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We used two-photon (2-p) Förster resonance energy transfer (FRET) microscopy to provide serial, noninvasive measurements of [Ca(2+)] in arterioles of living "biosensor" mice. These express a genetically encoded Ca(2+) indicator (GECI), either FRET-based exMLCK or intensity-based GCaMP2. The FRET ratios, Rmin and Rmax, required for in vivo Ca(2+) calibration of exMLCK were obtained in isolated arteries. For in vivo experiments, mice were anesthetized (1.5% isoflurane), and arterioles within a depilated ear were visualized through the intact skin (i.e., noninvasively), by 2-p excitation of exMLCK (at 820 nm) or GCaMP2 (at 920 nm). Spontaneous or agonist-evoked [Ca(2+)] transients in arteriolar smooth muscle cells were imaged (at 2 Hz) with both exMLCK and GCaMP2. To examine changes in arteriolar [Ca(2+)] that might accompany hypertension, five exMLCK mice were implanted with telemetric blood pressure transducers and osmotic minipumps containing ANG II (350 ng·kg(-1)·min(-1)) and fed a high (6%)-salt diet for 9 days. [Ca(2+)] was measured every other day in five smooth muscle cells of two to three arterioles in each animal. Prior to ANG II/salt, [Ca(2+)] was 246 ± 42 nM. [Ca(2+)] increased transiently to 599 nM on day 2 after beginning ANG II/salt, then remained elevated at 331 ± 42 nM for 4 more days, before returning to 265 ± 47 nM 6 days after removal of ANG II/salt. In summary, two-photon excitation of exMLCK and GCaMP2 provides a method for noninvasive, longitudinal quantification of [Ca(2+)] dynamics and vascular structure in individual arterioles of a particular animal over an extended period of time, a capability that should enhance future studies of hypertension and vascular function.

A method for noninvasive longitudinal measurements of [Ca2+] in arterioles of hypertensive optical biosensor mice

A method for noninvasive longitudinal measurements of [Ca²⁺] in arterioles of hypertensive optical biosensor mice