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Detecting membrane potentials is critical for understanding how neuronal networks process information. We report a vibrational spectroscopic signature of neuronal membrane potentials identified through hyperspectral stimulated Raman scattering (SRS) imaging of patched primary neurons. High-speed SRS imaging allowed direct visualization of puff-induced depolarization of multiple neurons in mouse brain slices, confirmed by simultaneous calcium imaging. The observed signature, partially dependent on sodium ion influx, is interpreted as ion interactions on the CH Fermi resonance peak in proteins. By implementing a dual-SRS balanced detection scheme, we detected single action potentials in electrically stimulated neurons. These results collectively demonstrate the potential of sensing neuronal activities at multiple sites with a label-free vibrational microscope.

Spectroscopic stimulated Raman scattering (SRS) microscopy is a label-free technique that generates chemical maps of live cells or tissues. A handheld SRS imaging system using an optical fiber for laser delivery will further enable in situ and in vivo compositional analysis for applications such as medical diagnosis and surgical guidance. In fiber-delivered SRS, the interaction of two ultrashort pulses in the confined mode area creates a significant background that overwhelms the stimulated Raman signal from a sample. Here, we report the first background-free fiber-delivered handheld SRS microscope for in situ chemical imaging. By temporally separating the two ultrafast pulses propagating in the fiber and then overlapping them on a sample through a highly dispersive material, we detected a stimulated Raman signal that is 200 times weaker than the background induced by the fiber. Broad applications of the handheld SRS microscope were demonstrated through in situ ambient-light chemical mapping of pesticide on a spinach leaf, cancerous tissue versus healthy brain tissue in a canine model, and cosmetic distribution on live human skin. A lab-built objective lens further reduced the size of the pen-shaped microscope to about one centimeter in diameter.

Stimulated Raman scattering microscopy (SRS) was deployed to quantify enamel demineralization in intact teeth. The surfaces of 15 bovine-enamel blocks were divided into four equal-areas, and chemically demineralized for 0, 8, 16, or 24 h, respectively. SRS images (spectral coverage from ∼850 to 1150 cm −1) were obtained at 10-μm increments up to 90 μm from the surface to the dentin-enamel junction. SRS intensities of phosphate (peak: 959 cm −1), carbonate (1070 cm −1), and water (3250 cm −1) were measured. The phosphate peak height was divided by the carbonate peak height to calculate the SRS-P/C-ratio, which was normalized relative to 90 μm (SRS-P/C-ratio-normalized). The water intensity against depth decay curve was fitted with exponential decay. A decay constant (SRS-water-content) was obtained. Knoop-hardness values were obtained before (SMH S) and after demineralization (SMH D). Surface microhardness-change (SMH-change) [ðSMH D − SMH S Þ∕SMH S ] was calculated. Depth and integrated mineral loss (ΔZ) were determined by transverse microradiography. Comparisons were made using repeated-measures of analysis of variance. For SRS-P/ C-ratio-normalized, at 0-μm (surface), sound (0-h demineralization) was significantly higher than 8-h demineralization and 24-h demineralization; 16-h demineralization was significantly higher than 24-h demineralization. For SRS-water-content, 24-h demineralization was significantly higher than all other demineralization-groups; 8-h demineralization and 16-h demineralization were significantly higher than 0-h demineralization. SRS-watercontent presented moderate-to-strong correlation with SMH-change and weak-to-moderate correlation with depth. These results collectively demonstrate the potential of using SRS microscopy for in-situ chemical analysis of dental caries.

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