Visualizing the Role of Lipid Dynamics during Infrared Neural Stimulation with Hyperspectral Stimulated Raman Scattering Microscopy

Infrared neural stimulation, or INS, is a method of using pulsed infrared light to yield label-free neural stimulation with broad experimental and translational utility. Despite its robust demonstration, the mechanistic and biophysical underpinnings of INS have been the subject of debate for more than a decade. The role of lipid membrane thermodynamics appears to play an important role in how fast IR-mediated heating nonspecifically drives action potential generation. Direct observation of lipid membrane dynamics during INS remains to be shown in a live neural model system. To directly test the involvement of lipid dynamics in INS, we used hyperspectral stimulated Raman scattering (hsSRS) microscopy to study biochemical signatures of high-speed vibrational dynamics underlying INS in a live neural cell culture model. Findings suggest that lipid bilayer structural changes are occurring during INS in vitro in NG108-15 neuroglioma cells. Lipid-specific signatures of cell SRS spectra were found to vary with stimulation energy and radiant exposure. Spectroscopic observations were verified against high-speed ratiometric fluorescence imaging of a conventional lipophilic membrane structure reporter, di-4-ANNEPS. Overall, the presented data supports the hypothesis that INS causes changes in the lipid membrane of neural cells by changing lipid membrane packing order - which coincides with likelihood of cell stimulation. Furthermore, this work highlights the potential of hsSRS as a method to study biophysical and biochemical dynamics safely in live cells.