Cathodoluminescence of green fluorescent protein exhibits the redshifted spectrum and the robustness

Abstract

Green fluorescent protein (GFP) and its variants are an essential tool for visualizing functional units in biomaterials. This is achieved by the fascinating optical properties of them. Here, we report novel optical properties of enhanced GFP (EGFP), which is one of widely used engineered variants of the wild-type GFP. We study the electron-beam-induced luminescence, which is known as cathodoluminescence (CL), using the hybrid light and transmission electron microscope. Surprisingly, even from the same specimen, we observe a completely different dependences of the fluorescence and CL on the electron beam irradiation. Since light emission is normally independent of whether an electron is excited to the upper level by light or by electron beam, this difference is quite peculiar. We conclude that the electron beam irradiation causes the local generation of a new redshifted form of EGFP and CL is preferentially emitted from it. In addition, we also find that the redshifted form is rather robust to electron bombardment. These remarkable properties can be utilized for three-dimensional reconstruction without electron staining in focused ion beam/scanning electron microscopy technology and provide significant potential for simultaneously observing the functional information specified by super-resolution CL imaging and the structural information at the molecular level obtained by electron microscope.

Electron‐Beam Induced Luminescence and Bleaching in Polymer Resins and Embedded Biomaterial

“…Our results provide perspective in the light of recent results on electron exposure induced CL from existing fluorescent probes such as fluorescent proteins. [ 15 ] Further analysis of electron‐induced luminescence origins may be required to confirm if these CL changes take place at the single probe level or result from chemical reactions between probes and/or the environment. For CLEM, the impact of electron‐induced luminescence is more subtle since FM inspection typically precedes EM imaging.…”

“…Interestingly, recent reports on electron‐irradiation of fluorescent proteins show electron‐enhanced fluorescence and cathodoluminescence (CL) that is robust under electron‐irradiation. [ 14,15 ] This could hold great promise for high‐resolution multimodal microscopy but it also raises the question whether luminescence can be more generally induced during EM in biological samples and their embedding polymer resins.…”

Superbunching in cathodoluminescence: a master equation approach

“…In electron microscopy, cathodoluminescence (CL) visualizes optical properties beyond diffraction limit of light. A wide range of materials can be investigated by this approach, for instance, defect/luminescence centers in semiconductors [1][2][3][4], quantum-confined structures [5][6][7], surface plasmon polaritons [8][9][10], and fluorescent proteins [11][12][13]. Thus, the electron microscopy-based CL measurement is a powerful tool to analyze various materials on nanoscale.…”