Bello M Angélica, Ruiz-León Yolanda, Sandoval-Sierra J Vladimir, Rezinciuc Svetlana, Diéguez-Uribeondo Javier
Biodiversity and Conservation Department, Real Jardín Botánico, CSIC;
Research Support Unit, Real Jardín Botánico, CSIC.
J Vis Exp. 2017 Feb 3(120):55031. doi: 10.3791/55031.
Common problems in the processing of biological samples for observations with the scanning electron microscope (SEM) include cell collapse, treatment of samples from wet microenvironments and cell destruction. Using young floral tissues, oomycete cysts, and fungi spores (Agaricales) as examples, specific protocols to process delicate samples are described here that overcome some of the main challenges in sample treatment for image capture under the SEM. Floral meristems fixed with FAA (Formalin-Acetic-Alcohol) and processed with the Critical Point Dryer (CPD) did not display collapsed cellular walls or distorted organs. These results are crucial for the reconstruction of floral development. A similar CPD-based treatment of samples from wet microenvironments, such as the glutaraldehyde-fixed oomycete cysts, is optimal to test the differential growth of diagnostic characteristics (e.g., the cyst spines) on different types of substrates. Destruction of nurse cells attached to fungi spores was avoided after rehydration, dehydration, and the CPD treatment, an important step for further functional studies of these cells. The protocols detailed here represent low-cost and rapid alternatives for the acquisition of good-quality images to reconstruct growth processes and to study diagnostic characteristics.
在使用扫描电子显微镜（SEM）进行观察时，生物样品处理过程中的常见问题包括细胞塌陷、来自潮湿微环境的样品处理以及细胞破坏。以幼嫩花组织、卵菌囊肿和真菌孢子（伞菌目）为例，本文描述了处理精细样品的具体方案，这些方案克服了在SEM下进行图像采集时样品处理中的一些主要挑战。用FAA（福尔马林-乙酸-酒精）固定并用临界点干燥器（CPD）处理的花分生组织未显示出细胞壁塌陷或器官变形。这些结果对于花发育的重建至关重要。对来自潮湿微环境的样品（如戊二醛固定的卵菌囊肿）进行类似的基于CPD的处理，对于测试不同类型底物上诊断特征（如囊肿刺）的差异生长是最佳的。在复水、脱水和CPD处理后，避免了附着在真菌孢子上的营养细胞的破坏，这是对这些细胞进行进一步功能研究的重要步骤。本文详细介绍的方案是获取高质量图像以重建生长过程和研究诊断特征的低成本且快速的替代方法。
