High rate, full field image acquisition in multiphoton imaging is achievable by parallelization of the excitation and of the detection paths. Via a Diffractive Optical Elements (DOEs) which splits a pulsed laser, and a spatial resolved descanned detection path, a new approach to microscopy has been developed. By exploiting the three operating mode, single beam, 16 beamlets or 64 beamlets, the best experimental conditions can be found by adapting the power per beamlet. This Multiphoton Multispot system (MCube) has been characterized in thick tissue samples, and subsequently used for the first time for Ca2+ imaging of acute heart slices. A test sample with fixed mice heart slices with embedded sub-resolution fluorescent beads has been used to test the capability of optical axial resolution up to ~200 microns in depth. Radial and axial resolutions of 0.6 microns and 3 microns have been respectively obtained with a 40X water immersion objective, getting close to the theoretical limit. Then images of heart slices cardiomyocites, loaded with Fluo4-AM have been acquired. The formation of Ca2+ waves during electrostimulated beating has been observed, and the possibility of easily acquire full frame images at 15 Hz (16 beamlets) has been demonstrated, towards the in vivo study of time resolved cellular dynamics and arrhythmia trigger mechanisms in particular. A very high speed two-photon Random Access system for in vivo electrophysiological studies, towards the correlation of voltage and calcium signals in arrhythmia phenomena, is now under developing at Light4tech.
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