Hexamethyldisilazane as a Drying Agent for Pollen Scanning Electron Microscopy

Use of hexamethyldisilazane (HMDS) as a final dehydrating solution provides robust, undistorted secondary electron images of a variety of angiosperm and gymnosperm pollen grains, including those considered to be susceptible to collapse in the scanning electron microscope. Ease of handling, low cost, lack of specialized equipment, minimal expenditure of time, and high rate of success are factors that favor HMDS over other drying agents for preparing pollen grains for scanning' electron microscopy.     

Hexamethyldisilazane for Scanning Electron Microscopy of Gastrotricha

We evaluated treatment with hexamethyldisilazane (HMDS) as an alternative to critical-point drying (CPD) for preparing microscopic Gastrotricha for scanning electron microscopy (SEM). We prepared large marine (2 mm) and small freshwater (100 μm) gastrotrichs using HMDS as the primary dehydration solvent and compared the results to earlier investigations using CPD. The results of HMDS dehydration are similar to or better than CPD for resolution of two important taxonomic features: cuticular ornamentation and patterns of ciliation. The body wall of both sculpted (Lepidodermeila) and smooth (Dolichodasys) gastrotrichs retained excellent morphology as did the delicate sensory and locomotory cilia. The only unfavorable result of HMDS dehydration was an occasional coagulation of gold residue when the solvent had not fully evaporated before sputter-coating. We consider HMDS an effective alternative for preparing of gastrotrichs for SEM because it saves time and expense compared to CPD.     

A Simple Drying Method for Field Emission Scanning Electron Microscopy for Chromosomes

Hexamethyldisilazane treatment and subsequent air drying of spread plant chromosomes is compared with critical point drying. The two procedures are equivalent for preparing chromosomes for examination by field emission scanning electron microscopy at low voltage.     

A New Technique of Preparing Pollen for Scanning Electron Microscopy

A new technique which avoids the distortion usually present in acetolyzed pollen is outlined. The main steps include hydration in Aerosol-OT, ultrasonication in acetone: water, dehydration in ethanol, transfer to amyl acetate, and critical point drying. Experimental studies, in which pollen treated by the new technique is compared with untreated and with acetolyzed pollen, show much better expansion and more observable detail of exine and colpar structures in the pollen prepared by the new method. Damage to Euphorbiaceous grains, especially those with “crotonoid” ornamentation, is extensive using conventional acetolysis but is circumvented entirely by the critical point drying method. It is concluded that SEM and light microscope studies of pollen should include at least some preparations by a non-acetolytic method such as the present one in order to record an optimal amount of structural information.     

Pollen Wall Structure Using a New Stripping-Sputtering Device for Scanning Electron Microscopy

A new method has been developed to elucidate pollen wall architecture by the separation of wall layers and the use of scanning electron microscopy (SEM). Separation of wall layers at natural boundaries, breakage across the wall and metal-coating of the specimen have been achieved by controlled ramming of free scattered ions produced by a novel “ion separating and coating – model A” instrument. The stripping treatment reveals interfaces and cross profiles of pollen walls and the sputtering treatment results in metal coating for examination with SEM. An advantage of the method is that it provides intact interfaces that are not eroded or damaged. The application of the method is exemplified by SEM analyses of pollen grains of Gossypium hirsutum L., Zea mays L., Sesamum indicum L. and Brassica napus L. var. oleifera. Interfaces between the tectum, column, foot, nexine-2 and intine layers of the pollen wall were all portrayed in G. hirsutum and to a great part in the other species. In G. hirsutum, it was possible to document the attachment point of surface spines, the appearance of individual baculae and the irregular labrum-operculum but regular inner labrum-aperture structure. No tectum was found in S. indicum. In all four species it was not possible to separate the intine from the sporoplast. The numbers of apertures were 20, 1, 10–14 and 3 in G. hirsutum, Z. mays, S. indicum and B. napus, respectively. The dumbell-shaped arrangement of apertures in G. hirsutum, the gear-shaped oblate sporoplast of S. indicum and the abundance of micropores on the intine of B. napus are characteristic features.     

Critical Point Drying of Liverwort Spores for Scanning Electron Microscopy

Preparation for scanning electron microscopy by the Freon-critical point method prevents the collapse of thin-walled liverwort spores and eliminates desiccation artifacts. The success of the method is demonstrated by stereomicrographs of 1) the thin-walled multicellular spore of Conocephalum conicum (Marchantiales) and 2) the thin-walled Jungermannialean spore of Lophocolea heterophylla.     

A New Method Using Hexamethyldisilazane for Preparation of Soft Insect Tissues for Scanning Electron Microscopy

A new rapid procedure for preparing soft internal tissues from insects that allows air drying was found to compare favorably with tissues prepared by critical point drying. In the new procedure, tissues were fixed in 1% glutaraldehyde, dehydrated through a graded ethanol series, immersed in hexamethyldisilazane (HMDS) for 5 minutes, and air dried. Tissues prepared by both the HMDS treatment and by critical point drying were coated with gold for scanning electron microscopy. Tissues prepared by the HMDS treatment did not shrink or distort upon air drying and excellent surface detail was preserved. The HMDS treatment required about 5 minutes, whereas the critical point drying procedure required about 1.5 hours.     

