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

Abstract

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 β (Aβ) 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 Aβ 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.

Slide-free clinical imaging of melanin with absolute quantities using label-free third-harmonic-generation enhancement-ratio microscopy

“…The 3D imaging depth was within 250 µm limited by the objective working distance. the A similar system was recently reported in Ref [24].…”

Investigating the optical clearing effects of 50% glycerol in ex vivo human skin by harmonic generation microscopy

“…The ultrashort pulse duration (38 fs) of this laser makes it ideal to generate efficient harmonic signals at the focal point. Double chirped mirrors 69 were utilized to compensate for the dispersion of the optical system for the shortest pulse width in human skin. A telescope (New port, KPX193 AR18) was used to collimate the laser light to maintain the beam size after a long traveling path.…”

“…Following Ref. 69 , the pulse group delay dispersion (GDD) at the illumination plane after the objective was − 60 fs 2 , corresponding to a pulsewidth of 28 fs. Since it has been reported that the chromatic dispersion value of water with lipid at 1300 nm is zero 70 and the group velocity dispersion value of water 71 at 1262 nm as − 47 fs 2 /mm, we thus expect that the GDD value of 300-μm-thick skin tissues at 1262 nm is on the order of − 10 fs 2 n so that its effect on lengthening the 1262 nm pulse duration is negligible.…”

“…However, the variation of thickness might be different in different layers of the skin (skin structure for the top to the bottom: the stratum corneum, viable epidermis, papillary dermis, and reticular dermis 75 ) and might result in different optical clearing efficacies. Five different parameters related to the layer thickness were investigated, which are the thickness of SC, the thickness of viable epidermis, depth of the basal layer, depth of the middle papillary dermis layer, and depth of the top reticular dermis layer 48 , 69 . On the top of the SC, the strongest THG signal will be generated.…”

The Progress of Label-Free Optical Imaging in Alzheimer’s Disease Screening and Diagnosis

“…Compared with the staining image, THG could visualize more detailed brain structures, such as neuronal soma and axon fiber bundles. By taking advantage of the emission of SHG from Aβ plaques and NFTs, and THG from Aβ plaques, the same research team used a combination of SHG and THG for differential visualization of Aβ plaques and NFTs ( Chakraborty et al, 2020a ). In this additive-color multi-harmonic generation imaging, the THG-green and SHG-red pseudo-colors were used to generate the images that visualize the yellow Aβ plaques, orange-red NFTs, and green axons and dendrites.…”