Abstract: Cryo-electron microscopy (cryo-EM), a major tool in structure biology, is used to reveal the three dimensional structures of important functional protein machineries.Since crystallization is not required, sample preparation in cryo-EM is less complicated and the sample is preserved in a more native state compared with other tools in structure biology such as X-ray crystallography. Recent progress in hardware and software development of this technique has not only improved the resolution of the reconstructions but also increased the range of applications from viral particles with a diameter above 100 nm to protein complexes with a diameter around 10 nm. Now, a 3~4 Å resolution can be regularly achieved, which is adequate for answering most biological questions. In this review we describe the theoretical basis as well as the current progress of high resolution single particle analysis. The remaining challenges are also discussed.