Zernike phase-contrast electron cryotomography applied to marine cyanobacteria infected with cyanophages

Advances in electron cryotomography have provided new opportunities to visualize the internal 3D structures of a bacterium. An electron microscope equipped with Zernike phase-contrast optics produces images with markedly increased contrast compared with images obtained by conventional electron microscopy. Here we describe a protocol to apply Zernike phase plate technology for acquiring electron tomographic tilt series of cyanophage-infected cyanobacterial cells embedded in ice, without staining or chemical fixation. We detail the procedures for aligning and assessing phase plates for data collection, and methods for obtaining 3D structures of cyanophage assembly intermediates in the host by subtomogram alignment, classification and averaging. Acquiring three or four tomographic tilt series takes ∼12 h on a JEM2200FS electron microscope. We expect this time requirement to decrease substantially as the technique matures. The time required for annotation and subtomogram averaging varies widely depending on the project goals and data volume.