Dual-Channel Confocal Ratioing of Calcium Dynamics in Living Eggs and Oocytes

Changes in intracellular calcium concentrations ([Ca²⁺ ]_i ) have been shown to regulate a wide variety of developmental processes. In particular, eggs that have completed meiosis or oocytes that are still in the process of undergoing meiotic maturation invariably display some form of a [Ca²⁺ ]_i elevation during fertilization (1 –3 ). In order to image fertilization-induced calcium responses, unfertilized eggs and oocytes can be loaded with photoproteins, (e.g., aequorin) for subsequent detection of the luminescence that is generated when free calcium ions bind to the photoprotein (4 ). Alternatively, eggs may either be injected with or simply incubated in various calcium-sensitive fluorophores (5 ). Such calcium indicators can then be used in conjunction with fluorescence microscopy to track calcium dynamics during fertilization. The relative benefits and disadvantages of aequorin vs calcium-sensitive probes have been discussed previously (6 ), and both types of calcium indicators remain widely employed in imaging analyses that involves conventional microscopy.