Hybrid fluorescence and electron cryo-microscopy for simultaneous electron and photon imaging.

Integration of fluorescence light and transmission electron microscopy into the same device would represent an important advance in correlative microscopy, which traditionally involves two separate microscopes for imaging. To achieve such integration, the primary technical challenge that must be solved regards how to arrange two objective lenses used for light and electron microscopy in such a manner that they can properly focus on a single specimen. To address this issue, both lateral displacement of the specimen between two lenses and specimen rotation have been proposed. Such movement of the specimen allows sequential collection of two kinds of microscopic images of a single target, but prevents simultaneous imaging. This shortcoming has been made up by using a simple optical device, a reflection mirror. Here, we present an approach toward the versatile integration of fluorescence and electron microscopy for simultaneous imaging. The potential of simultaneous hybrid microscopy was demonstrated by fluorescence and electron sequential imaging of a fluorescent protein expressed in cells and cathodoluminescence imaging of fluorescent beads.

Contrast enhancement in the phase plate transmission electron microscopy using an objective lens with a long focal length.

A new optical condition using an objective lens (OL) of a long focal length (objective mini lens: OM) was tested to enhance image contrast in phase plate transmission electron microscopy (P-TEM). A phase plate was set on the selected area aperture plane where diffraction patterns were formed under the optical condition using the OM. A phase shift by the phase plate was added to the electron waves to visualize phase objects. The application of the OM to the P-TEM should provide higher phase contrast than that obtained by the OL for the phase objects. One of the reasons for the contrast enhancement is that high-angle scattering electron waves which would give the background intensity were not used for image formation due to the large spherical aberration. Another reason is that the cut-on frequency above which the phase shift was added by the phase plate could be smaller using the OL with a long focal length. Experimental results and model calculations showed the contrast enhancement of the biological specimens using the OM.

Fast and accurate defocus modulation for improved tunability of cryo-EM experiments.

Current data collection strategies in electron cryo-microscopy (cryo-EM) record multiframe movies with static optical settings. This limits the number of adjustable parameters that can be used to optimize the experiment. Here, a method for fast and accurate defocus (FADE) modulation during movie acquisition is proposed. It uses the objective lens aperture as an electrostatic pole that locally modifies the electron beam potential. The beam potential variation is converted to defocus change by the typically undesired chromatic aberration of the objective lens. The simplicity, electrostatic principle and low electrical impedance of the device allow fast switching speeds that will enable per-frame defocus modulation of cryo-EM movies. Researchers will be able to define custom defocus 'recipes' and tailor the experiment for optimal information extraction from the sample. The FADE method could help to convert the microscope into a more dynamic and flexible optical platform that delivers better performance in cryo-EM single-particle analysis and electron cryo-tomography.

Understanding hydrogen-bonding structures of molecular crystals via electron and NMR nanocrystallography.

Understanding hydrogen-bonding networks in nanocrystals and microcrystals that are too small for X-ray diffractometry is a challenge. Although electron diffraction (ED) or electron 3D crystallography are applicable to determining the structures of such nanocrystals owing to their strong scattering power, these techniques still lead to ambiguities in the hydrogen atom positions and misassignments of atoms with similar atomic numbers such as carbon, nitrogen, and oxygen. Here, we propose a technique combining ED, solid-state NMR (SSNMR), and first-principles quantum calculations to overcome these limitations. The rotational ED method is first used to determine the positions of the non-hydrogen atoms, and SSNMR is then applied to ascertain the hydrogen atom positions and assign the carbon, nitrogen, and oxygen atoms via the NMR signals for 1H, 13C, 14N, and 15N with the aid of quantum computations. This approach elucidates the hydrogen-bonding networks in L-histidine and cimetidine form B whose structure was previously unknown.

Contrast enhancement of nanomaterials using phase plate STEM.

Visualizing materials composed of light elements is difficult, and the development of an imaging method that enhances the phase contrast of such materials has been of much interest. In this study, we demonstrate phase-plate scanning transmission electron microscopy (P-STEM), which we developed recently, and its application to nanomaterials. An amorphous carbon film with a small hole in its center was used to control the phase of incident electron waves, and the phase-contrast transfer function (PCTF) was modified from sine-type to cosine-type. The modification of the PCTF enhances image contrast with a spatial frequency below 1 nm-1. The PCTF for P-STEM with a spatial frequency below 1 nm-1 is about three times stronger than that of bright field STEM. The ratio obtained using power spectra is consistent with the result obtained from images of quantum dots. The image contrast of biological materials was also enhanced by P-STEM.

Computer simulations analysis for determining the polarity of charge generated by high energy electron irradiation of a thin film.

Detailed simulations are necessary to correctly interpret the charge polarity of electron beam irradiated thin film patch. Relying on systematic simulations we provide guidelines and movies to interpret experimentally the polarity of the charged area, to be understood as the sign of the electrostatic potential developed under the beam with reference to a ground electrode. We discuss the two methods most frequently used to assess charge polarity: Fresnel imaging of the irradiated area and Thon rings analysis. We also briefly discuss parameter optimization for hole free phase plate (HFPP) imaging. Our results are particularly relevant to understanding contrast of hole-free phase plate imaging and Berriman effect.

Phase-contrast scanning transmission electron microscopy.

This report introduces the first results obtained using phase-contrast scanning transmission electron microscopy (P-STEM). A carbon-film phase plate (PP) with a small center hole is placed in the condenser aperture plane so that a phase shift is introduced in the incident electron waves except those passing through the center hole. A cosine-type phase-contrast transfer function emerges when the phase-shifted scattered waves interfere with the non-phase-shifted unscattered waves, which passed through the center hole before incidence onto the specimen. The phase contrast resulting in P-STEM is optically identical to that in phase-contrast transmission electron microscopy that is used to provide high contrast for weak phase objects. Therefore, the use of PPs can enhance the phase contrast of the STEM images of specimens in principle. The phase shift resulting from the PP, whose thickness corresponds to a phase shift of π, has been confirmed using interference fringes displayed in the Ronchigram of a silicon single crystal specimen. The interference fringes were found to abruptly shift at the edge of the PP hole by π.