Use of confocal microscopy in comparative studies of vertebrate morphology

Laser scanning confocal microscopy provides a means to acquire and analyze images of complex morphological structures and to help place molecules or cells of interest in their proper morphological context. Confocal microscopy is a form of fluorescence microscopy that sharpens the images collected by visualizing the light from only one plane of focus. This allows for the collection of multiple focal planes in what is called a z-stack, which provides three-dimensional data. Five steps that any investigator using a confocal microscope should follow are described: (1) labeling and (2) mounting of specimens for viewing, (3) optimizing the image on the confocal, and (4) collecting and (5) analyzing of confocal image data. We describe three specific protocols incorporating these steps from our work on vertebrate inner ear development. The first two describe a collection of z-stacks in living, fluorescently labeled, and intact embryos. The second protocol is for time-lapse imaging of multiple focal planes at each time point. The third protocol describes confocal imaging of preserved material double labeled with antibodies and by retrograde labeling of neurons via axonal uptake. Finally, three alternative or complementary approaches to standard confocal microscopy are described and discussed.