Use of micromanipulator for continuous observation of frozen samples by cryoscanning electron microscopy and freeze replicas

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AbstractOur previous report described how a new cryoscanning electron microscope (cryo‐SEM) allowed for continuous observation of certain areas of any particular frozen sample, first, by cryo‐SEM and, after dissolution of the sample, by freeze replication using a transmission electron microscope. However, the latter procedure made continuous observation of individual areas which were selected by cryo‐SEM impossible, because the selective withdrawal of replicas, containing the desired areas could not be accomplished. Here we report how this problem was resolved using a cryo‐SEM equipped with a micromanipulator. Specimen surfaces, including areas of interest, were cut out by a thin needle attached to the micromanipulator while under observation with the SEM. Replicas from the cut areas alone were withdrawn and used for the structural correspondence studies. Using this process, continuous observation of the selected areas was successfully performed.