Electron microscopy of frozen hydrated eukaryotic flagella

Resting and active sea urchin sperm flagella have been examined by low-dose electron microscopy of frozen hydrated specimens. The flagella are unfixed, unstained, completely intact, and able to swim vigorously after going through the entire preparative procedure. The most prominent features of the image arise from the edges of the axonemal doublets and central-pair microtubules seen in projection. By comparison with these longitudinal markings, transverse features are less easy to discern, being camouflaged by superposition. However, Fourier transforms of digitized micrographs reveal a remarkable degree of crystalline order in quiescent flagella. Filtered images derived from these Fourier transforms show clearly features arising from the central-pair complex and radial spokes that were obscured in the original data. Potentially complicating effects of specimen thickness are shown to be quantitatively insignificant in the formation of images of unstained frozen hydrated flagella. Determination of native flagellar structure by 3-D reconstruction from multiple-tilted views appears to be feasible.