Three-Dimensional Observation of the Intracellular Membrane Structure in Human Myocardium : High-Resolution Scanning Electron Microscopy by the Osmium-DMSO-Osmium Method
S canning electron microscopy (SEM) can directly observe the 3D structures of a cell with high resolution. Tanaka and Naguro 1 reported a unique SEM method (osmium-DMSO-osmium method) to observe the 3D structure of the membrane system to remove cytoplasmic matrix much more effectively than any other method. With this method, the 3D features of the endoplasmic reticulum, Golgi complex, mitochondria, transverse tubules, intercalated disks, surface vesicles, and sarcolemma of a cell can be clearly
... observed. 1, 2 We first applied this method to tissue specimens of human left ventricular myocardium from 2 patients with dilated cardiomyopathy who underwent partial left ventriculectomy. The tissue specimens of the left ventricular wall resected during surgery were treated by the osmium-DMSO-osmium method as reported elsewhere. 1,2 Briefly, the small tissue specimens were fixed in 1% osmium tetroxide in 0.05 mol/L phosphate buffer (pH 7.2). After the tissue specimens had been immersed in DMSO, they were frozen on a metal plate, chilled with liquid nitrogen, and then cracked. After the cracked pieces had been rinsed in buffer solution, they were transferred into 0.1% osmium tetroxide in 0.05 mol/L PBS and left to stand at room temperature for 72 hours. After being postfixed with 1% osmic solution, they were treated with 2% tannic acid and 1% osmic solution for conductive staining. The specimens were dehydrated in graded concentrations of ethanol and freeze-dried in t-butyl alcohol. The tissue specimens were then coated with platinum 2 nm thick and observed under a Hitachi S-5000 scanning electron microscope. References 1. Tanaka K, Naguro T. High resolution scanning electron microscopy of cell organelles by a new specimen preparation method. Biomed Res. 1981;2(suppl):63-70. 2. Ogata T, Yamasaki Y. High-resolution scanning electron microscopic studies on the three-dimensional structure of the transverse-axial tubular system, sarcoplasmic reticulum and intercalated disc of the rat myocardium. Anat Rec. 1990;228:277-287.