Application of Multiphoton Imaging to Study of the Vasculature.

Abstract

Nonlinear or multiphoton fluorescence microscopy utilizes the simultaneous absorption of two (or three) longer wavelength photons to excite fluorophores (Denk et.al., 1990). For example, UV absorbing fluorophores such as DAPI or lndo-1 are excited using 700 nm near infrared light rather than 350 nm UV excitation. This relatively new form of laser scanning fluorescence microscopy has excellent optical sectioning capabilities in thick, highly scattering tissues. The high degree of intrinsic optical sectioning arises from the spatial nature of the excitation dependence, rather than from a confocal aperture or deconvolution algorithm. The fluorescence arising from any point in the specimen depends on the second or third power of the intensity (i.e. two and three photon excitation, respectively). This squaring (or cubing) of the illumination point spread function effectively confines the excitation to a tenth of a femtoliter optical volume when using a high NA lens. 680 to 1100 nm mode-locked lasers producing pulses of 100 femtosecond duration at a repetition rate of 80 Mhz are used to efficiently produce fluorescence excitation at average powers that are usually in the 0.5 to 10 mW range at the sample.