pHifty Shades of Green!

Fluorescent dyes play a crucial role in cell biology by enabling the visualization and tracking of specific intracellular environments and processes. One such dye, LysoSensor™ Green (LSG), is widely used to label acidic lysosomal compartments in live animal cells. To better understand the molecular behavior and functional interactions of LSG within the complex cellular environment, it is essential to investigate its structural and vibrational properties. The Optical Nanospectroscopy Group (Janina Kneipp) explored the vibrational spectra of LSG using surface-enhanced Raman scattering (SERS) at physiologically relevant concentrations and varying pH levels, employing biocompatible gold nanoprobes commonly used in live-cell applications. By comparing the SERS spectra of LSG with those of its aromatic components—benzimidazole and naphthalimide—they provide spectral assignments and insights into pH-dependent structural changes. Interestingly, off-resonance excitation at 785 nm offers enhanced sensitivity to pH-induced spectral shifts compared to excitation at 633 nm, highlighting the advantages of specific excitation wavelengths in bioanalytical settings. If you want to obtain a deeper molecular understanding of LSG behavior in biological systems, check out their Analysis & Sensing Article!

Abstract

Knowledge about the molecular structure and interactions of intracellular dyes is important to understand their function and possible effects in the biological environment. Here we discuss vibrational spectra of the dye LysoSensorTM (LSG) that is used as a fluorescent marker for the acidic lysosomal compartment in animal cells. Raman spectra of the molecule at concentrations that are typically applied in experiments with cell cultures were obtained by surface-enhanced Raman scattering (SERS) experiments at varying pH values, using biocompatible gold nanoprobes that are also used in live cell SERS. The signals in the SERS spectral profile of the dye were assigned based on spectra collected from its aromatic constituents benzimidazole and naphthalimide under the same conditions. The data indicate a strong pH dependence of the spectra of benzimidazole, less pronounced in the vibrational signature of the complete LSG molecule. Despite excitation of the SERS off-resonance with electronic transitions in the dye molecule, spectra obtained with the biocompatible wavelength of 785 nm have better sensitivity towards the pH-dependent structural changes, due to favorable electromagnetic enhancement of vibrational modes, than when using excitation at 633 nm. The characterization based on SERS will help to understand the interactions of the LSG molecule with the cellular environment and with other molecular probes, which will be useful for the design of efficient labels for bioanalysis.