The palette of fluorescent highlighters used to track the movement of messengers inside single cells has been dramatically expanded to include red and blue fluorescent indicators, which provide researchers a vivid full-color view of calcium ions moving about in their role as key intracellular signaling messengers. Until recently, cellular imaging of the calcium ion required the use of a green fluorescent indicator. Accordingly, imaging of calcium ions produced monochromatic images and movies in shades of green. Now, scientists at the University of Alberta have added red and blue indicators. Three-color fluorescence imaging of HeLa cells transfected with plasmids encoding R-GECO1 (red indicator) targeted to the nucleus, G-GECO1 targeted to the cytoplasm and GEM-GECO1 to the mitochondria. Courtesy of Robert E. Campbell et al. The well known selection of fluorescence protein colors has proved useful for live-cell imaging of multiple organelles and proteins, according to doctoral candidate Yongxin Zhao and Robert Campbell, associate professor of chemistry. They explained that a similar palette of calcium indicators will enable researchers to start designing experiments that involve visualizing multiple dynamic biochemical parameters simultaneously. Imaging of the calcium ion is commonly used by researchers to monitor cellular activity such as the firing of neurons. However, since calcium ions are colorless, it is necessary to introduce colored indicator proteins into the cell, which can either increase in fluorescent brightness or change the fluorescence color when they bind to the ions. These changes in brightness can be visualized easily using appropriate microscopy equipment. Examining the dynamics of calcium ions inside a single cell in better detail could help pharmaceutical researchers determine whether a drug designed to affect a specific cell is hitting its target. In addition, it could help scientists better visualize neuronal activity in model organisms such as transgenic worms or mice. Multicolor imaging with GECOs. HeLa cells transfected with nucleus-localized R-GECO1, cytoplasmic G-GECO1, and mitochondria-localized GEM-GECO1. (Top left) Red fluorescence. (Top right) Green fluorescence with cyan (~470 nm) excitation. (Bottom left) Pseudocolored ratio of blue to green fluorescence with UV (~380-nm) excitation. (Bottom right) Merge of the three images, with GEM-GECO1 ratio in magenta. The team, led by Zhao, engineered the new genetically encoded indicator proteins using bacterial cells. The indicator genes were programmed to be sent to the outside of the bacterial cytoplasmic membrane. The calcium ion concentration then could be experimentally altered to find the indicator variants that had the desired color and largest changes in brightness. Using this approach, the researchers performed directed laboratory evolution to ultimately provide the optimized indicator proteins. Campbell and Zhao said that their next priority is to expand the color palette of the indicators to include the yellow, orange and far-red regions of the spectrum. In addition, they plan to distribute the genes to as many research groups as possible through Addgene, a nonprofit plasmid repository. The research appeared online Sept. 8 in Science (doi: 10.1126/science.1208592).