Abstract.SignificanceTo prevent meningioma recurrence, it is necessary to detect and remove all corresponding tumors intraoperatively, including those in the adjacent dura mater.AimCurrently, the removal of meningiomas from the dura mater depends solely on cautious visual identification of lesions by a neurosurgeon. Inspired by the requirements for resection, we propose multiphoton microscopy (MPM) based on two-photon-excited fluorescence and second-harmonic generation as a histopathological diagnostic paradigm to assist neurosurgeons in achieving precise and complete resection.ApproachSeven fresh normal human dura mater samples and 10 meningioma-infiltrated dura mater samples, collected from 10 patients with meningioma, were acquired for this study. First, multi-channel mode and lambda mode detection were utilized in the MPM to characterize the architectural and spectral features of normal and meningioma-infiltrated dura mater, respectively. Three imaging algorithms were then employed to quantify the architectural differences between the normal and meningioma-infiltrated dura mater through calculations of the collagen content, orientation, and alignment. Finally, MPM was combined with another custom-developed imaging algorithm to locate the meningioma within the dura mater and further delineate the tumor boundary.ResultsMPM not only detected meningioma cells in the dura mater but also revealed the morphological and spectral differences between normal and meningioma-infiltrated dura mater, providing quantitative information. Furthermore, combined with a self-developed image-processing algorithm, the precise borders of meningiomas in the dura mater could be accurately delineated.ConclusionsMPM can automatically detect meningiomas in the dura mater label-free. With the development of advanced multiphoton endoscopy, MPM combined with image analysis can provide decision-making support for histopathological diagnosis, as well as offer neurosurgeons more precise intraoperative resection guidance for meningiomas.