Preparation of normal and ischemic myocardial tissue for scanning electron microscopy

Normal and ischemic myocardium was prepared by slicing and cryofracture techniques for examination with the SEM. The tissues were dehydrated in ethanol and Freon 113 (slicing method) or were cryofractured in Freon 113 cooled with liquid nitrogen, followed by critical point drying and coating with gold and palladium. Some osmicated tissue pieces were treated with thiocarbohydrazide for examination without metal coating. Some hearts were infiltrated with silver particles and were examined with backscattered electron imaging technique (BSI). The cryofractured tissue provided the most useful and consistent surface features of the cell organelles with a minimum of mechanical disorder compared to other methods. Although the myocardium prepared by slicing method revealed the interior of the cells, it contained several frayed edges and loose myofibrils making interpretation of the ischemic myocardium difficult. The normal cells exhibited myofibrils covered by transverse tubules at the level of Z bands as confirmed by BSI using silver particles as an extracellular tracer and numerous oval to elongated mitochondria located between the myofibrils. An extensive network of tubules (sarcoplasmic reticulum) over the sarcomeres and nuclei were observed. The changes observed by SEM in the ischemic hearts were consistent with those seen in TEM. With prolonged coronary ligation the changes became obvious. The T-tubules were often broken and nuclei were distorted. The myofibrils were disorganized and formed homogeneous bands. These SEM studies suggest that myocardium prepared by cryofracture technique yields information on normal and ischemic cell structure consistent with data obtained by TEM.