Diamond knife

Abstract: Summary 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.

Abstract: The process of serial sectioning for electron microscopy has been refined such that loss of thin sections is kept below 0.1% and the series is continued at will. The method relies on microscopic control of all manipulative steps, Formvar casting on plate glass for coated slot grids, coating of the block with contact cement for reliable ribboning, pickup by a one-step method with grid support in the diamond knife trough, staining in LKB grid holders, gentle treatment of grids in the electron microscope, and a slight modification to the microscope for safe grid withdrawal. The results are particularly applicable to the reconstruction of neuronal microcircuits and larger volumes of neuropil.