Hirofumi Iijima

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.

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.

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.

Development of Amorphous Carbon Thin Film Phase Plate

However, thin film Zernike phase plate had some problems, those are, their reliability, lifetime (due to charging and aging) and cost (due to craft production including hole forming by a focused ion beam). To solve these problems, we have been challenging to fabricate several kinds of the thin film phase plates with various materials and structures by a high throughput fabrication method utilizing a micro electro mechanical systems (MEMS) technology. As a first trial, we have fabricated titanium (Ti) / silicon nitride (SiN) / Ti sandwich type thin film Zernike phase plates [3] and we improved the manufacturing yield. However, we could not achieve sufficient stability, due to charging of the Ti/SiN/Ti thin film Zernike phase plate.

Contrast Enhancement of Long-Range Periodic Structures using Hole-Free Phase Plate

Phase contrast transmission electron microscopy is a powerful tool to enhance the image contrast of transparent materials such as ice-embedded biological specimens and polymer materials. In this method, a phase plate, which is placed at the back-focal plane of the objective lens, gives a phase shift for scattered electron waves, resulting in a significant enhancement contrast of specimens in images. Zernike phase plate (ZPP), consisting of a thin carbon film with a small central hole, is first tested practical phase plate [1]. However, ZPP has disadvantages for image quality. Phase contrast of specimens in low spatial frequency does not improve, since the scattered electron passing through a central hole of ZPP does not change their phases. The threshold frequency at the center hole edge is called cut-on frequency. The additional disadvantageous effect of the abrupt cut-on frequency reveals that strong fringes appear.

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.

Development of Phase Contrast Scanning Transmission Electron Microscopy and its application

An interaction between electron waves and biological molecules composed of light elements is very weak. This makes it difficult to obtain their high contrast images in transmission electron microscopy (TEM). A phase plate (PP) for TEM was realized by Danev and Nagayama in 2001[1] and contrast of the biological molecules can be enhanced. They made Zernike-type phase plate which is a thin carbon film and has a small hole in its center. It is placed at a back focal plane (BFP) of the objective lens (OL). It gives a phase shift of a half  to scattering waves by means of the mean inner potential of the thin film and the unscattered wave was passed through the hole without giving a phase shift. Thus, the phase difference was provided among the scattering waves and un-scattering waves. It acts as a filter in the Fourier space and modifies the phase contrast transfer function (PCTF) of the OL form the sine-type to the cosine-type. Accordingly, phase contrasts of the biomolecules would be visualized.

High Throughput Fabrication Process of a Zernike Phase Plate

In transmission electron microscopy (TEM) for biological and polymer samples, it is difficult to image with high contrast, since they are mostly composed of light elements and have similar density. One solution to enhance the contrast is utilization of phase contrast microscopy, which is realized in optical microscopy. Accordingly, many types of phase plates for electron microscopy have been proposed. Zernike phase contrast TEM (ZPC-TEM) with a Zernike phase plate (ZPP) provides higher contrast at Af (defocus) = 0 with respect to one in conventional TEM [1]. ZPC-TEM attracts much attention in Cryo-TEM applications such as cryo-electron tomography and single-particle analysis [2], because their specimens are easy to be damaged with electrons and they need high contrast with minimum dose on the specimen.

First Demonstration of Phase Contrast Scanning Transmission Electron Microscopy

Biological molecules are composed of light elements and can be considered as phase objects for the electron waves. Therefore, images of their unstained specimens show low contrast in transmission electron microscopy (TEM). Phase contrast TEM (P-TEM) is one of the powerful techniques to enhance image contrast of the phase objects [1]. In P-TEM, the phase plate (PP) is placed at a back focal plane (BFP) of the objective lens (OL), and its role is giving a phase shift on a portion of transmitted electron waves, which are scattered or unscattered depending on the type of the PP.