Whole cryo-FIB lamella acquisition at high magnification with rectangular beam montage cryo-electron tomography

The visualisation of large biological samples in cryo-electron tomography requires both few-nanometre resolution and a wide, ideally >10 µm, field of view. For a single image, increasing magnification trades increased resolution for a smaller field of view. Montaging, where high-magnification images are stitched together to increase field of view, addresses the trade-off but is dose-inefficient with round beams that are standard in transmission electron microscopy (TEM), since this illumination shape does not tessellate. Our workflow greatly simplifies montaging schemes for dose-sensitive specimens, allowing large montages of any shape – from individual cells or other features of interest up to whole lamellae – to be collected at high resolution without compromising electron dose efficiency. We demonstrate the workflow by acquiring montage tomograms of whole yeast ( Saccharomyces cerevisiae) cells in cryo-FIB lamellae and of human neurons.