\textit{In vivo} fluorescence fiber-optic microscopy of superfast Ca$^{2+}$ transients in syrinx muscles

Optical techniques in conjunction with fluorescent markers have revolutionized the investigation of dynamical cellular processes, such as studies of calcium ion (Ca$^{2+})$ dynamics in muscles. Recently, it was shown that songbirds have superfast syringeal muscles, which can modulate song acoustics up to 250~Hz. Such rapid contraction cycles most likely require very rapid Ca$^{2+}$ kinetics. We developed a technique to measure Ca$^{2+}$ transients in syringeal muscles of anesthetized birds with a custom-built endoscope. The fluorescence measurements were carried out before and after applying a calcium indicator dye and while muscles were stimulated electrically. The results show fast ($\sim $50-ms FWHM) and superfast~($\sim $7-ms FWHM) Ca$^{2+}$ transients. The strongest signals were observed 30-40 minutes after applying the dye. This study confirms that rapid Ca$^{2+}$ transients in syringeal muscles facilitate superfast contraction kinetics and demonstrates the feasibility of this optical technique as a biosensor for detecting fluorescence signals of muscular calcium activity on a ms timescale of living animals.