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.
Standard histopathology is well accepted as the gold standard for the diagnosis a wide range of diseases. Despite continuing advances in tissue staining automation, typical histological processing such as formalin-fixed paraffin-embedded are also labour- and time-intensive for treatment decisions in intraoperative histopathologic diagnosis. Multiphoton microscopy (MPM), based on second harmonic generation (SHG) and two-photon excited fluorescence (TPEF), can be a versatile tool that enables label-free mapping of endogenous fluorophores within a fresh specimen, which provides pathology-like images with cellular and subcellular details. Here, we describe the use of label-free MPM for visualizing rat and human ex vivo brain tissue without tissue fixation, processing, and staining. Moreover, MPM is able to identify 6 types of cells in rat cerebrum and cerebellum, including cortical neurons, glia cells, Purkinje cells, pyramidal neurons and granule neurons in hippocampus, as well as epithelial cells in lateral ventricle. In addition, we further demonstrate that MPM can provide definitive pathological features in cerebral ischemia and focal cortical dysplasia (FCD) for assisting pathologic diagnosis. Our work establishes the methodology and augments the diagnostic accuracy of traditional frozen section histopathology. With the development of the miniature two-photon microscope, MPM will show more potential as a practical clinical tool for providing intraoperative reference image guidance of resection in neurosurgery.
Cortical structures in the central nervous system exhibit an ordered laminar organization. Defined cell layers are significant to our understanding of brain structure and function. In this work, multiphoton microscopy (MPM) based on second harmonic generation (SHG) and two-photon excited fluorescence (TPEF), which was applied for qualitatively visualizing the structure of cerebral and cerebellar cortex from the fresh, unfixed, and unstained specimen. MPM is able to effectively identify neurons and neurites in cerebral cortex, as well as glial cells, Purkinje cells, and granule cells in cerebellar cortex at subcellular resolution. In addition, the use of automated image processing algorithms can quantify the circularity of neurons and the density distribution of neurites based on the intrinsic nonlinear optical contrast, further providing quantitative characteristics for automatically analyzing the laminar structure of cortical structures. These results suggest that with the development of the feasibility of two-photon fiberscopes and microendoscope probes, the combined MPM and image analysis holds potential to provide supplementary information to augment the diagnostic accuracy of neuropathology and in vivo identification of various neurological illnesses in clinic.