We describe the basic principles for imaging frozen-hydrated specimens in a transmission electron microscope and provide a step-by-step guide to a new user, from starting up and aligning the microscope, to loading a cryo-grid into the specimen holder and inserting the holder into the microscope, to setting up the low dose mode for imaging, and eventually to shutting down the microscope. The procedure is based on a JEOL TEM; however, it is applicable to microscopes from other manufacturers with small modifications. We also give several tips on how to minimize specimen exposure before taking pictures; how to minimize the specimen drift and charging during exposure; how to estimate the thickness of the vitreous ice, and how to quickly estimate the electron exposure dose. Despite recent advances in instrumentation, the microscopist's patience and attention to detail may still be the key to acquiring a high quality cryo-electron micrograph.
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