Effect of the surrounding environment on electron beam irradiation damage of enhanced green fluorescent protein

Abstract

Fluorescent proteins exhibit fluorescence and photoconversion, which are used to study biological phenomena. Among these, enhanced green fluorescent protein (EGFP) emits cathodoluminescence when irradiated with electron beams; this phenomenon has numerous applications in new research tools for biological phenomena. However, bleaching during electron irradiation is a major problem. Generally, the presence of water is important for biological samples, and structural observations are often performed under cryogenic conditions. One of the advantages of cryogenic conditions is the stabilization of the sample due to cooling. However, it is unclear which factor is more effective: the presence of water molecules or cryogenic preservation. To explore the stabilizing factors of the sample structure, we prepared four environments around the sample–dry at room temperature, wet at room temperature, dry at low temperature, and under cryogenic conditions–and investigated the electron beam irradiation damage by measuring the fluorescence emission spectra. Emission intensity from EGFP was attenuated, and the peak was red-shifted by electron beam irradiation; however, the intensity attenuation was fast under dry conditions at low temperature and slow under wet conditions at room temperature. These results imply that sample cooling has no significant effect on the stability of the EGFP chromophore and that the presence of water molecules is extremely important.