To screen the temperature and pH conditions of THF (186562, Sigma-Aldrich) for dehydration, we first mixed recombinant EGFP (0.05 mg/ml, final concentration) with 30 volume % THF/dH2O solution and incubated the mixture for 24 hours under different conditions. The recombinant EGFP was a histidine-tagged fusion protein of EGFP that was expressed in E. coli and purified by Ni-affinity chromatography. The THF solution was adjusted to pH 9.0 with triethylamine (80134318, Sinopharm Chemical Reagent Co. Ltd., China). The pH was measured by a pH meter (Ohaus). The resulting fluorescence signals were measured with a custom-made wide-field optical imaging system over time. The absorption spectra were measured by a LAMBDA 950 UV/Vis/NIR spectrophotometer (PerkinElmer, USA). All absorption spectra were normalized to the absorbance measured at 430 nm in each sample for further analysis. The fluorescence spectrum excited by a 488-nm laser was measured on an inverted confocal fluorescence microscope (LSM 710, Zeiss, Germany) in lambda mode.

We also mixed the recombinant EGFP with PBS (pH 7.4, room temperature), acetic acid (dissolved in H2O with pH 5.0, room temperature), 8 M urea (60°C; 10023218, Sinopharm Chemical Reagent Co., Ltd., China), and 6 M guanidine hydrochloride (GH; room temperature; 30095516, Sinopharm Chemical Reagent Co., Ltd., China). The absorption spectra were measured after incubation for 2 hours, when the fluorescence of EGFP treated by HAc, urea, and GH almost disappeared.

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