A Scanning Electron Microscopy Survey of Some Maydeae Pollen

Scanning electron microscopy was used to examine the wall sculpturing of pollen from Zea mays L. ssp. mays (maize), Zea mays ssp. mexicana (Schrad.) Iltis (teosinte), Zea perennis (Hitchc.) Reeves and Mangelsdorf (perennial teosinte), and two species of Tripsacum L. The Zea taxa are shown to possess similar pollen types, with spinules scattered regularly over the exine surface. Tripsacum exhibits a distinctly reticuloid pattern, with spinules clumped into isolated lacunae. Hybrids between Zea and Tripsacum are either intermediate in exine pattern or similar to Tripsacum, depending on the genome combination.     

Sputtering,a New Method for Coating Pollen Grains in Scanning Electron Microscopy

Sputtering is an easy, rapid and effective method for metal coating of pollen grains for examination in the scanning electron microscope. A very thin, regular and stable metal layer is obtained by bombarding a metal target with ions under a low vacuum, so that it ejects atoms on to the specimen. This allows of the observation of exine sculpturing which would be completely masked by a coating resulting from evaporation of carbon and metal. The sputtering method was tested on pollen of Deschampsia flexuosa, Molinia caerulea and Betula pubescens. Very narrow perforations could be discerned in the exine, similar to those seen in the images of carbon replicas obtained by many authors with the transmission electron microscope.     

Silver Methenamine Staining for Scanning Electron Microscopy of Bone Sections Containing Biomaterials

Sections of tissue containing orthopedic materials are currently used to study the compatibility of those materials and to perform electron probe microanalysis at the material-tissue interface. Identification of the cells in contact with the material by Scanning electron microscopy (SEM) is of interest. We have developed a method for staining cells and tissue structures embedded in polymethyl methacrylate with silver methenamine once the sections have been obtained. Sections were prepared by grinding, and the silver methenamine was applied after oxidation with periodic acid. The procedure was carried out in a microwave oven. Backscatter SEM showed staining of the cell nucleus membrane, chromatin, the nuclear organizers, and the chromosomes of dividing cells. The cytoplasm and the cytoplasmic membrane were also stained. Collagen fibers of the extracellular matrix and the mineralized matrix of bone were labeled. Material particles in the macrophages were easily recognizable and Energy-Dispersive Spectrometer were not impaired by the presence of silver in the preparation.     

A Method for Isolating and Preparing Silica Bodies in Grasses for Scanning Electron Microscopy

Silica bodies are discrete deposits of dehydrated silica within epidermal cells. To describe these bodies completely, surrounding organic and unsilicified material must be removed. Methods generally used for isolating and preparing silica bodies were unsuitable for most grass species. An effective method for studying grasses is described here. After ashing the plant tissue, the ash was repeatedly rinsed with HCl in a specialized multiple funnel manifold and collected on Nuclepore filters. In addition, the silica bodies were sonicated for a few minutes to remove any remaining mineral impurities. Compared to conventional procedures, this method has a number of advantages: unsilicified material and mineral impurities were removed effectively, smaller quantities of plant tissue could be used, and the loss of silica bodies was minimized.     

Transmission Electron Microscopy of Tissue Prepared for Scanning Electron Microscopy by Ethanol-Cryofracturing

Tissue processed for scanning electron microscopy by ethanol-cryofracturing combined with critical point drying was embedded and sectioned for transmission electron microscopy. Study of sections cut in a plane passing through the fracture edge indicated that preservation of cellular fine structure of fractured cells was excellent. Even at the most peripheral edge of the fracture there was no evidence that movement of cytoplasmic components occurred to distort the original structural organization of fractured cells. Lack of cytoplasmic detail in ethanol-cryofractographs has been due more to the nature of the fracturing of the tissue and to the obscuring effects of the metal coating than to structural deformation at the fracture edge or to limitations in resolving power of the scanning electron microscope used.     

Cryo-Preservation of Roots for Scanning Electron Microscopy

Fully hydrated roots can be examined in the scanning electronmicroscope after cryo-preservation. Shrinkage associated withdehydration by freeze-drying or critical point drying, to whichroot hairs and secreted mucigel are particularly vulnerable,is avoided. Roots, Lepidium sativum, scanning electron microscopy, cryo-preservation, fully hydrated     

Processing Pollen and Spore Exines for Electron Microscopy

A resin mixture containing Araldite M, 15 ml; Epon 812, 25 ml; dodecenyl succinic anhydride, 55 ml; and dibutyl phthlate, 2 ml, was found to be the optimal embedding resin for both fresh and acetylated pollen exines. Diamond knives greatly facilitated sectioning. Exine fine structure, and stratification patterns in fresh pollen were most clearly revealed by section staining of glutaraldehyde-fixed (2 hr), OsO₄-stained (2 hr) specimens. Acetylated exines (acetic anhydride-H₂SO₄ 9:1; 100 C, 5 min) did not require additional treatment prior to embedding, but section staining of exines so treated greatly enhanced stain differentiation of exine subunits. Successfully used section stains included Reynold's lead hydroxide, Millonig's lead citrate and aqueous KMnO₄. Additional procedures were tried but were found to have serious disadvantages, e. g. exines treated with KMnO₄ before embedding shattered badly during sectioning.     

