The use of the confocal microscope to study livingXenopuseggs affords the opportunity to obtain four-dimensional (4-D) data (three-dimensional [3-D] data over time) throughout early development of these large cells. Microscopy of living cells often reveals important information about dynamic cellular events that cannot be gleaned from analyses of fixed cells; however, certain compromises must be made to assure cell viability. Several approaches are typically used for examining living cells. One approach is to collect successive images at fixed time intervals over extended periods of time (e.g., time-lapse data collection), examining either the same optical plane or multiple different planes (3-D data sets, referred to as a z-series). Another approach is to collect, as rapidly as possible, multiple images over relatively short periods of time, again from a single optical plane or many different optical planes. In either case, it is imperative that the data be collected without altering normal cell function. Although multiphoton microscopy is the preferred approach for studying living cells, as it results in less damage to the cell, it is difficult withXenopusoocytes and embryos because the energy of the longer wavelengths used is absorbed by the pigment granules, causing intense local heating (Larabell, personal observations). We have found, however, that standard confocal microscopy using a krypton-argon laser can be used to examineXenopuseggs and embryos with undetectable adverse effects as determined by the ability of the eggs to develop into swimming tadpoles (1 ,2 ).
