for light and heavy elementsrrespectively. The results are demonstrated for a specimen consisting of platinum grains on a holey carbon film and for granulocytes stained with osmium tetroxide.     

Photooxidation technology for correlated light and electron microscopy

The combination of the capabilities of light microscopical techniques with the power of resolution of electron microscopy along with technical advances has led to a gradual decline of the gap between classical light and electron microscopy. Among the correlative techniques using the synergistic opportunities, photooxidation methods have been established as valuable tools for visualizing cell structures at both light and electron microscopic level. Fluorescent dyes are used to oxidize the substrate diaminobenzidine, which in its oxidized state forms fine granular precipitates. Stained with osmium, the diaminobenzidine precipitates are well discernible in the electron microscope, thus labelling and defining the cellular structures, which at light microscopy level are recorded by fluorescent probes. The underlying photooxidation reaction is based on the excitation of free oxygen radicals that form upon illumination of fluorochromes; this is a central step in the procedure, which mainly influences the success of the method. This article summarizes basic steps of the technology and progresses, shows efforts and elaborated pathways, and focuses on methodical solutions as to the applicability of different fluorochromes, as well as conditions for fine structural localizations of the reaction products.     

Quantitative DNA measurements in an instrument combining scanning electron microscopy and light microscopy

An instrument for combined scanning electron microscopy (SEM) and light microscopy (LM) to which a photometer unit is attached is described. A special stage in the vacuum chamber of a scanning electron microscope incorporates light microscope optics (objective and condenser) designed for transmission and epi-illumination fluorescence LM. An optical bridge connects these optics to a light microscope, without objective and condenser. The possibility of performing quantitative DNA measurements in this combined microscope (the LM/SEM) was tested using preparations of either chicken erythrocytes, human lymphocytes, or mouse liver cells. The cells were fixed, brought on a cover-glass, quantitatively stained for DNA, dehydrated, and critical point dried (CPD). After mounting the cells were coated with gold. The specimens were brought into the vacuum chamber of the combined microscope and individual cells were studied with SEM and LM. Simultaneously DNA measurements were performed by means of the photometer unit attached to the microscope. It is shown in this study that DNA measurements of cells in the combined microscope give similar results when compared to DNA measurements of embedded cells performed with a conventional fluorescence microscope. Furthermore, it is shown that although the gold layer covering the LM/SEM specimens weakens the fluorescence signal, it does not interfere with the DNA measurements.     

Rapid method for resectioning of light microscopy sections for electron microscopy

A rapid method is described for obtaining ultrathin sections from light microscopy sections. Five-micrometre epoxy sections, heat-flattened to slides, were affixed to the tips of plastic blocks by light-curable dental bond, and cured while still on the microscope stage by illumination with blue light for 2 min. Sections were detached from the slides by rapid cooling and then resectioned for electron microscopy.     

Correlative light and electron microscopy for the analysis of cell division

Correlative light and electron microscopy (CLEM) has recently gained increasing attention, because it enables the acquisition of dynamic as well as ultrastructural information about subcellular processes. It is the power of combining the two imaging modalities that gives additional information as compared to using the imaging techniques separately. Here, we briefly summarize two CLEM approaches for the analysis of cells in mitosis and cytokinesis.     

Techniques for combining light microscopy and scanning electron microscopy: a survey of the literature

A survey of methods combining light microscopy and scanning electron microscopy is presented. A simple correlation is made when two preparations from adjacent parts of one specimen are investigated in two different microscopes. A more sophisticated method is the consecutive investigation of one specimen with two microscopes. A major problem in this method is the relocation of the area of interest. Several authors have presented solutions for this problem. It is preferable when one preparation is investigated in only one instrument, combining the two microscopical (LM and SEM) techniques, thus making relocation redundant.     

Identification of monocot flora using pollen features through scanning electron microscopy

Pollen used to track structural and functional evolution in plants as well as to investigate the problems relative to plant classification. Pollen characters including ornamentation, shape, apertural pattern, pollen symmetry, colpus length, width, and margins used to detect the similarities and dissimilarities between genera and also species of the same genus. In this study pollen features of 20 monocot species belonging to 15 genera of the Amaryllidaceae, Asparagaceae, Iridaceae, Ixioliriaceae, Liliaceae, and Xanthorrhoeaceae were studied using scanning electron microscopy (SEM) and light microscopy (LM). In this study two species that is Zephyranthes citrina and Tulbaghia violacea were reported for the first time from Pakistan. Pollen grains were visualized with LM. Non‐acetolyzed and acetolyzed pollen were examined using SEM. A taxonomic key was developed to highlight the variation in pollen features in order to make their systematic application for correct species identification.     

Comparison between light and electron microscopy in canine and feline renal pathology: a preliminary study

The aim of this study is to compare the accuracy and clinical use of light and transmission electron microscopy in detecting the early stages of renal pathologies in domestic animals. We examined 30 samples of renal tissue from cats and dogs referred to the Veterinary Hospital of the Department of Animal Pathology for different systemic diseases. The progressions of the kidney pathologies were classified using the scheme system proposed by the International Renal Interest Society. All samples were submitted for conventional histology and ultrastructural examination. Our study shows that electron microscopy is necessary to complete the histological examinations, especially to define early stages of kidney diseases (minimal changes disease, epithelial tubular pathologies, tubular basement membrane and glomerular basement membrane changes). Electron microscopy can be more accurate in defining the level of focal lesion, and permits discrimination between different clinical and pathological alterations such as fibrillary deposits. In conclusion, transmission electron microscopy associated with clinical, histological, histochemical and immunological examinations, is an essential method for diagnosis and prognosis of renal disease.     

Preparation of fetal rat brains for light and electron microscopy

To study cellular shapes, growth patterns, and fine structure during early stages of CNS development in rat embryos, preparative procedures were evaluated and modified to meet two criteria: (1) Coronal semithin sections should reveal undeformed telencephalic hemispheres that were symmetrically expanded on both sides of midline structures and were surrounded by contiguous mesenchyme. (2) In electron micrographs, cells should have intact, undistorted surface membranes, evenly distributed nucleoplasm and well preserved cytoplasmic organelles. To meet these criteria, 378 fetuses with a gestational age of 11–20 days (E11–E20) were used to test and modify procedures for anesthesia, embryo removal and handling, dissection, fixation, dehydration, and embedding of the embryonic CNS. Most specimens were in an early stage of development (E11–E13), which, in case of the neopallial wall, is the preneural period. The tests produced methods that met the above criteria and identified the most common artifacts and their causes. Deformities of the cerebral hemispheres and separations between the brain and its coverings were usually caused by trauma during embryo removal and during handling before fixation. Changes in cellular volumes, especially swelling during fixation and dehydration, were the most important causes of histological artifacts. The procedures and methods that consistently produced the best light and electron microscopic preservation of the E11–E13 rat CNS are described. Fixation was best when the brains were treated with glutaraldehyde and s-collidine buffer, followed by osmium tetroxide in s-collidine buffer. A surprisingly beneficial effect of sodium chloride in the dehydrating alcohol was noted.