Applications of Freeze-substitution to Electron Microscope Studies of Invertebrate Oocytes

A modified freeze-substitution process is described which gives a low percentage (less than 5 per cent) of preparations of invertebrate eggs which appear to be ice crystal-free at the resolution of the electron microscope. The mitochondria show no membranes in these preparations but can be recognized by internal spaces with the size and the distribution of the cristae. The Golgi bodies resemble those seen with diffusion fixatives, but the limiting membranes are here double; that is, they appear to be a triple-layered sandwich with two outer dark approximately 25A layers and an inner light layer of the same thickness. The endoplasmic reticulum is clearly present and resembles that seen with diffusion fixatives. Here again, the limiting membranes are double with the same dimensions as those in the Golgi bodies. The membranes of the Golgi bodies and ER are seen after permanganate but not lead hydroxide staining. The hyaloplasm (or "cell sap") is crowded with 150 to 200A particles and these are also seen lining the ER membranes. In general, the structures as seen with the present technique show considerable similarity to those seen with diffusion fixatives.