Sybers H D, Myre C D, Myre M V
Scan Electron Microsc. 1983(Pt 2):769-76.
X-ray microanalysis of biological material is best accomplished on unfixed frozen tissue sectioned on a cryoultramicrotome. The need for ultra-rapid freezing to minimize ice crystal artifact has been well documented but is difficult to achieve without the use of potentially hazardous Isopentane or Freon 22 in liquid nitrogen slush. In the present study we employ a slurry of powdered graphite in liquid nitrogen to obtain rapid freezing of cardiac tissue. The results obtained were as good as those which are achieved with Freon 22 in liquid nitrogen, and subsequent cryoultramicrotomy produced thin sections relatively free of ice crystal artifact. Microanalysis revealed a slight increase in mitochondrial calcium as compared with cytoplasmic calcium in normal myocytes. Mitochondrial calcium concentration rose significantly after 30 min of ischemia while only a slight rise in cytoplasmic calcium occurred. These findings suggest that the techniques employed are adequate for detecting ion shifts which occur during ischemia and may be useful for determining the effects of therapeutic agents at the organellar level.
对生物材料进行X射线微分析，最好在使用冷冻超薄切片机切片的未固定冷冻组织上进行。超快速冷冻以尽量减少冰晶伪像的必要性已有充分记录，但如果不在液氮浆中使用潜在危险的异戊烷或氟利昂22，很难实现。在本研究中，我们使用液氮中的石墨粉浆来快速冷冻心脏组织。获得的结果与在液氮中使用氟利昂22所取得的结果一样好，随后的冷冻超薄切片术产生了相对没有冰晶伪像的薄片。微分析显示，与正常心肌细胞的细胞质钙相比，线粒体钙略有增加。缺血30分钟后，线粒体钙浓度显著升高，而细胞质钙仅略有升高。这些发现表明，所采用的技术足以检测缺血期间发生的离子转移，并且可能有助于确定治疗药物在细胞器水平上的作用。
