Figure 6. Detection of nitric oxide–induced vascular cyclic GMP (cGMP) signals associated with vasorelaxation in vivo in anesthetized R26-CAG-cGMP indicator with an EC50 of 500 nmol/L (cGi500)(L1) mice.
Blood vessels in the skin were imaged in dorsal skinfold chambers by multiphoton fluorescence resonance energy transfer (FRET) microscopy. A and C, The distribution of sensor fluorescence (cyan fluorescent protein [CFP] channel) indicates strong expression in the skin, including the vasculature. Scale bars, 100 µm. B, cGMP imaging of the region outlined in A (yellow). Intravenous injection of 100 µL saline did not result in a FRET ratio change. Repeated injection of 100 µL of 1 mmol/L 2-(N,N-diethylamino)-diazenolate-2-oxide diethylammonium salt (DEA/NO) induced clear cGMP transients (black trace). Note that both saline and DEA/NO injection resulted in instantaneous alterations of CFP and yellow fluorescent protein (YFP) emissions (cyan and yellow traces) in the same direction (indicated by asterisks). These changes barely affected the FRET ratio signal and were most likely artifacts induced by movements during the injection procedure. Only DEA/NO, not saline, resulted in sustained changes of CFP and YFP emission signals in opposite directions, indicative of cGMP binding to the sensor. D, cGMP imaging of the region outlined in C (yellow). Mice were injected intravenously 3 times with 100 µL of 0.1 mmol/L DEA/NO followed by 2 injections of 100 µL of 1 mmol/L DEA/NO. In parallel to the cGMP FRET signals (black trace), the vessel diameter (red trace) was measured at the location indicated by the red dashed line in C. Δd/d indicates relative diameter change.