Lissajous Scanning Two-photon Endomicroscope for In vivo Tissue Imaging

An endomicroscope opens new frontiers of non-invasive biopsy for in vivo imaging applications. Here we report two-photon laser scanning endomicroscope for in vivo cellular and tissue imaging using a Lissajous fiber scanner. The fiber scanner consists of a piezoelectric (PZT) tube, a single double-clad fiber (DCF) with high fluorescence collection, and a micro-tethered-silicon-oscillator (MTSO) for the separation of biaxial resonant scanning frequencies. The endomicroscopic imaging exhibits 5 frames/s with 99% in scanning density by using the selection rule of scanning frequencies. The endomicroscopic scanner was compactly packaged within a stainless tube of 2.6 mm in diameter with a high NA gradient-index (GRIN) lens, which can be easily inserted into the working channel of a conventional laparoscope. The lateral and axial resolutions of the endomicroscope are 0.70 µm and 7.6 μm, respectively. Two-photon fluorescence images of a stained kidney section and miscellaneous ex vivo and in vivo organs from wild type and green fluorescent protein transgenic (GFP-TG) mice were successfully obtained by using the endomicroscope. The endomicroscope also obtained label free images including autofluorescence and second-harmonic generation of an ear tissue of Thy1-GCaMP6 (GP5.17) mouse. The Lissajous scanning two-photon endomicroscope can provide a compact handheld platform for in vivo tissue imaging or optical biopsy applications.