The award goes to: Jacques Dubochet, 75, a Swiss national who is honourary professor of biophysics at the University of Lausanne; Briton Richard Henderson, 72, who was born in Scotland and is now programme Leader at MRC labouratory of Molecular Biology, in Cambridge; and Joachim Frank, 77, an American born in Germany, who is currently professor of biochemistry and molecular sciences at Columbia University in NY.
"It's like frames in a movie".
The 2017 prize, worth 9 million kronor ($1.1 million), is being announced Wednesday by the Royal Swedish Academy of Sciences.
The cooling technique was first demonstrated by Dubochet, while Frank developed image processing techniques that allowed complex protein molecules to be viewed in 3D; Henderson moved the technique on by imaging a bacteria molecule at atomic resolution.
Previously, electron microscopy imaging was only suitable for studying dead matter, because the electron beam destroys any biological material it is applied to.
In the 1980s, Dubochet was able to cool water so quickly that it solidified without forming ice crystals, instead forming around a biological sample so the biomolecules retained their shape, getting a "freeze frame" of the action.
He added that cryo-electron miscropy "is about to completely transform structural biology", saying the technology is being taken up by a new generation of medical researchers.
Speaking of his childhood fear of darkness and scientific curiosity, Dubochet said in an earlier interview: "It was important for me to face my fears and understand the frightening things".
A year ago the Nobel prize in chemistry received three European scientists for the creation of "molecular machines" - the smallest cars in the world. Together these discoveries have made today's cryo-electron microscopy possible.
It was also put to work a year ago in the fight against Zika, when the mosquito-borne virus was linked to an epidemic of brain-damaged babies in Brazil.
"By solving more and more structures at the atomic level we can answer biological questions, such as how drugs get into cells, that were simply unanswerable a few years ago", Jim Smith, science director at the London-based biomedical research charity Wellcome, said in a statement.
The detailed images may pave the way for developing new medicines, vaccines and industrial chemicals, but experts said such payoffs are largely in the future. Electron microscopy is also conducted in a vacuum, which is problematic for imaging cells in their natural state. "This breakthrough proved the technology's potential", the Royal Swedish Academy of Sciences says in a press release.