Label-free identification of the hippocampus and surrounding structures by multiphoton microscopy

Abstract

The hippocampus is one of the essential neuroanatomical substrates and plays an important role in different neurological illnesses. In this work, multiphoton microscopy (MPM) based on intrinsic nonlinear optical processes two-photon excited fluorescence (TPEF) and second harmonic generation (SHG), was applied to label-freely detect the entire hippocampus and surrounding structures in high-magnification imaging, as well as acquire large-scale MPM images at subcellular resolution. It was found that MPM has the capability to identify cornu ammonis, dentate gyrus (DG), alveus, and fimbria of the entire hippocampus, choroid plexus in lateral ventricles, and white matter tracts. MPM also can be used to quantitatively describe the differences of the cellular nucleus in the cornu ammonis and the DG, further identify the morphological features of hippocampal subfields. In addition, the surrounding structures of the hippocampus including the lateral ventricles and white matter serve as useful information to determine the position of the hippocampus. Our results suggest that with the development of the clinical feasibility of two-photon fiberscopes and microendoscope probes, MPM has the potential for in vivo intraoperative identification and monitoring of hippocampus-related lesions without the need for tissue labelling or fluorescent markers.