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(Circulation. 2000;101:2328.)
© 2000 American Heart Association, Inc.

Images in Cardiovascular Medicine

Three-Dimensional Observation of the Intracellular Membrane Structure in Human Myocardium

High-Resolution Scanning Electron Microscopy by the Osmium-DMSO-Osmium Method

Makoto Okabe, MD; Yumiko Kanzaki, MD; Hiroaki Shimomura, MD; Fumio Terasaki, MD; Tetsuya Hayashi, MD; Yasushi Kitaura, MD

From the Third Division, Department of Internal Medicine, Osaka Medical College, Takatsuki City, Osaka, Japan.

Correspondence to Makoto Okabe, MD, Third Division, Department of Internal Medicine, Osaka Medical College, 2–7, Daigaku-machi, Takatsuki City, Osaka, 569-8686, Japan. E-mail in3013{at}poh.osaka-med.ac.jp

Scanning electron microscopy (SEM) can directly observe the 3D structures of a cell with high resolution. Tanaka and Naguro¹ 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}

View larger version (106K): [in this window] [in a new window]   Figure 1. Scanning electron micrographs of membrane system in left ventricular myocardium from patients with dilated cardiomyopathy. A, Meshwork of sarcoplasmic reticulum that develops beneath sarcolemma of cardiomyocyte. It occasionally forms flat cisternal regions (arrows). Rows of mitochondria (M) are seen among meshwork. Bar=1.0 µm. Magnification x13 000. B, Mitochondria in a cardiomyocyte show various sizes and shapes. Note that some of these mitochondria have circular cristae (arrows). Bar=1.0 µm. Magnification x15 000. C, Fibroblast in a focus of myocardial fibrosis is filled with stacks of cisternae of rough endoplasmic reticulum (RER). Outer surface of cisternae is densely studded with ribosomes (arrows). Golgi complex (G) is formed by stacks of saccules associated with small vesicles (arrowheads). CF, collagen fibrils. Bar=1.0 µm. Magnification x13 000.

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.

Footnotes

The editor of Images in Cardiovascular Medicine is Hugh A. McAllister, Jr, MD, Chief, Department of Pathology, St Luke’s Episcopal Hospital and Texas Heart Institute, and Clinical Professor of Pathology, University of Texas Medical School and Baylor College of Medicine.

Circulation encourages readers to submit cardiovascular images to the Circulation editorial office, St Luke’s Episcopal Hospital and Texas Heart Institute, 6720 Bertner Ave, MC1-267, Houston, TX 77030.

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.[Medline] [Order article via Infotrieve]
   

This Article Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Okabe, M. Articles by Kitaura, Y. Search for Related Content PubMed PubMed Citation Articles by Okabe, M. Articles by Kitaura, Y. Related Collections Structure Cell biology/structural biology Heart failure - basic studies Imaging