Long duration three−dimensional imaging of calcium waves in zebrafish using multiphoton fluorescence microscopy

Both luminescent (1, 2) and fluorescent (2) reporters have been used to image periodic large−scale intercellular calcium waves that begin during zebrafish gastrulation, at about 65% epiboly, and continue for at least 12 hours. These waves arise every 5 to 10 min from a variety of locations and traverse the blastoderm margin and main body axis (1). During somitogenesis they appear as a series of pulses of elevated calcium levels centered on the tailbud region. The waves travel at approximately 5−10 μm per s and thus fall into the category of fast calcium waves that likely propagate by a positive feedback mechanism involving calcium−induced calcium release from intracellular stores, possibly including diffusion of calcium or IP3 through gap junctions (3). Likely targets of the waves include calcium−sensitive proteins involved in epiboly (3) and convergent extension (4), and others such as calreticulin that may play a role in the temporal regulation of nuclear receptor activity (5). Long−distance signaling by rhythmic calcium waves is an appealing mechanism for synchronizing calcium−triggered events throughout the embryo with high temporal precision. Since the zebrafish embryo is a roughly spherical body approximately 600 μm in diameter, imaging these waves in a single optical plane, as in previous studies, can only approximate their three−dimensional trajectories. Moreover, other calcium signals that may be occurring at the same time, but in different optical planes within the embryo, cannot be documented. Ideally, free calcium levels should be imaged for many hours throughout the entire volume of the embryo at intervals shorter than the lifetimes of individual signaling events. Luminescent techniques require considerable temporal integration to achieve adequate spatial resolution and thus cannot approach this goal. Conversely, scanning laser microscopy using visible light has the necessary spatial and temporal resolution, but cannot be used for prolonged imaging at high sampling rates due to phototoxic damage to the embryo (E. Gilland, pers. obs.). The present study demonstrates that multiphoton fluorescence microscopy has the potential to achieve the goal of sampling calcium dynamics throughout the entire zebrafish embryo for long durations with sufficient spatial and temporal resolution to reveal complex three−dimensional signaling event