Oolong tea extract as a substitute for uranyl acetate in staining of ultrathin sections

In conventional transmission electron microscopy, uranyl acetate staining is used to enhance the cellular components. However, uranyl acetate is considered a radioactive material that is very toxic if ingested or inhaled and subject to restrictions in many countries. In an attempt to introduce a substitute for uranyl acetate, we evaluated oolong tea extract (OTE) for staining of ultrathin sections. Tissue sections from normal rat liver representing an ideal model organ were processed according to a routine electron microscopic fixation and embedding procedure. Serial ultrathin sections were cut and processed with either routine double electron staining or 0.2% OTE staining for 30–40 min at room temperature followed by lead citrate staining (OTE staining method). Transmission electron microscopy observations revealed that all sub‐cellular structures in hepatocytes were clearly visible with OTE staining and the quality of staining was highly compatible with those of routine double staining methods. It is suggested that OTE could be used as a non‐radioactive and hazard‐free substitute for uranyl acetate in transmission electron microscopy staining.     

X-ray microanalysis of ultrathin frozen and freeze-dried sections of human sperm cells

X-ray microanalysis of air dried and ultrathin frozen and frozen dried sections of human sperm cells has indicated a large deviation of elemental composition between cells in a single sample and between samples. Analysis of air dried cells indicates a similar subcellular elemental distribution to that found in sectioned material. Sodium to potassium ratios are found to be similar in both preparations. Air drying is considered a valid method for the preparation of sperm cells for analysis.     

A double lead stain method for enhancing contrast of ultrathin sections in electron microscopy: a modified multiple staining technique

A modification of the conventional method for the staining of ultrathin sections resulted in an increase in contrast of ultrastructural detail in tissues. Tissues embedded in Spurr's low viscosity embedding medium were stained with freshly centrifuged Reynolds' lead citrate for 1–5 min, rinsed in double distilled water and dried prior to staining with a saturated solution of uranyl acetate for 40 min, and freshly centrifuged Reynolds' lead citrate for 20 min. Sections treated by this procedure showed enhanced staining of cellular organelles and cytoplasmic matrix. This procedure is recommended for tissues with poor staining qualities resulting from either prolonged fixation or from inadequacies in the buffer or embedding medium used.     

Ultrastructure of unfixed plasma membranes in ultrathin frozen sections

Ultrathin sections of unfixed kidney tubular cells were obtained by cryo-ultramicrotomy using the 'wet' method. The results showed that extremely thin sections permit high resolution studies on unfixed plasma membranes. Structural units on the inner surface of the plasma membrane could be detected.     

Polychromatic Giemsa staining for Epon semi-thin sections

A polychromatic staining procedure which uses the Giemsa stain and differentiates between several cell and tissue structures with distinct colours has been developed for semi-thin sections of glutaraldehyde-fixed, Epon-embedded animal and plant tissues. The method does not require removal of the plastic and can be applied in a simple and rapid way for routine staining of semi-thin sections with good results.     

An oscillating cryo-knife reduces cutting-induced deformation of vitreous ultrathin sections

A new oscillating cryo‐knife for producing uncompressed vitreous sections is introduced. The knife is a modified cryo diamond knife that is driven by a piezo translator. Optimal setting for the oscillation was found to be in the inaudible frequency range of 20–25 kHz. Yeast cells and polystyrene spheres were used as model systems to describe compression in the vitreous sections. We found that compression could be reduced by a factor of about 2 when the knife was oscillating. When the oscillator was turned off, sections were compressed by 40–45%. However, only 15–25% compression was obtained when the knife was oscillating. In some cases completely uncompressed sections of yeast cells were produced. It was also found that the amount of compression depends on the specimen itself and on its embedding medium. With the results shown here, we demonstrate that the oscillating knife can produce high‐quality vitreous sections with minimum cutting artefacts.