Axial localization of multiphoton excitation to a single plane is achieved by temporal focusing of an ultrafast pulsed excitation. We take advantage of geometrical dispersion in an extremely simple experimental setup, where an ultrashort pulse is temporally stretched and hence its peak intensity is lowered outside the focal plane of the microscope. Using this strategy, out-of-focus multiphoton excitation is dramatically reduced, and the achieved axial resolution is comparable to line-scanning multiphoton microscopy for wide-field excitation and to point-scanning multiphoton microscopy for line excitation. In this introduction, we provide a detailed description of the considerations in choosing the experimental parameters, as well as the alignment of a temporal focusing add-on to a multiphoton microscope. We also review current advances and applications for this technique.
ASJC Scopus subject areas
- Biochemistry, Genetics and Molecular Biology(all)