A stopped-flow/rapid-freezing machine with millisecond time resolution to prepare intermediates in biochemical reactions for electron microscopy.
We have developed an instrument capable of freezing transient intermediates in rapid biochemical reactions for subsequent freeze-fracturing, replication, and viewing by transmission electron microscopy. The machine combines a rapid mixing unit similar to one widely used in chemical kinetics (Johnson, 1986) with a propane jet freezing unit previously used to prepare static samples for freeze-fracturing (Gilkey and Staehelin, 1986). The key element in the system is a unique thin-walled flow cell of copper that allows for injection and aging of the sample, followed by rapid freezing. During freeze-fracturing, a tangential cut is made along the wall of the flow cell to expose the sample for etching and replication. The dead time required for mixing and injection of the reactants into the flow cell is less than 5 ms. Electronic controls allow one to specify, on a millisecond time scale, any time above 5 ms between initiation of the reaction and quenching by rapid freezing.
Department of Cell Biology and Anatomy, Johns Hopkins Medical School, Baltimore, Maryland 21205.
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Pollard
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Maupin
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Sinard
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Huxley
D000199:Actins / Q000648:ultrastructure; D000818:Animals; D004867:Equipment Design; D005614:Freeze Fracturing / Q000295:instrumentation*; D008854:Microscopy, Electron / Q000379:methods*; D009218:Myosins / Q000648:ultrastructure
D000199:Actins; D009218:Myosins