Rapid Freezing using Sandwich Freezing Device for Good Ultrastructural Preservation of Biological Specimens in Electron Microscopy.

Chemical fixation has been used for observing the ultrastructure of cells and tissues. However, this method does not adequately preserve the ultrastructure of cells; artifacts and extraction of cell contents are usually observed. Rapid freezing is a better alternative for the preservation of cell structure. Sandwich freezing of living yeast or bacteria followed by freeze-substitution has been used for observing the exquisite natural ultrastructure of cells. Recently, sandwich freezing of glutaraldehyde-fixed cultured cells or human tissues has also been used to reveal the ultrastructure of cells and tissues. These studies have thus far been carried out with a handmade sandwich freezing device, and applications to studies in other laboratories have been limited. A new sandwich freezing device has recently been fabricated and is now commercially available. The present paper shows how to use the sandwich freezing device for rapid freezing of biological specimens, including bacteria, yeast, cultured cells, isolated cells, animal and human tissues, and viruses. Also shown is the preparation of specimens for ultrathin sectioning after rapid freezing and procedures for freeze-substitution, resin embedding, trimming of blocks, cutting of ultrathin sections, recovering of sections, staining, and covering of grids with support films.

Sandwich freezing device for rapid freezing of viruses, bacteria, yeast, cultured cells and animal and human tissues in electron microscopy.

We have been using sandwich freezing of living yeast and bacteria followed by freeze-substitution for observing close-to-native ultrastructure of cells. Recently, sandwich freezing of glutaraldehyde-fixed cultured cells and human tissues have been found to give excellent preservation of ultrastructure of cells and tissues. These studies, however, have been conducted using a handmade sandwich freezing device and have been limited in a few laboratories. To spread the use of this method to other laboratories, we fabricated and commercialized a new sandwich freezing device. The new device is inexpensive, portable and sterilizable. It can be used to rapid-freeze viruses, bacteria, yeast, cultured cells and animal and human tissues to a depth of 0.2 mm if tissues are prefixed with glutaraldehyde. The commercial availability of this device will expand application of rapid freezing to wide range of biological materials.

Use of rocuronium-sugammadex, an alternative to succinylcholine, as a muscle relaxant during electroconvulsive therapy.

We compared the recovery time from neuromuscular blockade induced by rocuronium combined with sugammadex versus succinylcholine during electroconvulsive therapy (ECT). Anesthesia was induced using propofol, followed by succinylcholine (1 mg/kg) or rocuronium (0.6 mg/kg). Immediately after the seizure stopped, 16 mg/kg sugammadex was infused. Neuromuscular monitoring was performed and continued until recovery of the train-of-four ratio to 0.9. We compared the recovery time of T1 to 10 and 90% between groups. Patients were also assessed for clinical signs, such as time to first spontaneous breath from the administration of muscle relaxant and eye opening to verbal commands. Although recovery time of T1 to 10 and 90% in the rocuronium-sugammadex group was shorter than in the succinylcholine group, the difference was not statistically significant. Further, the seizure duration with succinylcholine (33 ± 8 s) was shorter than that with rocuronium-sugammadex (39 ± 4 s). In conclusion, this study demonstrates the potential benefit of use of rocuronium-sugammadex as an alternative to succinylcholine for muscle relaxation during ECT.