Third-harmonic generation microscopy in highly scattering media.

The performance of third-harmonic generation (THG) microscopy in highly scattering media is analyzed with the Monte Carlo technique. The three-dimensional point-spread function (PSF) of the laser-scanning THG microscope with a pulsed excitation light source is derived for both isotropic and anisotropic scattering media and at different h/d(s) values, where h is the scattering depth as measured from the geometric focus of the objective lens and d(s) is the mean free path of the scattering medium. The generated THG signal is detected by a large-area photodetector. The PSF of the THG microscope is given by the third power of the normalized distribution of the excitation beam near the beam focus. The behavior of the temporal broadening of the excitation pulse on the generated THG signal is also analyzed as a function of h/d(s). The relative advantages and disadvantages of the THG microscope relative to the two-photon fluorescence microscope are discussed thoroughly.