β-Amyloid (Aβ) plaque, representing the progressive accumulation of the protein that mainly consists of Aβ, is one of the prominent pathological hallmarks of Alzheimer’s disease (AD). Label-free imaging of Aβ plaques holds the potential to be a histological examination tool for diagnosing AD. We applied label-free multiphoton microscopy to identify extracellular Aβ plaque as well as intracellular Aβ accumulation for the first time from AD mouse models. We showed that a two-photon-excited fluorescence signal is a sensitive optical marker for revealing the spatial–temporal progression and the surrounding morphological changes of Aβ deposition, which demonstrated that both extracellular and intracellular Aβ accumulations play an important role in the progression of AD. Moreover, combined with a custom-developed image-processing program, we established a rapid method to visualize different degrees of Aβ deposition by color coding. These results provide an approach for investigating pathophysiology of AD that can complement traditional biomedical procedures.

Accurate histopathological diagnosis is essential for facilitating the optimal surgical management of intracranial germinoma. Current intraoperative histological methods are time- and labor-intensive and often produce artifacts. Multiphoton microscopy (MPM) is a label-free imaging technique that can produce intraoperative histological images of fresh, unprocessed surgical specimens. We employ an MPM based on second-harmonic generation and two-photon excited fluorescence microscopy to image fresh, unfixed, and unstained human germinoma specimens. We show that label-free MPM is not only capable of identifying various cells in human germinoma tissue but also capable of revealing the characteristics of germinoma such as granuloma, stromal fibrosis, calcification, as well as the abnormal and uneven structures of blood vessels. In conjunction with custom-developed image-processing algorithms, MPM can further quantify and characterize the extent of stromal fibrosis and calcification. Our results provide insight into how MPM can deliver rapid diagnostic histological data that could inform the surgical management of intracranial germinoma.