Multidimensional custom-made non-linear microscope: from ex-vivo to in-vivo imaging

Abstract

We have built a custom-made multidimensional non-linear microscope equipped with a combination of several non-linear laser imaging techniques involving fluorescence lifetime, multispectral two-photon and second-harmonic generation imaging. The optical system was mounted on a vertical honeycomb breadboard in an upright configuration, using two galvo-mirrors relayed by two spherical mirrors as scanners. A double detection system working in non-descanning mode has allowed both photon counting and a proportional regime. This experimental setup offering high spatial (micrometric) and temporal (sub-nanosecond) resolution has been used to image both ex-vivo and in-vivo biological samples, including cells, tissues, and living animals. Multidimensional imaging was used to spectroscopically characterize human skin lesions, as malignant melanoma and naevi. Moreover, two-color detection of two photon excited fluorescence was applied to in-vivo imaging of living mice intact neocortex, as well as to induce neuronal microlesions by femtosecond laser burning. The presented applications demonstrate the capability of the instrument to be used in a wide range of biological and biomedical studies.