In situ fiducial markers for 3D correlative cryo-fluorescence and FIB-SEM imaging

iScience

Nadav Scher¹  Ori Avinoam¹  Katya Rechav²  Perrine Paul-Gilloteaux³ 

[1] Department of Biomolecular Sciences, Weizmann Institute of Science, Rehovot, Israel;Department of Chemical Research Support, Weizmann Institute of Science, Rehovot, Israel;Structure Fédérative de Recherche François Bonamy, INSERM, CNRS, Université de Nantes, Nantes, France;

关键词: Cell biology;    Technical aspects of cell biology;    Structural biology;   

DOI  :  

来源: DOAJ

【 摘 要 】

Summary: Imaging of cells and tissues has improved significantly over the last decade. Dual-beam instruments with a focused ion beam mounted on a scanning electron microscope (FIB-SEM), offering high-resolution 3D imaging of large volumes and fields-of-view are becoming widely used in the life sciences. FIB-SEM has most recently been implemented on fully hydrated, cryo-immobilized, biological samples. Correlative light and electron microscopy workflows combining fluorescence microscopy (FM) with FIB-SEM imaging exist, whereas workflows combining cryo-FM and cryo-FIB-SEM imaging are not yet commonly available. Here, we demonstrate that fluorescently labeled lipid droplets can serve as in situ fiducial markers for correlating cryo-FM and FIB-SEM datasets and that this approach can be used to target the acquisition of large FIB-SEM stacks spanning tens of microns under cryogenic conditions. We also show that cryo-FIB-SEM imaging is particularly informative for questions related to organelle structure and inter-organellar contacts, nuclear organization, and mineral deposits in cells.

【 授权许可】

Unknown