Optical Visualization of Cerebral Cortex by Label-Free Multiphoton Microscopy

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 and two-photon excited fluorescence, is applied for visualizing the mouse and human cerebral cortex from fresh, unfixed, and unstained specimen. We show that label-free MPM is able to effectively visualize microscopic scale of cortical neurons, six-layer cortical lamination, and functional cortex that correlates with the corresponding stained histological images. Moreover, we further demonstrate that MPM is feasible for identifying focal cortical dysplasia (FCD) in the human cerebral cortex. The border between gray and white matter, abnormal radial cortical lamination, immature small diameter neurons, pia mater, and blood vessels are also clearly visualized as the typical histopathologic hallmark of FCD. In addition, to diagnose FCD more accurately and rapidly, an image processing method is employed to extract MPM image features for auxiliary identification of FCD specimen. These results offer additional metrics to pathologists and surgeons, which hold the translational potential to be used as a diagnostic tool into routine clinical use.