Physical and biological limitations of in vivo multiphoton fluorescence microscopy

In this paper we present experimental and theoretical study of limitations on deep imaging of biological tissue structure using multiphoton fluorescence microscopy (MFM), that are imposed by scattering of short NIR pulses in optically turbid medium. The time stretching of the laser pulse and its transversal widening limit the maximum imaging depth of NFM since the magnitude of fluorescence response from the medium is the power function of energy flux density in the excitation laser beam. Increasing the imaging depth by raising the laser power may result in phototoxic damage of biological objects. We have theoretically and experimentally studied temporal structure of collimated femtosecond laser pulse scattered in an optically turbid model medium with controlled concentration of micron-sized spherical beads. In parallel we investigated phototoxic effect on biological liquid at high-intense femtosecond NIR pulse irradiation for various expositions by structural analysis of dehydrated blood plasma droplet. The irradiation regimes were found with complete recovery of the droplet structure, as well as with its slight and severe stable modification.