Fluorescent Protein Photobleaching: From Molecular Processes to Spectromicroscopy
Abstract
Fluorescent proteins (FPs) are essential tools for biological imaging, but their use is limited by photobleaching, a light-induced loss of fluorescence intensity that reduces spatial and temporal resolution. The molecular mechanisms underlying FP photobleaching remain poorly understood due to the diversity of FPs and the complexity of their photochemistry. Existing approaches either monitor fluorescence decrease in live cells, reflecting imaging conditions but lacking molecular detail, or rely on in vitro spectroscopy of purified proteins, providing mechanistic insight but often limited to individual FPs. We introduce a quantitative workflow bridging these approaches by combining live-cell measurements with in vitro spectroscopy. In vitro measurements are performed on a dedicated setup that simultaneously monitors absorption, emission, as well as fluorescence variations during photobleaching. Applied to six FPs spanning different chromophores, emission ranges and sequences, this approach reveals that photobleaching involves multiple chemical pathways, including oxidation, dimerization, and backbone cleavage. Spectroscopic analysis uncovers a heterogeneous ensemble of photoproducts with distinct photophysical properties that can remain optically active during irradiation, including shortened fluorescence lifetimes or altered absorption spectra. These findings demonstrate that FP photobleaching cannot be described as a simple ON–OFF process but involves complex transformations affecting both fluorescence intensity and lifetime. Such transformations can introduce significant biases in quantitative imaging, particularly in advanced techniques such as FLIM and FRET. Finally, we introduce quantitative indicators enabling robust comparison of FP photostability across experimental conditions. This framework provides a comprehensive approach for understanding and quantifying photobleaching and its implications for fluorescence imaging.
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BibTeX
@article{beguin2026fluorescent,
title = {{Fluorescent Protein Photobleaching: From Molecular Processes to Spectromicroscopy}},
author = {Theo Beguin and Kaixin Wang and Yasmina Bousmah and Ninette Abou Mrad and Frédéric Halgand and Hélène Pasquier and Marie Erard},
journal = {ACS Sensors},
year = {2026},
month = aug,
doi = {10.1021/acssensors.6c01504},
url = {https://hal.science/hal-05759638v1},
note = {CC BY-NC-SA 4.0},
}
RIS
TY - JOUR
TI - Fluorescent Protein Photobleaching: From Molecular Processes to Spectromicroscopy
AU - Theo Beguin
AU - Kaixin Wang
AU - Yasmina Bousmah
AU - Ninette Abou Mrad
AU - Frédéric Halgand
AU - Hélène Pasquier
AU - Marie Erard
PY - 2026
DA - 2026/08/04
JO - ACS Sensors
DO - 10.1021/acssensors.6c01504
UR - https://hal.science/hal-05759638v1
AB - Fluorescent proteins (FPs) are essential tools for biological imaging, but their use is limited by photobleaching, a light-induced loss of fluorescence intensity that reduces spatial and temporal resolution. The molecular mechanisms underlying FP photobleaching remain poorly understood due to the diversity of FPs and the complexity of their photochemistry. Existing approaches either monitor fluorescence decrease in live cells, reflecting imaging conditions but lacking molecular detail, or rely on in vitro spectroscopy of purified proteins, providing mechanistic insight but often limited to individual FPs. We introduce a quantitative workflow bridging these approaches by combining live-cell measurements with in vitro spectroscopy. In vitro measurements are performed on a dedicated setup that simultaneously monitors absorption, emission, as well as fluorescence variations during photobleaching. Applied to six FPs spanning different chromophores, emission ranges and sequences, this approach reveals that photobleaching involves multiple chemical pathways, including oxidation, dimerization, and backbone cleavage. Spectroscopic analysis uncovers a heterogeneous ensemble of photoproducts with distinct photophysical properties that can remain optically active during irradiation, including shortened fluorescence lifetimes or altered absorption spectra. These findings demonstrate that FP photobleaching cannot be described as a simple ON–OFF process but involves complex transformations affecting both fluorescence intensity and lifetime. Such transformations can introduce significant biases in quantitative imaging, particularly in advanced techniques such as FLIM and FRET. Finally, we introduce quantitative indicators enabling robust comparison of FP photostability across experimental conditions. This framework provides a comprehensive approach for understanding and quantifying photobleaching and its implications for fluorescence imaging.
SN - 2379-3694
ER -