Frank L, Nebesářová J, Vancová M, Paták A, Müllerová I
Institute of Scientific Instruments AS CR, v.v.i., Královopolská 147, 61264 Brno, Czech Republic.
Biology Centre AS CR, v.v.i., Branišovská 31, 37005 České Budějovice, Czech Republic.
Ultramicroscopy. 2015 Jan;148:146-150. doi: 10.1016/j.ultramic.2014.10.009. Epub 2014 Oct 27.
The examination of thin sections of tissues with electron microscopes is an indispensable tool. Being composed of light elements, samples of living matter illuminated with electrons at the usual high energies of tens or even hundreds of kiloelectronvolts provide very low image contrasts in transmission or scanning transmission electron microscopes. Therefore, heavy metal salts are added to the specimen during preparation procedures (post-fixation with osmium tetroxide or staining). However, these procedures can modify or obscure the ultrastructural details of cells. Here we show that the energy of electrons used for the scanned transmission imaging of tissue sections can be reduced to mere hundreds or even tens of electronvolts and can produce extremely high contrast even for samples free of any metal salts. We found that when biasing a sufficiently thin tissue section sample to a high negative potential in a scanning transmission electron microscope, thereby reducing the energy of the electrons landing on the sample, and collecting the transmitted electrons with a grounded detector, we obtain a high contrast revealing structure details not enhanced by heavy atoms. Moreover, bombardment with slow electrons sensitively depolymerises the resin in which the tissue is embedded, thereby enhancing the transmitted signal with no observable loss of structure details. The use of low-energy electrons requires ultrathin sections of a thickness of less than 10nm, but their preparation is now possible. Ultralow energy STEM provides a tool enabling the observation of very thin biological samples without any staining. This method should also be advantageous for examination of 2D crystals, thin films of polymers, polymer blends, etc.
使用电子显微镜检查组织薄片是一种不可或缺的工具。由于生物样品由轻元素组成，在通常数十甚至数百千电子伏特的高能量下用电子照射时，在透射电子显微镜或扫描透射电子显微镜中提供的图像对比度非常低。因此，在制备过程中（用四氧化锇后固定或染色）会向标本中添加重金属盐。然而，这些程序可能会改变或掩盖细胞的超微结构细节。在这里，我们表明用于组织切片扫描透射成像的电子能量可以降低到仅数百甚至数十电子伏特，即使对于不含任何金属盐的样品也能产生极高的对比度。我们发现，当在扫描透射电子显微镜中将足够薄的组织切片样品偏置到高负电位，从而降低落在样品上的电子能量，并用接地探测器收集透射电子时，我们获得了高对比度，揭示了未被重原子增强的结构细节。此外，用慢电子轰击会使包埋组织的树脂敏感地解聚，从而增强透射信号，而不会观察到结构细节的损失。使用低能电子需要厚度小于10nm的超薄切片，但现在可以制备。超低能量扫描透射电子显微镜提供了一种工具，能够在不进行任何染色的情况下观察非常薄的生物样品。这种方法对于二维晶体、聚合物薄膜、聚合物共混物等的检查也应该是有利的。
