 Scientists at the Mox Planck Florida Institute for Neuroscience and Stanford University have developed new proteins that radically improve the ability to see inside neurons. The advance expands the number of colors researchers can use, allowing them to better understand what's going on at the molecular level when a memory is formed and giving them a new tool to study the mechanisms of neurological diseases. Until now, scientists have been limited to using just one color when tracking signaling molecules in neurons. Green fluorescent protein, or GFP, is a good workhorse, but it's been difficult to find other colors that can work with it. New proteins would allow researchers to study more than one molecule at a time and would also let them keep track of any physical changes to synapses. Structural changes are a key part of synaptic plasticity or how synapses strengthen or weaken over time and are thought to be the underpinnings of memory. To expand their options, the team developed a new red fluorescent protein named SiRFP that can be visualized at the same time as GFP. They then tested whether it would be useful as a structural marker while using a GFP-based sensor to monitor calcium flow. They found that the new red protein performed remarkably well, even allowing them to observe dendritic spines in the brains of live mice. The team then turned its attention to making a new red biosensor for a technique called FRET, or fluorescence resonance energy transfer. In FRET, one light-sensitive molecule donates energy to another light-sensitive molecule if they're close together. By tracking the energy exchange between the pair, scientists can image the activation of signaling molecules during spine structural changes. The team created a red FRET donor by making other tweaks to SiRFP, including making sure it didn't dimerize. They then identified a far-red FRET acceptor named M. Maroon, which provided the highest FRET efficiency with the donor. Critically, this red FRET pair lets researchers image a green FRET pair at the same time. The scientists verified this technique in slices of the hippocampus, a brain region that is crucial for many forms of learning and memory. They labeled two different signaling molecules known to be important for synaptic plasticity with the green and red sensors. Then, using a specialized imaging technique, they triggered structural plasticity in specific dendritic spines. This allowed the researchers to track, for the first time, two signaling molecules simultaneously in individual spines undergoing structural plasticity. Now, scientists can accurately quantify how multiple proteins behave in neurons over time and space, opening up opportunities to learn much more about how we learn and use memory.