3D electron microscopic imaging using Focused-Ion Beam Scanning Electron Microscopy (FIB-SEM)
This technique enables the acquisition of 3-dimensional (volume) information about the distribution of organelles and cellular structure at nanometer resolution.
Samples are prepared much as they are for standard transmission electron microscopy (epoxy resin embedding) but with the incorporation of more heavy metals to facilitate imaging under the scanning electron beam. A small area of interest must be identified either by optical microscopy or scanning EM of the block surface. A trench is then created with the ionizing beam so that a vertical face through the depth of the sample is exposed. That face is then imaged using the scanning electron beam. The ionizing beam is then used to remove material from the block and the newly exposed face is again imaged. This process is repeated for hundreds or thousands of iterations. The cumulative set of images can then be reconstructed to create a 3-D data set.
Samples for this technique can be prepared in the Electron Microscopy & Histology Core at WCM.
For discussing sample preparation for FIB-SEM, please email Lee Cohen-Gould at firstname.lastname@example.org.
Imaging is carried out using FEI Helios 650, located at the NEW YORK STRUCTURAL BIOLOGY CENTER, of which Weill Cornell Medicine is a full member.
An example of a 3D dataset obtained with FIB-SEM
The movie below shows extracellular compartments at the macrophage-adipocyte interface that are able to hold a pH gradient.
Reference: "Exocytosis of macrophage lysosomes leads to digestion of apoptotic adipocytes and foam cell formation" by Haka et al., J Lipid Res. 2016 Jun; 57(6): 980–992.