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. HDP-STEM images of multi-walled carbon nanotubes showed higher contrast than those obtained by conventional bright-field STEM. Since the PP of the HDP-STEM is nonsymmetrical, several different images were obtained by changing the detection conditions. A two-dimensional electron detector was also used to remove the scattering contrast component in the same way as with the Zernike PP and obtain an image containing only (differential) phase contrast.

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. We applied a filter function to the Fourier transform of image to perform PCTF correction and evaluated the phase modulation of the electron waves, which was quantitatively agreement with the values expected from the thickness estimated from the scattering contrast within 20% error. So far, few quantitative discussions on phase modulation have been conducted. Although the accuracy needs to be improved, this method is the first step toward quantitative complex observations.

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. We consider that both images have scattering effects that provide a dark contrast. Therefore, scattering contrast was derived by summing these two images, and scattering effects were subtracted from each image to display negative and positive phase contrast. The resultant images are consistent with the weak phase object approximation. These results propose separating scattering (electron amplitude) and phase-contrast (electron phase) using P-STEM, along with a two-dimensional electron detector.

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

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.

Investigation of hydrated smectite microstructure through wet environmental transmission electron microscopy.

Water is an essential constituent of all biological materials as well as many non-biological materials. Not only the removal of water may result in undesirable morphological and structure change, the inability to sustain the hydrated conditions in the microscope also prevents the study of reactions which take place in aqueous environment. In order to overcome these problems we used wet environmental-cell transmission electron microscopy TEM (WETEM). Conventional TEM of dry smectite showed well-defined particle outlines (but without a specific shape) and typical smectite aggregates. Selected area electron diffraction (SAD) of dry particles showed stacking of smectite particles (i.e., aggregate) in very clear dot and ring patterns. In contrast, WETEM depicted well-dispersed clay particles showing a variety of different particle shapes. Analysis of SAD patterns obtained from dry and hydrated states illustrated a lattice change in different environments. The small lattice expansion in (h k 0) resulted from the expansion of the (0 0 l) plane resulting from the addition of water molecules in the crystal along the c-axis.

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.

Cathodoluminescence and Electron-Induced Fluorescence Enhancement of Enhanced Green Fluorescent Protein.

Becaues the spatial resolution of fluorescence microscopy is not high enough to study the molecular level of relationship between the structure and function of biological specimens, correlative light and electron microscopy has been used for this purpose. Another possibility for a high-resolution light microscopy is cathodoluminescence microscopy. Here, we report a new phenomenon, the electron-induced activation of luminescence (cathodoluminescence) and electron-enhanced fluorescence for the enhanced green fluorescent protein (EGFP). This was found using our recently developed hybrid fluorescence and electron microscopy. Contrary to the past reports, which showed a degradation of organic compounds by electron irradiation, stable cathodoluminescence emitted from an organic molecule, EGFP, has been observed using the hybrid microscopy. Addition of the glycerol promoted the fluorescence enhancement of EGFP probably due to the change in the electronic state density of excitation channels from the ground to the excited state or of relaxation channels from the excited to the emission state. Stable cathodoluminescence and enhanced fluorescence of the EGFP may introduce a cathodoluminescence microscopy, which will increase the variety of the imaging to investigate the biological compounds.

Electron microscopic recording of myosin head power stroke in hydrated myosin filaments.

Muscle contraction results from cyclic attachment and detachment between myosin heads and actin filaments, coupled with ATP hydrolysis. Despite extensive studies, however, the amplitude of myosin head power stroke still remains to be a mystery. Using the gas environmental chamber, we have succeeded in recording the power stroke of position-marked myosin heads in hydrated mixture of actin and myosin filaments in a nearly isometric condition, in which myosin heads do not produce gross myofilament sliding, but only stretch adjacent elastic structures. On application of ATP, individual myosin heads move by ~3.3 nm at the distal region, and by ~2.5 nm at the proximal region of myosin head catalytic domain. After exhaustion of applied ATP, individual myosin heads return towards their initial position. At low ionic strength, the amplitude of myosin head power stroke increases to >4 nm at both distal and proximal regions of myosin heads catalytic domain, being consistent with the report that the force generated by individual myosin heads in muscle fibers is enhanced at low ionic strength. The advantages of the present study over other in vitro motility assay systems, using myosin heads detached from myosin filaments, are discussed.

