The contracting muscle: a challenge for freeze-substitution and low temperature embedding

Frog sartorius and semitendinosus muscles are quick-frozen either in the resting state or during contraction by means of a LN2 cooled falling copper block. The frozen specimens are freeze-substituted (acetone + OsO4 + uranyl acetate) in a REICHERT JUNG CS auto and either embedded in Spurr's resin and polymerised at a high temperature (60 degrees C) or embedded and polymerised in the Lowicryls K4M, K11M or HM23 at low temperatures (below -30 degrees C). Excellent morphological results are obtained when freeze-substitution, embedding and polymerisation are all carried out below -50 degrees C. Muscles in which a major portion of cellular K+ ions has been replaced by electron dense Cs+ or Tl+ ions are also cryofixed at rest or during contraction, freeze-substituted in pure acetone for 1 week at -80 degrees C and polymerised in K11M at -60 degrees C. A characteristic uneven distribution of the electron dense ions--known from earlier published control experiments--can be observed in sections of resting muscles. Electrically stimulated muscles show ion redistribution. It is concluded that freeze-substitution and low temperature embedding of quick-frozen contracting muscle may be used to investigate changes of ultrastructure, redistribution of cellular water and intracellular movements of mobile ions during muscle contraction.