Leforestier Amélie, Levitz Pierre, Preat Thomas, Guttmann Peter, Michot Laurent J, Tchénio Paul
Laboratoire de Physique des Solides, UMR8502 CNRS-Université Paris 11, Bâtiment 510, 91405 Orsay Cedex, France.
PHENIX Laboratory, UMR 8234, CNRS-Université Pierre et Marie Curie, 75252 Paris, France.
J Struct Biol. 2014 Nov;188(2):177-82. doi: 10.1016/j.jsb.2014.09.012. Epub 2014 Oct 6.
Cryo-soft X-ray microscopy is an emerging imaging tool complementary to cryo-electron microscopy, allowing to image frozen hydrated specimens ten to hundred times thicker, but at lower resolution. We describe how the method, so far restricted to isolated small cells or cell monolayers, can be extended to large cells and tissue. We image the synapses of the Kenyon cells in frozen hydrated Drosophila brains combining cryo-soft X-ray microscopy of thick vitreous sections, and cryo-electron microscopy of ultrathin vitreous sections. We show how to obtain frozen hydrated sections of thicknesses ranging from 40 nm up to 2.5 μm, by tuning the sectioning speed of the cryo-microtome. A fluorescent stereo-microscope mounted on the cryo-microtome allowed us to target the regions of interest after GFP-labeling of synapses. Thick cryo-sections were imaged by cryo-soft X-ray microscopy at a resolution better than 25 nm, while ultrathin cryo-sections of the same regions were explored in parallel at the nanometre level of resolution by cryo-electron microscopy.
低温软X射线显微镜是一种新兴的成像工具，可作为低温电子显微镜的补充，能够对厚度比其厚10到100倍的冷冻水合标本进行成像，但分辨率较低。我们描述了迄今为止仅限于分离的小细胞或细胞单层的该方法如何扩展到大型细胞和组织。我们结合厚玻璃切片的低温软X射线显微镜和超薄玻璃切片的低温电子显微镜，对冷冻水合果蝇大脑中的肯扬细胞突触进行成像。我们展示了如何通过调整低温切片机的切片速度来获得厚度从40纳米到2.5微米不等的冷冻水合切片。安装在低温切片机上的荧光立体显微镜使我们能够在对突触进行绿色荧光蛋白标记后靶向感兴趣的区域。厚冷冻切片通过低温软X射线显微镜以优于25纳米的分辨率成像，而同一区域的超薄冷冻切片则通过低温电子显微镜在纳米分辨率水平上并行进行探索。
