Label-Free Vibrational Spectroscopic Imaging of Neuronal Membrane Potential

Abstract

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.

Label-free characterization of Amyloid-β-plaques and associated lipids in brain tissues using stimulated Raman scattering microscopy

“…The optical setup and principle of SRS microscopy are illustrated in Supplemental Figure S1 and described in Materials and Methods. In recent years, CRS has emerged as a powerful approach for label-free, biochemical imaging of neuronal structures and for dynamic monitoring of functional parameters such as membrane potential 4 and neurotransmitter concentrations 5 . In addition, CRS has been used in fundamental research on neurodegenerative diseases, to characterize Alzheimer's pathology 6,7,8 , Parkinson's Disease 9 and multiple sclerosis 10 , or to study peripheral nerve degeneration in ALS 11 .…”

Raman spectroscopy probing of redox states and mechanism of flavin coenzyme

“…Here, we found that surface‐enhanced Raman spectroscopy (SERS) is a powerful experimental approach for the fine characterization of redox states of flavin coenzymes and redox reaction schemes in different pH environments. The SERS technique is already established as a powerful analytical approach for the label‐free characterization and analysis of biomolecules such as heme protein, porphyrins, membrane protein, and highly organized systems such as membrane preparation and photosynthetic bacteria, and photosynthetic reaction centers . In this work, we have utilized the SERS technique for the analysis and characterization of electrochemically generated redox states of FMN, which helps to determine the electrochemical reaction pathway of FMN in different pH environments.…”

Applications of vibrational tags in biological imaging by Raman microscopy

“…More importantly, the rich spectral features that vary between different molecules makes it possible for hyperspectral imaging of multiple chemical species that are always of great interest to biologists. In recent advances with label-free techniques, for example, metabolic heterogeneity of live Euglena gracilis has been studied 10 , providing insight to microalgal research; in other studies, neurotransmitter 11 and neuronal membrane potential 12 have been visualized, adding new tools to investigate neurobiology; and brain tumor infiltration diagnostics 13, 14 has been successfully demonstrated in mouse brain and human patient samples, and an intraoperative set-up using fiber laser was then engineered 15 to assist surgery. However, although label-free vibrational imaging has been demonstrated a useful tool in biological studies, the detection specificity is usually limited – any molecules that harbor the same chemical bond of target will have severely overlapping spectrum and make it extremely hard to image a specific molecule of interest.…”