Serial section immunoelectron microscopy of algal cells

Abstract

Electron microscopy when combined with immunogold labeling provides a 2D image of intracellular protein distribution. Cells are however 3D structures. We describe a method of serial section immunogold electron microscopy that allows a 3D cellular image to be reconstructed from a series of electron micrographs. Cells are fixed to preserve cellular ultrastructure and they are embedded in plastic allowing ultrathin sections to be obtained. The ribbon of ultrathin serial sections produced as the microtome sequentially cuts through the sample is labeled with a monospecific antibody to the protein of interest and then with protein-A gold making the antigen-antibody complex visible in the electron microscope. A common field of view from each serial section is photographed in the electron microscope. Using image analysis software, each digitized micrograph is sequentially aligned; immunolabel and cellular structures of interest are traced onto each micrograph; the micrographs are stacked; and the structures of interest are rendered as solid surfaces producing a 3D image of protein distribution within the cell.