Performances of high numerical aperture water and oil immersion objective in deep-tissue, multi-photon microscopic imaging of excised human skin.

Multi-photon fluorescence microscopy (MPFM) is a powerful technique for imaging scattering, biological specimens in depth. In addition to the sectioning effect generated by the point-like excitation volume, the near-infrared wavelengths used for multi-photon excitation allow deeper penetration into optically turbid specimens. In physiological specimens, the optical properties such as the scattering coefficients and refractive indices are often heterogeneous. In these specimens, it is not clear which type of immersion objective can provide optimized images in-depth. In particular, in-depth dermatological imaging applications using MPFM requires such optimization to obtain qualitative and quantitative information from the skin specimens. In this work, we address this issue by comparing the performances of two common types of high numerical aperture (NA) objectives: water-immersion and oil-immersion. A high-quality water-immersion objective (Zeiss, 40 x C-Apochromat, NA 1.2) and a comparable oil-immersion objective (Zeiss, 40 x Fluar, NA 1.25) were used for in-depth imaging of autofuorescent excised human skin and sulforhodamine B treated human skin specimens. Our results show that in the epidermal layers, the two types of immersion objectives perform comparably. However, in the dermis, multi-photon imaging using the oil immersion objective results in stronger fluorescence detection. These observations are most likely due to the degraded point-spread-function (PSF) caused by refractive index mismatch between the epidermis and the dermis.