Illuminating RPMI-1640: Fluorescence dynamics and optical insights for biological applications

RPMI-1640 is a commonly utilized cell culture medium that serves a variety of purposes in both biological research and clinical investigations. Given that Phenol Red constitutes a significant part of RPMI-1640, it is crucial to evaluate the medium’s optical characteristics in fluorescence-based assays to facilitate precise data interpretation in biological studies. This research examines the fluorescence properties of RPMI-1640 within the visible spectral range through the application of laser-induced fluorescence (LIF) spectroscopy and differential spectral analysis (DSA). The absorption spectrum indicates a broad distribution across the visible region, which can be attributed to constituents such as Vitamin B12, Phenol Red, and Riboflavin; however, notable fluorescence emission was observed when excited by wavelengths ranging from 400 to 450 nm. The stability of fluorescence, as measured with a 405 nm laser, exhibited minimal signal degradation over a 30-minute period. Additionally, the angular-dependent fluorescence characteristics and the asymmetries observed in the difference spectra provided evidence for the spatially non-homogeneous inner filter effect (2nd-IFE), wherein the reabsorption of emitted fluorescence modifies both the intensity and spectral profile. DSA further elucidated the 2nd-IFE, identifying isosbestic-like points where fluorescence remains largely unaffected by inner filter effects, thus rendering them appropriate for optical assessments of cellular properties within the culture medium.