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Abstract

In biological imaging of fluorescent molecules, multiphoton
laser scanning microscopy MPLSM has become the favorite method
of fluorescence microscopy in tissue explants and living animals. The
great power of MPLSM with pulsed lasers in the infrared wavelength
lies in its relatively deep optical penetration and reduced ability to
cause potential nonspecific phototoxicity. These properties are of crucial
importance for long time-lapse imaging. Since the excited area is
intrinsically confined to the high-intensity focal volume of the illuminating
beam, MPLSM can also be applied as a tool for selectively
manipulating fluorophores in a known, three-dimensionally defined
volume within the tissue. Here we introduce localized multiphoton
photoactivation MP-PA as a technique suitable for analyzing the dynamics
of photoactivated molecules with three-dimensional spatial
resolution of a few micrometers. Short, intense laser light pulses uncage
photoactivatable molecules via multiphoton excitation in a defined
volume. MP-PA is demonstrated on photoactivatable paGFP in
Drosophila wing imaginal discs. This technique is especially useful for
extracting quantitative information about the properties of photoactivatable
fusion proteins in different cellular locations in living tissue as
well as to label single or small patches of cells in tissue to track their
subsequent lineage.