AbstractHuntington’s disease, a major neurodegenerative disorder, involves deposition of aggregation-prone proteins with long polyglutamine (polyQ) expansions. The ability to non-perturbatively visualize the formation of aggregates could offer new molecular insight for their pathologic roles. Here, we propose stimulated Raman scattering imaging of deuterium-labeled glutamine to investigate native polyQ aggregates in live cells with subcellular resolution. Through the enrichment of deuterated glutamine in the polyQ sequence of mutant Huntingtin (mHtt) proteins, we first achieved sensitive and specific SRS imaging of carbon-deuterium bonds (C-D) from aggregates without GFP labeling. These aggregates become 1.8-fold denser compared to those with GFP. Second, we performed ratiometric quantification, which revealed a dependence of protein compositions on aggregation sizes. Moreover, we calculated the absolute concentrations for sequestered mHtt and non-mHtt proteins within the same aggregates. Our method may readily reveal new features of polyQ aggregates and could be suited for in vivo investigations on multicellular organisms.
In Vivo Chemical and Structural Analysis of Plant Cuticular Waxes Using Stimulated Raman Scattering Microscopy