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The researchers tested numerous variants—first in bacteria, then in animals. The best was GCaMP3, which differs from its predecessor by just four amino acids. But that slight alteration makes a big difference. GCaMP3 is 3 times brighter in the absence of intracellular calcium (labeled cells can be found even in the absence of calcium signaling) and about 10 times brighter in its presence, Bargmann estimates. The protein also is more finely attuned to calcium fluctuations—a crucial detail, Looger says, because “If calcium goes up and the protein doesn't [respond], it's as if a tree fell in the woods and no one heard it.”
“What Loren did, in an intellectually sophisticated way, was to drive a system that worked okay into a system that worked 10 times as well,” Bargmann says. “He essentially made 10 times as many experiments interpretable.”
For Drosophila researchers, the sensor fills a key technology gap, says Jayaraman: implanting electrodes in a fly isn't exactly easy. “Although we cannot hope to abandon electrophysiology, there's no question that, for a certain set of questions, this is a tool that can go pretty far,” he says. Jayaraman's lab is using GCaMP3 to learn what happens in the central brain of tethered walking and flying fruit flies as they respond to the sensory world around them.
Svoboda's team is using GCaMP3 to illuminate brain circuits that respond when mice walk on a treadmill or sense contact through their whiskers. A video of the primary motor cortex in live animals dramatically illustrates the approach: the scene resembles a Christmas display, with individual neurons flickering gaily.
Bargmann receives regular requests for her GCaMP3 worms—and not just from neurobiologists. “People who weren't doing this kind of experiment in the past have asked for them,” she says, “because now... it looks like something that a normal person could do, and not some superhuman feat.”
One goal at Janelia Farm is to develop and share broadly useful imaging technologies. Looger estimates some 200 researchers have used GCaMP3, including Joe Fetcho, a researcher at Cornell University who studies the neurobiology of zebrafish movements. “This is the first [GECI] that is good enough that it will allow us to [detect individual action potentials] with some confidence,” Fetcho says.
“GCaMP3 is certainly the best thing out there right now,” says Looger. But he isn't finished. “There's still some headroom,” he says. “GCaMP3 is not the ultimate GECI.”