We have developed GluSnFRs, genetically encoded glutamate-sensing fluorescent reporters based on cyan and yellow fluorescent proteins surrounding a bacterial glutamate binding protein. Our newest GluSnFR, in which linker sequences and glutamate affinities have been systematically optimized, exhibits a 6.8-fold improvement in response magnitude over its predecessor and a dissociation constant of 11 µM. We demonstrate quantitative optical measurements of the timecourse of synaptic glutamate release, spillover and reuptake in cultured hippocampal neurons with millisecond resolution. These results indicate that significant glutamate spillover can occur between synapses. Active re-uptake is shown to be the dominant mechanism of glutamate clearance from the dendritic surface. A simple kinetic model of GluSnFR measurements suggests that frequency of stimulation modulates peak and average spillover glutamate concentrations, but does not change the proportion of glutamate recovered. Because many forms of long-lasting learning and memory are associated with expansion or de novo formation of synapses, visualizing recently expanded or formed synapses may shed light on where memories are stored. Time-lapse microscopy can track synaptic growth in sparsely labeled neurons in superficial brain regions, but identifying growing synapses throughout a functional nervous system is currently not possible. One approach may be to label newly synthesized synaptic proteins as markers of synaptic birth or growth, analogous to labeling newly synthesized DNA to identify newly born cells. Existing methods for visualizing newly synthesized proteins that rely on sequential biarsenical labeling of tetracysteines or photoconversion of fluorescent proteins are not compatible with deep tissues or freely behaving animals, and suffer from problems with toxicity and sensitivity. Here we report TimeSTAMP, a time-specific tag for the age measurement of proteins, which allows small-molecule-triggered epitope tagging of newly synthesized proteins of interest. We use TimeSTAMP to relate new postsynaptic density protein accumulation to synaptic growth in cultured neurons and to visualize protein synthesis in living animals. Our results open up the possibility of retrospective identification of sites of synaptic plasticity anywhere in the nervous systems of freely behaving animals.