BEAM: A Custom‐Built and Versatile Optical Setup for Real‐Time Monitoring of Absorption and Emission Spectra During Photobleaching of Fluorescent Proteins

The article presents bleaching with emission and absorption monitoring (BEAM), a cost‐effective and customizable optical setup designed for real‐time monitoring of photochemical reactions through simultaneous measurement of absorption and emission spectra. BEAM utilizes readily available optical components such as lamp or laser diodes for excitation, cuvettes for sample containment, and spectrometers for spectral acquisition. Its modular architecture enables precise control over excitation wavelength, sample volume, temperature, and stirring conditions, making it highly adaptable to various experimental needs. The article also proposes a detailed protocol for measuring the effective photon flux density and irradiance across different optical configurations. The system's capabilities are demonstrated through the study of Citrine's photobleaching, a widely used but poorly photostable fluorescent protein (FP). BEAM enables continuous monitoring of Citrine's spectral evolution under different conditions, including excitation laser powers, solution volumes, and protein concentrations. The results revealed complex changes in both absorption and emission spectra during the bleaching process. Nevertheless, the fluorescence intensity of Citrine follows a first‐order decay profile, facilitating straightforward kinetic modeling. This allowed determination of key photophysical parameters, such as the photobleaching molar cross section and quantum yield. These findings underscore BEAM's utility for characterizing fluorescent proteins and open pathways for comparative studies with other FPs.