A Simple Method for Quick-Freezing

In conventional freeze-fracture replicas produced from tissue cryoprotected with glycerol, the hydrophobic inner surfaces of membranes are revealed, but hydrophillic structures are obscured in the surrounding ice. Quick-freezing of tissue obviates the need for glycerol, which prevents the removal of this ice by etching or freeze-drying, but the major problem in freezing without glycerol cryoprotection is ice crystal formation. We describe here a simple method for quick-freezing tissue, in the absence of glycerol, on a nitrogen-cooled copper block with a hand-held specimen holder. This method freezes samples well enough to preserve molecular detail that can be revealed by subsequent etching. We show some examples of the quality of this freezing with respect to the visualization of molecular detail in isolated protein molecules such as ferritin and catalase. Furthermore, we show examples of in situ cellular structures that are revealed by this method, and we compare the structure seen in these replicas with structures preserved by quick-freezing at liquid helium temperatures.