Somlyo A V, Shuman H, Somlyo A P
J Cell Biol. 1977 Sep;74(3):828-57. doi: 10.1083/jcb.74.3.828.
A method of rapid freezing in supercooled Freon 22 (monochlorodifluoromethane) followed by cryoultramicrotomy is described and shown to yield ultrathin sections in which both the cellular ultrastructure and the distribution of diffusible ions across the cell membrane are preserved and intracellular compartmentalization of diffusabler ions can be quantitated. Quantitative electron probe analysis (Shuman, H., A.V. Somlyo, and A.P. Somlyo. 1976. Ultramicros. 1:317-339.) of freeze-dried ultrathin cryto sections was found to provide a valid measure of the composition of cells and cellular organelles and was used to determine the ionic composition of the in situ terminal cisternae of the sarcoplasmic reticulum (SR), the distribution of CI in skeletal muscle, and the effects of hypertonic solutions on the subcellular composition if striated muscle. There was no evidence of sequestered CI in the terminal cisternae of resting muscles, although calcium (66mmol/kg dry wt +/- 4.6 SE) was detected. The values of C1 determined with small (50-100 nm) diameter probes over cytoplasm excluding organelles over nuclei or terminal cisternae were not significantly different. Mitochondria partially excluded C1, with a cytoplasmic/ mitochondrial Ci ratio of 2.4 +/- 0.88 SD. The elemental concentrations (mmol/kg dry wt +/- SD) of muscle fibers measured with 0.5-9-mum diameter electron probes in normal frog striated muscle were: P, 302 +/- 4.3; S, 189 +/- 2.9;C1, 24 +/- 1.1;K, 404 +/- 4.3, and Mg, 39 +/- 2.1. It is concluded that: (a) in normal muscle the "excess CI" measured with previous bulk chemical analyses and flux studies is not compartmentalized in the SR or in other cellular organelles, and (b) the cytoplasmic C1 in low K solutions exceeds that predicted by a passive electrochemical distribution. Hypertonic 2.2 X NaCl, 2.5 X sucrose, or 2.2 X Na isethionate produced: (a) swollen vacuoles, frequently paired, adjacent to the Z lines and containing significantly higher than cytoplasmic concentrations of Na and Cl or S (isethionate), but no detectable Ca, and (b) granules of Ca, Mg, and P = approximately (6 Ca + 1 Mg)/6P in the longitudinal SR. It is concluded that hypertonicity produces compartmentalized domains of extracellular solutes within the muscle fibers and translocates Ca into the longitudinal tubules.
本文描述了一种先在过冷的氟利昂22（二氟一氯甲烷）中快速冷冻，然后进行冷冻超薄切片术的方法，结果显示该方法能获得超薄切片，其中细胞超微结构以及可扩散离子跨细胞膜的分布得以保留，并且可对可扩散离子的细胞内区室化进行定量分析。通过对冻干的超薄冷冻切片进行定量电子探针分析（舒曼，H.，A.V. 索姆利奥，和A.P. 索姆利奥。1976年。《超微结构》1:317 - 339），发现其能有效测量细胞和细胞器的成分，并用于确定肌浆网（SR）原位终末池的离子成分、氯离子在骨骼肌中的分布以及高渗溶液对横纹肌亚细胞成分的影响。静息肌肉的终末池中没有氯离子隔离的证据，尽管检测到了钙（66mmol/kg干重±4.6标准误）。用小直径（50 - 100纳米）探针在排除细胞核或终末池上的细胞器的细胞质上测定的Cl值没有显著差异。线粒体部分排除氯离子，细胞质/线粒体氯离子比值为2.4±0.88标准差。在正常青蛙横纹肌中，用直径0.5 - 9微米的电子探针测量的肌纤维元素浓度（mmol/kg干重±标准差）为：磷，302±4.3；硫，189±2.9；氯，24±1.1；钾，404±4.3，以及镁，39±2.1。得出以下结论：（a）在正常肌肉中，先前通过大量化学分析和通量研究测得的“过量氯离子”并非隔离在肌浆网或其他细胞器中；（b）在低K溶液中，细胞质中的氯离子超过被动电化学分布所预测的值。高渗的2.2倍氯化钠、2.5倍蔗糖或2.2倍羟乙基磺酸钠会产生：（a）与Z线相邻的肿胀液泡，常成对出现，其中钠、氯或硫（羟乙基磺酸钠）的浓度明显高于细胞质，但未检测到钙；（b）纵行肌浆网中有钙、镁和磷的颗粒，其比例约为(6钙 + 1镁)/6磷。得出结论，高渗会在肌纤维内产生细胞外溶质的区室化区域，并使钙转运到纵管中。
