Simplified Sample Embedding and Polishing Methods for Preparing Hydrophilic, Fragile, or Solvent-Susceptible Materials for Thin Sections for Microscopic Analyses

2019 ◽  
Vol 25 (1) ◽  
pp. 257-265 ◽  
Author(s):  
Masato Ueshima ◽  
Hirofumi Sakanakura

AbstractIn the preparation of thin sections for microscopy, embedding and polishing processes in particular can change the composition and morphologies of samples. Soils and ashes are very fragile and solvent-susceptible, and appropriate sample preparation procedures have not been well-established. To improve the existing preparation methods and make them easier and faster, we embedded freeze-dried blocks, polished, and then examined these thin-section samples using polarization microscopy, laser microscopy, and field emission scanning electron microscopy with energy-dispersive X-ray spectrometry, and electron backscattered diffraction (EBSD). Appropriate thin-section samples can be prepared by: (1) rinsing with acetone and then embedding with Spurr resin along with repeated evacuation and ventilation, rather than conventional dehydration/replacement; (2) polishing using silicon carbide paper and diamond slurries, and then wiping with a cloth and a synthetic oil; and (3) slightly rinsing with 100% ethanol to remove the oil. The preparation method minimized contamination and pores, and showed flat surfaces and sometimes EBSD patterns. Freeze-drying has been claimed to cause the development of cracks due to ice crystal formation upon freezing, however, our method not only overcomes such problems for microscopic observation but saves substantial time, taking only 2 days in total to process a specimen, and requiring less than 1 g of resin and ~1 g of sample.

Author(s):  
I. Taylor ◽  
P. Ingram ◽  
J.R. Sommer

In studying quick-frozen single intact skeletal muscle fibers for structural and microchemical alterations that occur milliseconds, and fractions thereof, after electrical stimulation, we have developed a method to compare, directly, ice crystal formation in freeze-substituted thin sections adjacent to all, and beneath the last, freeze-dried cryosections. We have observed images in the cryosections that to our knowledge have not been published heretofore (Figs.1-4). The main features are that isolated, sometimes large regions of the sections appear hazy and have much less contrast than adjacent regions. Sometimes within the hazy regions there are smaller areas that appear crinkled and have much more contrast. We have also observed that while the hazy areas remain still, the regions of higher contrast visibly contract in the beam, often causing tears in the sections that are clearly not caused by ice crystals (Fig.3, arrows).


1970 ◽  
Vol 9 (57) ◽  
pp. 385-390 ◽  
Author(s):  
T. M Tobin ◽  
K. Itagaki

The top surface of an accurately aligned ice crystal is melted by an aluminum surface and then frozen to a warm “Lucite” plate ant! tapped free. Etch-pit development shows that the dislocation density on the resulting surface is similar to die bulk dislocation density determined by X-ray topographic methods.


1970 ◽  
Vol 9 (57) ◽  
pp. 385-390 ◽  
Author(s):  
T. M Tobin ◽  
K. Itagaki

The top surface of an accurately aligned ice crystal is melted by an aluminum surface and then frozen to a warm “Lucite” plate ant! tapped free. Etch-pit development shows that the dislocation density on the resulting surface is similar to die bulk dislocation density determined by X-ray topographic methods.


1979 ◽  
Vol 27 (11) ◽  
pp. 1520-1523 ◽  
Author(s):  
P M Frederik ◽  
W M Busing

Frozen thin sections and sections from freeze-dried and embedded tissue are used for the autoradiographic localization of diffusible substances at the electron microscope level. The presence of ice crystals in such sections may limit the autoradiographic resolution. Ice crystals are formed during freezing and may grow during subsequent processing of tissue. The contribution of ice crystal growth to the final image was estimated by measuring the distribution of the ice crystal sizes in freeze-etch replicas and in sections from freeze-dried and embedded tissues. A surface layer (10-15 mu) without visible ice crystals was present in both preparations. Beneath this surface layer the diameter of ice crystals increased towards the interior with the same relationship between crystal size and distance from the surface in the freeze-etch preparation as in the freeze-dry preparation. Ice crystal growth occurring during a much longer time during freeze-drying compared to freeze-etching does not significantly contribute to the final image in the electron microscope. The formation of ice crystals during freezing determines to a large extent the image (and therefore the autoradiographic resolution) of freeze-dry preparations and this probably holds also for thin cryosections of which examples are given.


Author(s):  
J. B. Hall

Zeolite Y, a remarkably stable zeolite widely used in catalyst formulations, is a synthetic aluminosilicate with the faujaslte structure. It is distinguished from zeolite X by having fewer Al atoms per unit cell and greater stability. Y can be made even more stable by further reducing the number of Al atoms per unit cell to form ultrastable Y (U.S.Y). Because the Al/Si ratio of a zeolite is an important parameter affecting its properties, knowledge of the Al and Si distribution across individual particles is also important. A study of that distribution was therefore undertaken by electron microscopy. A catalyst preparation of 40% U.S.Y and 60% alumina was also analyzed.The samples were prepared for electron microscopy by embedding the particles in epoxy and cutting thin sections using an ultramicrotome. The thin section microanalysis was performed on a Philips 400T electron microscope equipped with a Tracor Northern TN2000 x-ray analyzer with digital beam control.


2011 ◽  
Vol 21 (01n02) ◽  
pp. 25-30 ◽  
Author(s):  
SHINO HOMMA-TAKEDA ◽  
KYOKO SUZUKI ◽  
KEIKO HARUMOTO ◽  
TOMOYASU YOSHITOMI ◽  
HIROYUKI ISO ◽  
...  

