Additive-color multi-harmonic generation microscopy for simultaneous label-free differentiation of plaques, tangles, and neuronal axons.

Multicolor fluorescence imaging has been widely used by neuroscientists to simultaneously observe different neuropathological features of the brain. However, these optical modalities rely on exogenous labeling. Here, we demonstrate, for the first time, a label-free additive-color multi-harmonic generation microscopy to elucidate, concurrently with different hues, Alzheimer's disease (AD) neuropathological hallmarks: amyloid (A) plaques and neurofibrillary tangles (NFT). By treating third harmonic generation (THG) and second harmonic generation (SHG) as two primary colors, our study can simultaneously label-free differentiate AD hallmarks by providing different additive colors between A plaques, NFT, and neuronal axons, with weaker THG presentation from NFT in most places of the brain. Interestingly our pixel-based quantification and Pearson's correlation results further corroborated these findings. Our proposed label-free technique fulfills the unmet challenge in the clinical histopathology for stain-free slide-free differential visualization of neurodegenerative disease pathologies, with a sub-femtoliter resolution in a single image field-of-view.