Dubochet, who is now retired, is Swiss; Frank, of Columbia University, New York, is German, and Henderson, of the MRC laboratory of molecular biology in Cambridge, is Scottish.
This development is "decisive for both the basic understanding of life's chemistry" and the development of drugs, the Nobel committee said.
Scientists' knowledge evolved steadily over the last six decades - beginning with the pioneering crystallographic studies of the structures of globular proteins that fetched Max F Perutz and John C Kendrew the Chemistry Nobel in 1962, to mastering cryo-electron microscopy (cryo-EM) for which the 2017 Prize has been awarded.
"This method started in the biochemistry of a new era", said jury.
Previously, electron microscopy imaging was only suitable for studying dead matter, because the electron beam destroys any biological material it is applied to. "I think that this discovery that's being recognized has huge potential and is broadly applicable across all scientific disciplines", says Allison Campbell, the president of the American Chemical Society. The achievement was to preserve the structure of liquid water at a temperature low enough so that it would not evaporate. Henderson imaged a protein in three dimensions down to the atomic level.
This new microscope technology has enabled scientist to have detailed images of life's complex machinery in atomic resolution. They swapped water with a sugar cocktail, which could withstand the vacuum and systematically tweaked the settings of their microscope to limit the damage caused by the electrons. Toward the end of Wednesday's event, a journalist from Chinese radio asked Royal Swedish Academy of Sciences Secretary General Göran K. Hansson about the prevalence of Americans, and what it says about USA research environment and policies.
Magdalena Zernicka-Goetz, professor of mammalian development and stem cell biology at the University of Cambridge, said that she thought the win was wonderful. He used this technique to model the surface of the ribosome, the cell's protein synthesis factory.
Joachim Frank of Columbia University in NY developed an image-processing method that merges many two-dimensional images of a biomolecules to create a sharp three-dimensional picture.
The Nobel Prize in literature will be announced on October 5 and the peace prize on October 6.
Chemistry is the third of this year's Nobel Prizes after the winners of the medicine and physics prizes were announced earlier this week. The three winners of the $1.1 million (9 million kronor) prize adapted another technique, electron microscopy, which uses a beam of electrons rather than ordinary light to inspect samples.