Effect of excitation wavelength on penetration depth in nonlinear optical microscopy of turbid media

Abstract

We present a comparative study of two-photon excited fluorescence (TPEF) and second harmonic generation (SHG) imaging in turbid media at 800- and 1300-nm excitation. The depth-dependent decay of TPEF and SHG signals in turbid tissue phantoms is used to estimate the impact of light scattering on excitation intensity at each wavelength. A 50 to 80% increase in scattering length is observed using 1300-nm excitation, while peak TPEF emission intensity is obtained 10to20μm beneath the surface for both sources. The increased penetration depth at 1300nm is confirmed by TPEF and SHG microscopy of tissue phantoms composed of gelatin/microspheres and 3-D organotypic collagen-fibroblast cultures, respectively. Our results establish the feasibility of 1.3-μm excitation in nonlinear optical microscopy. © 2009 Society of Photo-Optical Instrumentation Engineers.