ENERGY-FILTERED ELECTRON TOMOGRAPHY OF ICE-EMBEDDED ACTIN AND VESICLES

Semiautomatic single-axis tilt electron tomography has been used to vi sualize the three-dimensional organization of actin filaments in ''pha ntom cells,'' i.e. lipid vesicles, The instrumentation consisted of a 120-kV electron microscope equipped with a postcolumn energy filter, w hich was used in the zero-loss imaging mode, Apart from changing the t ilt angle, all steps required for automated tomography, such as recent ering the image area, refocusing, and centering the energy-selecting s lit, were performed by external computer control, This setup permitted imaging of ice-embedded samples up to a thickness of 800 nm with impr oved image contrast compared with that produced by tomography with a c onventional electron microscope, In spite of the missing-wedge effect that is especially obvious in the study of membrane-filament interacti on, single-axis tilt tomography was found to be an appropriate (in fac t the only available) method for this kind of investigation, In contra st to random actin networks found in actin gels, actin filaments in an d on vesicles with a bending radius of less than similar to 2 mu m ten d to be arranged in single layers of parallel filaments and often indu ce an elongated shape of the vesicles. Actin filaments located on the outside usually associate with the vesicle membrane.