Comparative analysis of viral particles using different electron microscopy methodologies

Semliki Forest Virus (SFV) can enter a cell by fusing with the host membrane. To obtain detailed insight of the enveloped viruses, it is necessary to follow the progression in a sensitive and continuous fashion. In this study, the process of budding SFV was analyzed by combining techniques of electron tomography and high pressure frozen (HPF)/freeze-substitution (FS). The tomograms were examined by comparing them to a three dimensional (3D) reconstruction of purified SFV obtained from single particle reconstruction techniques and the size variation of budding SFV particles was examined. Here we report a comparison between six experimental parameters in samples imaged for tomography: osmium fixation condition (Fig. 1), section thickness, post-staining, usage of support film, single vs. double tilt and specimen temperature during data collection. The resulting measurements were compared with average measurements of frozen-hydrated purified specimens. We found that the structure of the virus particles reconstructed tomographically correlated well with the electron density determined from frozen-hydrated samples. The data showed that different experimental methods can be used to observe specific details (membrane vs. neucleocapsid). Our experiment showed that electron tomography (ET) is a reliable method for 3D modeling. The data also revealed biologically important information such as the insertion of the viral protein into the target membrane. By using ET for reconstruction together with HPF & FS for sample preparation, the measurement of whole viral particles showed the consistency of virus dimension with a systemic reduction of 15% in diameter, to that of the isolated single particle reconstruction with cryomicoscopy (Fig. 2).