For quantitative measurements of light elements by micro-PIXE (particle-induced X-ray emission) analysis, suitable external standards have been expected. In this paper thin sections of polyvinyl alcohol solution containing phosphorus ( P ) and potassium ( K ) were assessed for their purpose as standards by micro-PIXE analysis. Homogeneity of P and K added to the standard materials were validated by 1 μm and 10 μm-step scanning of the standard. The relative standard deviations of the X-ray intensity of the standards P and K were < 25% and <16%, respectively. The calibration line between the X-ray intensity obtained from a 100 × 100 μm2 area and the elements concentration was also acceptable, indicating that the thin section standards are adequate for an external standard of micro-PIXE measurements for light elements.


1978 ◽  
Vol 56 (17) ◽  
pp. 2050-2061 ◽  
Author(s):  
John N. A. Lott ◽  
Mark S. Buttrose

Protein bodies from almond (Prunus dulcis), Brazil nut (Bertholletia excelsa), and quandong (Santalum acuminatum) have been studied in thin sections of fixed and embedded tissue, in freeze-fracture replicas of unfixed tissue, by chemical analysis of tissue for P, K, Mg, and Ca, and by energy dispersive x-ray (EDX) analysis of both sections of glutaraldehyde-fixed tissue and freeze-dried tissue powders. The protein bodies in all three species contained globoid crystals, protein crystalloids, and proteinaceous matrix regions. Results of EDX analyses were consistent with globoid crystals being rich in phytin. Variation in both the structure and the elemental composition of globoids was common. In almond some globoids were lobed rather than spherical, and large globoid crystals often contained considerable calcium whereas small globoid crystals contained little if any calcium. The globoid crystals of Brazil nut often contained barium in addition to P, K, Ca, and Mg. Protein crystalloids of Brazil nut were compound crystals. Protein bodies of quandong seed, which is largely endosperm rather than embryo, were unexceptional.


Clay Minerals ◽  
1982 ◽  
Vol 17 (2) ◽  
pp. 259-262 ◽  
Author(s):  
A. Meunier ◽  
B. Velde

Precise identification of clay minerals found in granular rocks has always posed a great problem to the clay petrographer. Even if it is possible to locate the position of authigenic clay mineral formation in a thin section, subsequent identification of this same material by X-ray diffractometry is usually very difficult. Attempts have been made using selected-area radiation of thin sections (Pawluck & Dumanski, 1973; Wicks & Zussman, 1975; Wilson & Clark, 1978) but the area analysed remains relatively large, i.e. of the order of several mm2. The other solution is micro-picking of material from a thin section and subsequent identification by Debye-Scherrer camera methods (Wallace, 1955; Rickwood, 1977). This method, however, does not allow preferred orientation, and thus precise identification, of many clay species. The ideal method is to combine micro-picking from thin sections from areas of several hundreds of square microns with an oriented sample preparation, which can then be treated in the traditional way (glycolation, heating, etc.) for characterization by X-ray diffractometry.


1985 ◽  
Vol 33 (10) ◽  
pp. 1073-1079 ◽  
Author(s):  
J Appleton ◽  
R Lyon ◽  
K J Swindin ◽  
J Chesters

In order to undertake meaningful high-resolution x-ray microanalysis of tissues, methods should be used that minimize the introduction of artefacts produced by loss or translocation of ions. The most ideal method is rapid freezing but the subsequent sectioning of frozen tissues is technically difficult. An alternative method is to freeze dry the tissues at a low temperature, and then embed them in resin. This facilitates the rapid production of reproducible thin sections. With freeze-dried, embedded hypertrophic cartilage, the morphology was similar to that seen using aqueous fixatives even when no additional electron density is introduced by the use of osmium vapor. Energy-dispersive analysis of specific areas show that little or no loss or migration of ions occurs from structures such as mitochondria. Mitochondrial granules consisting of calcium and phosphorus precipitates were not observed except where the cells were damaged as a result of the freezing process. This may suggest that these granules only appear when tissue is damaged because of inadequate preservation.


1976 ◽  
Vol 21 (1) ◽  
pp. 119-127
Author(s):  
G.M. Roomans ◽  
L.A. Seveus

In yeast cells, of which the intracellular potassium had been partly replaced by rubidium or caesium, the intracellular ion distribution was studied by means of energy-dispersive X-ray microanalysis. The cells were rapidly frozen and thin sections were cut at low temperature on a cryo-ultramicrotome without the use of a trough liquid. By this dry cryosectioning procedure, complete retention of the diffusbile ions in the cells was obtained. Unless the sections had been exposed to moisture, no signs of redistribution were apparent. For quantitative determinations a gelatin standard, containing known amounts of the elements of interest, was prepared in the same way as the cells. The concentrations of potassium, rubidium, caesium and chloride in the nucleus, the cytoplasm and the vacuole could be measured. The intracellular distributions of potassium, rubidium and caesium were very similar. The concentrations of these ions in the cytoplasm were about equal to those in the nucleus and twice those in the vacuole. The total concentration in the cytoplasm was 180–190 mmol/kg fresh weight, in the nucleus 190–200 mmol/kg fresh weight and in the vacuole 75–90 mmol/kg fresh weight. The permeability of the yeast cell for chloride is markedly lower than for the cations.


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