Correlated light and electron microscopy of the cytoskeleton

The cytoskeleton of cultured cells can be most easily visualized in the electron microscope by simultaneous extraction and fixation with Triton-glutaraldehyde mixtures, followed by negative staining. Actin filaments are better preserved by stabilization with phalloidin, either during or after the primary fixation step. A technique is described for the combination of this procedure with live cell microscopy. Optimal conditions for light microscopy are achieved by culturing cells on coverslips coated with formvar film. For cell relocation a gold finder grid pattern is embossed on the film by evaporation through a tailor-made mask. After video microscopy and fixation, the film is floated from the coverslip and an electron microscope grid added to the film with the central hole of the grid over the region of interest. Accurate positioning is achieved under a dissecting microscope, using forceps mounted in a micromanipulator. Examples are shown of the changes in organization of actin filaments in the lamellipodia of migrating melanoma cells resulting from changes in protrusion rate. The technique is applicable to alternative processing procedures after fixation, including cryoelectron tomography.