Second harmonic generation for brain imaging: pathology-related studies

Microscopy of the brain has been facing problems of contrast and thick tissue imaging. Second harmonic generation (SHG) is a non-linear effect of the light interaction with the imaged material, resulting in photon emission at half the wavelength of the absorbed light. SHG microscopy provides an unprecedented opportunity for imaging collagen and other noncentrosymmetric protein fibrils in unstained thick tissue samples and in the live brain via a regular multiphoton setup. This opens a remarkable methodological window for imaging pathological processes of high importance, including brain trauma, fibrosis, tumorigenesis, and neuroimplant-induced foreign body response. Moreover, SHG is a valuable tool for imaging astrocytes and nerve fiber microtubules. Third harmonic generation enhanced by three-photon resonance with the Soret band of hemoglobin is combined with SHG to resolve the microstructure of blood vessel walls and astrocyte-process endfeet on gliovascular interfaces. Here, we review current state-of-the-art methods in the field of brain imaging applications of SHG, including research on brain and spinal cord injury, glioma, ischemia, Alzheimer's disease, neuroimplantation, and brain meninges. We then address the method development perspective in the broader context of other tissue pathologies. Finally, we account for recent progress in artificial intelligence applications for SHG microscopy data analysis.