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 10 to 20 microm beneath the surface for both sources. The increased penetration depth at 1300 nm 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-microm excitation in nonlinear optical microscopy.

Fig. 1

Schematic of the experimental setup. HWP: half-wave plate; GLP: Glan-laser polarizer; M: mirror (M1 and M5 mirrors held in flip mounts); BS: beam sampler; P: prism; AC: autocorrelator/spectrometer; SL: scanning lens; TL: tube lens; DM: dichroic mirror; CL: collection lens; F: filter; and PMT: photomultiplier tube.

Fig. 2

TPEF of fluorescent polystyrene beads in scattering agarose gels. (a) Fluorescent signal versus penetration depth for 800-nm (◻) and 1300-nm ([●) excitation wavelengths. The solid curves are a guide to the eye. (b) TPEF intensities (logarithmic scale) versus penetration depth for data starting from 25 μm. Inset: image of the phantom at 15 μm depth, using 1300-nm excitation wavelength.

Fig. 3

TPEF and SHG images of the RAFT model. 3-D reconstruction of the volume scanned within the sample excited with (a) 800 nm and (b) 1300 nm. The dimensions are 100×100×160 μm in the xyz directions (in the figure, x:z=1:5).

Fig. 4

SHG signal of collagen. (a) SHG signal versus penetration depth for 800- (◻) and 1300-nm (●) excitation wavelengths. The solid curves are a guide to the eye. (b) SHG intensities (logarithmic scale) versus penetration depth.