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

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.

Application of Hilbert-differential phase contrast to scanning transmission electron microscopy.

We report a novel class of scanning transmission electron microscopy with Hilbert-differential phase contrast (HDP-STEM) that displays nanostructures of thin samples in a topographical manner. A semicircular π-phase plate (PP) was used as an optical device for manipulating electron waves in HDP-STEM. This is the different design from the Zernike PP used in our previous phase plate STEM (P-STEM), but both must be placed in the front focal plane of the condenser lens.

First step toward complex observations by 4D-STEM with phase plate.

Quantitative measurements by electron microscopy are becoming increasingly important because we are often concerned with establishing quantitative relationships between the properties and structures of materials. This paper presents a method to derive the scattering and phase contrast components from scanning transmission electron microscope (STEM) images using a phase plate and two-dimensional electron detector and to quantitatively evaluate the amount of phase modulation. The phase-contrast transfer function (PCTF) modifies the phase contrast because it is not unity over all spatial frequency regions; therefore, the amount of phase modulation observed in the image becomes smaller than the actual value.

Toward complex observation in electron microscopy using two-dimensional electron detector coupled with phase plate STEM.

A two-dimensional (2D) detector was used to construct phase plate STEM (P-STEM) images. Phase-contrast can be enhanced by the electron intensity inside the hole region of a thin film phase plate. The electron intensity outside the hole region also provides a dark image contrast, which is inconsistent with the weak phase object approximation.

Evaluation of electron radiation damage to green fluorescent protein.

Green fluorescent protein (GFP) emits light when irradiated by not only light but also electrons. This electron-induced light emission called cathodoluminescence (CL) can be used to realize a high-resolution light emission microscopy based on the irradiation of a very narrow electron beam. To implement CL mapping in life sciences the investigation of the damage resistance of GFP to electron irradiation needs to be clarified.