In situ fiducial markers for 3D correlative cryo-fluorescence and FIB-SEM imaging
Abstract
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 mi-croscope (FIB-SEM), offering high-resolution 3D imaging of large volumes andfields-of-view are becoming widely used in the life sciences. FIB-SEM has mostrecently been implemented on fully hydrated, cryo-immobilized, biological sam-ples. Correlative light and electron microscopy workflows combining fluores-cence microscopy (FM) with FIB-SEM imaging exist, whereas workflowscombining cryo-FM and cryo-FIB-SEM imaging are not yet commonly available.Here, we demonstrate that fluorescently labeled lipid droplets can serve asinsitufiducial markers for correlating cryo-FM and FIB-SEM datasets and that thisapproach can be used to target the acquisition of large FIB-SEM stacks spanningtens of microns under cryogenic conditions. We also show that cryo-FIB-SEM im-aging is particularly informative for questions related to organelle structure andinter-organellar contacts, nuclear organization, and mineral deposits in cells.