An improved cryofixation method: cryoquenching of small tissue blocks during microwave irradiation

The metal contact method of rapid freezing is greatly improved by irradiating the specimen with microwaves at 2.45 GHz for a short period of time (50 ms), while pushing the specimen onto the surface of the copper block cooled by liquid N2. The microwave irradiation, together with two technical improvements (a light-mass plunger and a recently developed beta-gel shock absorber) for preventing bounce, produces a good freezing zone for squid retina, with high reproducibility for each experimental trial, extending from the contact surface to a depth of about 15 microns, which is comparable to the depth obtained by the metal contact method using liquid He in the absence of microwave irradiation. A good freezing zone was also experimentally demonstrated in specimens of rat liver and heart muscle. Microwave irradiation does not have appreciable effects on the ultrastructure of squid retina. The mechanism underlying the improvement in the rapid freezing under the microwave irradiation is discussed.