Freeze‐substitution

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Abstract
Freeze-substitution is a technique suitable for the preparation of unicellular and multicellular plant and animal specimens for conventional light microscopy, TEM and SEM. It is also widely used as a means of preparing animal and plant tissues for the localization of water soluble substances by analytical electron microscopy, autoradiography or visual detection of precipitates. The technical requirements of preparation, together with an evaluation of the procedures, are presented for various applications. Careful selection and evaluation of freezing technique, substitution solvent and regime are required for meaningful results.