Studies of fixed neutrophils using an antibody against cAMP showed a uniform Phellodendrine distribution of cAMP throughout the cytoplasm of unstimulated cells. Upon phagocytic challenge with an opsonized target, higher concentrations of cAMP were localized to the forming phagosome. The FRET microscopic method introduced here has the advantages of providing high specificity for cAMP and good temporal and spatial resolution. Its disadvantages include a weak signal and a small dynamic range. Mutation of YFP to Citrine in Epac-camps improved the probe��s specificity for cAMP by reducing potential artifacts resulting from local fluctuations in cytoplasmic pH. The probe was bright enough to permit image acquisition every 30 seconds, allowing measurement of localized increases in cAMP throughout the 7- to 8-minute process of phagocytosis. However, like most linked FRET biosensors, the Epac-camps probe exhibited a limited dynamic range. Binding of cAMP to Epac-camps resulted in a small decrease in FRET efficiency: the fluorescent proteins are bright but the measurable shift in FRET is a weak signal. Such small differences in FRET efficiency can be problematic when trying to detect localized signals surrounded by cytoplasm, 8-Prenylchrysin especially in thick cells. Moreover, the maximum and minimum FRET efficiencies reported by the biosensor are restricted to the small range of cAMP concentrations above and below the binding affinity of Epac. The weak signal and small dynamic range of Epac-camps explain why the decrease in FRET signals from cells treated with EdTx or forskolin were less dramatic than the increases in signals reported by the biochemical assay. Likewise, the failure of the probe to report decreases in cAMP concentrations indicates that concentrations of cAMP in unstimulated macrophages are at or below the lower limit of detection by Epac-camps. Thus, the probes were adequate to report transient increases in cAMP near forming phagosomes but would have likely missed smaller foci of elevated cAMP or any local decrease in cAMP. Although we did not observe any differences in bulk cAMP during macrophage phagocytosis by RAW macrophages, a transient increase in cAMP was localized to the forming phagosome.