Multispot multiphoton Ca²⁺ imaging in acute myocardial slices

Multiphoton microscopy has become essential for dynamic imaging in thick living tissues. High-rate, full-field image acquisition in multiphoton microscopy is achievable by parallelization of the excitation and detection pathways. We developed our approach via a diffractive optical element which splits a pulsed laser into 16 beamlets and exploits a descanned detection system consisting of an array of beamlet-associated photomultiplier tubes. The optical performance of the multiphoton multispot system (MCube) has been characterized in cardiac tissue sections and subsequently used for the first time for fluorescence imaging of cardiomyocyte Ca²⁺ dynamics in viable acute cardiac slices. Multispot multiphoton microscopy (MMM) has never been used before to monitor Ca²⁺ dynamics in thick, viable tissue samples. Acute heart slices are a powerful close-to-in vivo model of Ca²⁺ imaging allowing the simultaneous observation of several cells in their own tissue environment, exploiting the multiphoton excitation ability to penetrate scattering tissues. Moreover, we show that the concurrent high spatial and temporal resolutions afforded by the parallel scanning in MMM can be exploited to simultaneously assess subcellular Ca²⁺ dynamics in different cells in the tissue. We recorded local Ca²⁺ release events including macrosparks, travelling waves, and rotors.