In-vivo tissue imaging using a compact mobile nonlinear microscope

We have built a compact flexible non-linear microscope equipped with a combination of different non-linear laser imaging techniques including two-photon fluorescence, second-harmonic generation, fluorescence lifetime imaging microscopy, and multispectral two-photon emission detection. The system is composed of a microscope head, containing both scanning and detection system, as well as the electronic and electro-mechanical devices, optically relayed to the laser source with a seven-mirror articulated arm. The particular mirror positioning inside the arm allows to move the microscope head maintaining the optical alignment of the system. The microscope head is composed by two ErGaAl anodized boards, one for laser scanning and the other for signal detection. System performances were characterized by means of point spread function and instrument response function measurements as well as by spatial, temporal, and spectral calibration. The instrument, offering high spatial (up to 300 nm) and temporal (up to 300 ps) resolution, was tested on in-vivo skin imaging of both cellular epidermis and connective dermis. Lifetime and spectral features of fluorescence were used for differentiating epidermal layers by means of fluorescence lifetime and for scoring skin ageing through spectral detection of both second-harmonic and two-photon fluorescence.