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.

A carbon sandwich environmental cell for wet specimens.

A carbon sandwich environmental cell for wet specimens was developed for environmental transmission electron microscopy (E-TEM). A plastic film with many holes was used to form carbon capsules. The carbon sandwich environmental cells were composed of the spacer and two carbon films and enclosing the samples and experimental solution. The thickness of the water layer can be controlled by changing the conditions used to prepare the plastic spacers. The quality of the images was improved over our previous E-TEM, which could circulate gas around samples. A resolution of 2 nm was obtained by using loosely condensed DNAs.

Direct observation of biological molecules in liquid by environmental phase-plate transmission electron microscopy.

We have been developing a combination method for environmental TEM (E-TEM) and phase-plate TEM (P-TEM) that enables direct observations of the structure of biological molecules in aqueous solution. It is clearly demonstrated that the biological molecules in a water layer can be imaged by the combined method without any stain. The spatial resolution obtained in the present study was about 10nm. This should be improved by using energy filtering. The image contrast of the specimen in water was reduced in comparison with that in vacuum. A model calculation that includes the effects of beam broadening, intensity decrease, and background increase caused by scattering from the water layer around the specimen shows that an increase in the thickness of the water layer reduces the contrast, intensity, and resolution of the image.

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.

Electron microscopic evidence for the myosin head lever arm mechanism in hydrated myosin filaments using the gas environmental chamber.

Muscle contraction results from an attachment-detachment cycle between the myosin heads extending from myosin filaments and the sites on actin filaments. The myosin head first attaches to actin together with the products of ATP hydrolysis, performs a power stroke associated with release of hydrolysis products, and detaches from actin upon binding with new ATP. The detached myosin head then hydrolyses ATP, and performs a recovery stroke to restore its initial position. The strokes have been suggested to result from rotation of the lever arm domain around the converter domain, while the catalytic domain remains rigid. To ascertain the validity of the lever arm hypothesis in muscle, we recorded ATP-induced movement at different regions within individual myosin heads in hydrated myosin filaments, using the gas environmental chamber attached to the electron microscope. The myosin head were position-marked with gold particles using three different site-directed antibodies. The amplitude of ATP-induced movement at the actin binding site in the catalytic domain was similar to that at the boundary between the catalytic and converter domains, but was definitely larger than that at the regulatory light chain in the lever arm domain. These results are consistent with the myosin head lever arm mechanism in muscle contraction if some assumptions are made.

Direct demonstration of the cross-bridge recovery stroke in muscle thick filaments in aqueous solution by using the hydration chamber.

Despite >50 years of research work since the discovery of sliding filament mechanism in muscle contraction, structural details of the coupling of cyclic cross-bridge movement to ATP hydrolysis are not yet fully understood. An example would be whether lever arm tilting on the myosin filament backbone will occur in the absence of actin. The most direct way to elucidate such movement is to record ATP-induced cross-bridge movement in hydrated thick filaments. Using the hydration chamber, with which biological specimens can be kept in an aqueous environment in an electron microscope, we have succeeded in recording ATP-induced cross-bridge movement in hydrated thick filaments consisting of rabbit skeletal muscle myosin, with gold position markers attached to the cross-bridges. The position of individual cross-bridges did not change appreciably with time in the absence of ATP, indicating stability of time-averaged cross-bridge mean position. On application of ATP, individual cross-bridges moved nearly parallel to the filament long axis. The amplitude of the ATP-induced cross-bridge movement showed a peak at 5-7.5 nm. At both sides of the filament bare region, across which the cross-bridge polarity was reversed, the cross-bridges were found to move away from, but not toward, the bare region. Application of ADP produced no appreciable cross-bridge movement. Because ATP reacts rapidly with the cross-bridges (M) to form complex (M x ADP x Pi) with an average lifetime >10 s, the observed cross-bridge movement is associated with reaction, M + ATP --> M x ADP x Pi. The cross-bridges were observed to return to their initial position after exhaustion of ATP. These results constitute direct demonstration of the cross-bridge recovery stroke.