Focused Ion Beam Scanning Electron Microscopy (FIB-SEM) Imaging of intracellular Ciliogenesis in RPE-1 Cells

cssi_iCiliary

Retinal pigmented epithelial cells (RPE-1) expressing GFP-EHD1 and SMO-tRFP were grown on alpha numerical grided coverglass. A cell containing GFP-EHD1 in the ciliary pocket membrane was visualize by fluorescence microscopy and after fixing, staining and resin embedding, this cells was located and imaged by focused ion beam scanning electron microscopy (FIB-SEM). The raw image stack was aligned, binned and contrast-inverted to yield an image volume with XX nm isotropic (cubic) pixel sampling. These image volumes were examined for the presence of membrane tubules and were used for segmentation and 3-D reconstruction of these structures.