The Use of Osmium-Thiocarbohydrazide for Structural Stabilization and Enhancement of Secondary Electron Images in Scanning Electron Microscopy of Pollen

Pollen grains stained in a sequence of osmium (O) and thiocarbohydrazide (T) solutions (collectively known as OTOTO) appear structurally stable and undistorted in the scanning electron microscope (SEM), and usually do not require special drying. In fact, OTOTO can be regarded as another special drying method in palynology. This sequential incubation also strikingly increases the electrical conductivity of pollen grains in the SEM. Compared to standard sputter-coating or vacuum evaporative procedures, OTOTO reduces charging and yields secondary electron images with significantly higher resolution.     

Pollen Morphology of Polemoniaceae in Relation to Systematics and Pollination Systems: Scanning Electron Microscopy

Representative pollen grains of each genus and section of the family Polemoniaceae were examined with scanning electron microscopy. Exine pattern diversity within the family is discussed in relation to pollination biology. Pollen data support some previously recognized relationships within the family; in other instances the use of pollen morphology suggests the institution of new associations. The value of pollen morphology as an index to possible phylogenetic interpretations within the Polemoniaceae is discussed.     

The Dimension of Trichomonas vaginalis as Measured by Scanning Electron Microscopy

It is known that physicochemical conditions (e.g., pH, temperature, and ionic strength) affect the size of trichomonads. In this study, the sizes of 4 isolates of Trichomonas vaginalis cultured for more than a year (called "old T") and 3 isolates freshly isolated from vaginitis cases (called "fresh T") were compared by scanning electron microscopy. Although the fresh T had shorter body length, body width, and flagellar length than old T, total length (about 26 µm), including body length, flagella length, and axostyle length was almost the same in the 2 groups. A striking difference was observed between the axostyles of the 2 groups; the axostyle length of the fresh T (8.2 µm) was more than twice as long as that of the old T (4.0 µm). However, in several parasitology textbooks, the length of T. vaginalis is said to vary widely from 7 to 32 µm, and its undulating membrane is said to extend about half way (53.5%) to the posterior end of the body. On the other hand, in our study, the undulating membrane was observed to extend more than 3/4 of the body length (72.1%) in old T, whereas in fresh T it could not be measured. Taken together, we suggest that T. vaginalis averages 26 (21-32) µm in total length, with 9.5 (7.4-11.4) µm of body length and 6.8 (5.3-7.7) µm of width, and its undulating membrane extending 3/4 of its body length. Therefore, these findings may provide useful information for morphological characteristics of T. vaginalis.     

Fertilization Cone of Carp Eggs as Revealed by Scanning Electron Microscopy

The process of formation of the fertilization cone in carp eggs was examined by scanning electron microscopy. The fertilized eggs responded to penetration of one sperm by primary and secondary steps of formation of a fertilization cone of unique morphology. In the primary step, the earliest fertilization cone was seen at the superior or anterosuperior part of a fused sperm head in inseminated eggs fixed 20 sec after immersion in fresh water. The cone reached a maximum of more than 10 μm in length and 3–4 μm in thickness by 40 sec, resulting in a transient plugging of the micropylar canal. In the secondary step, usually seen at 105–120 sec, a conformation reminiscent of a very small caldera volcano was formed, with the shortened earlier cone and part of the sperm tail at its top. By 2.5 min, the fertilization cone had become conical, and the sperm tail still extended from its top. At 3 min, the sperm tail was often not detectable, but a cytoplasmic eminence was still seen as a trace of the fertilization cone. The role of the earlier fertilization cone in blocking polyspermy is discussed.     

Dry Ice Fixation of Myofibrils for Scanning Electron Microscopy

A rapid method of fixation of myofibrils using dry ice is reported. A glass slide or coverslip containing a drop of glutaraldehyde-fixed suspension of myofibrils is placed on dry ice causing the myofibrils to adhere to the glass surface. The specimens are then dehydrated through the alcohols, air dried and metal coated. This technique gives the myofibrils a corrugated appearance under the scanning electron microscope corresponding to the sarcomere banding.     

Fixation of Platelets for Scanning and Transmission Electron Microscopy

A double fixation method of preparing platelet suspensions for both scanning and transmission electron microscopy is outlined. Prefixation in 0.1% glutaraldehyde allows for immediate preservation of morphologic characteristics induced by experimental procedures, but does not completely destroy platelet surface stickiness. Preservation of surface stickiness allows subsequent production of a platelet pellet for processing for transmission electron microscopy. This pelleting cannot be achieved when higher initial concentrations of glutaraldehyde are used for prefixation. Prefixation in 0.1% glutaraldehyde is also an appropriate initial step for preservation of platelets in suspension for scanning electron microscopy.