Using Multiphoton Excitation to Explore the Murky Depths of Developing Embryos

Fluorescence microscopy has become the method of choice for imaging living specimens, as it offers high signal-to-background and the ability to discriminate between multiple fluorophores. Recently developed techniques, such as confocal (1 ) or multiphoton imaging (2 ), permit optical sectioning of intact specimens. These may be collected as stacks of images at different focal depths to obtain three-dimensional (3D) structural data. Stacks of images may be collected at regular time intervals in order to reveal the dynamics of 3D structures in living tissue (3 ). However, living tissue is generally of poor optical quality; microinhomogeneities in refractive index caused by cytosol/membrane interfaces scatter light, thereby limiting the depth from which optical sections may be obtained. Multiphoton laser-scanning microscopy (MPLSM) is becoming increasingly favored for in vivo imaging because of its superior ability to obtain images from deep within specimens (4 ) and the improved viability that can be obtained (5 ).