The methodology for preparing specimens in the frozen, hydrated state has been assessed using crystals and T4 bacteriophages. The methods have also been demonstrated with lambda bacteriophages, purple membrane of Halobacterium halobium and fibres of DNA. For particles dispersed in an aqueous environment, it is shown that optimum structural preservation is obtained from a thin, quench-frozen film with the bulk aqueous medium in the vitreous state. Crystallization of the bulk water may result in solute segregation and expulsion of the specimen from the film. Contrast measurements can be used to follow directly the state of hydration of a specimen during transition from the fully hydrated to the freeze-dried state and permit direct measurement of the water content of the specimen. By changing the concentration and composition of the aqueous medium the contrast of particles in a vitreous film can be controlled and any state of negative, positive or zero contrast may be obtained. At 100 K, frozen-hydrated, freeze-dried or sugar embedded crystals can withstand a three- to four-fold increase in electron exposure for the same damage when compared with similar sugar-embedded or freeze-dried samples at room temperature.