Multispot multiphoton Ca²⁺ imaging in acute myocardial slices.

Area of Science:

• Biomedical Optics
• Cellular Physiology
• Cardiovascular Research

Background:

• Multiphoton microscopy is crucial for imaging in thick, living tissues.
• High-rate imaging requires parallelized excitation and detection.
• Previous methods limited simultaneous observation of cellular dynamics in complex tissues.

Purpose of the Study:

• To develop and validate a multispot multiphoton microscopy (MMM) system for high-speed, full-field imaging.
• To apply MMM for the first time to study cardiomyocyte calcium (Ca²⁺) dynamics in viable acute cardiac slices.
• To demonstrate the capability of MMM for simultaneous subcellular Ca²⁺ dynamics assessment in multiple cells.

Main Methods:

• Utilized a diffractive optical element to split a pulsed laser into 16 beamlets.
• Employed a descanned detection system with photomultiplier tubes for parallel detection.
• Characterized the system's optical performance in cardiac tissue sections.
• Applied the system to image Ca²⁺ dynamics in acute cardiac slices.

Main Results:

• Successfully implemented a multiphoton multispot system (MCube) for high-rate imaging.
• Achieved first-time fluorescence imaging of cardiomyocyte Ca²⁺ dynamics in viable acute cardiac slices using MMM.
• Demonstrated simultaneous assessment of subcellular Ca²⁺ dynamics in different cells.
• Recorded various Ca²⁺ release events, including macrosparks, travelling waves, and rotors.

Conclusions:

• Multispot multiphoton microscopy (MMM) is a powerful tool for studying Ca²⁺ dynamics in thick, viable tissues like acute cardiac slices.
• MMM offers high spatial and temporal resolution for simultaneous observation of subcellular Ca²⁺ events in multiple cells.
• This technique advances the understanding of cardiac electrophysiology and cellular signaling in a near-physiological context.