Temporal dynamics of charge buildup in cryo-electron microscopy.

It is well known that insulating samples can accumulate electric charges from exposure to an electron beam. How the accumulation of charge affects imaging parameters and sample stability in transmission electron microscopy is poorly understood. To quantify these effects, it is important to know how the charge is distributed within the sample and how it builds up over time. In the present study, we determine the spatial distribution and temporal dynamics of charge accumulation on vitreous ice samples with embedded proteins through a combination of modeling and Fresnel diffraction experiments. Our data reveal a rapid evolution of the charge state on ice upon initial exposure to the electron beam accompanied by charge gradients at the interfaces between ice and carbon films. We demonstrate that ice film movement and charge state variations occur upon electron beam exposure and are dose-rate dependent. Both affect the image defocus through a combination of sample height changes and lensing effects. Our results may be used as a guide to improve sample preparation, data collection, and data processing for imaging of dose-sensitive samples.

Low-dose electron energy-loss spectroscopy using electron counting direct detectors.

Since the development of parallel electron energy loss spectroscopy (EELS), charge-coupled devices (CCDs) have been the default detectors for EELS. With the recent development of electron-counting direct-detection cameras, micrographs can be acquired under very low electron doses at significantly improved signal-to-noise ratio. In spectroscopy, in particular in combination with a monochromator, the signal can be extremely weak and the detection limit is principally defined by noise introduced by the detector. Here we report the use of an electron-counting direct-detection camera for EEL spectroscopy. We studied the oxygen K edge of amorphous ice and obtained a signal noise ratio up to 10 times higher than with a conventional CCD.We report the application of electron counting to record time-resolved EEL spectra of a biological protein embedded in amorphous ice, revealing chemical changes observed in situ while exposed by the electron beam. A change in the fine structure of nitrogen K and the carbon K edges were recorded during irradiation. A concentration of 3 at% nitrogen was detected with a total electron dose of only 1.7 e-/Å2, extending the boundaries of EELS signal detection at low electron doses.

Role of subsurface diffusion and Ostwald ripening in catalyst formation for single-walled carbon nanotube forest growth.

Here we show that essentially any Fe compounds spanning Fe salts, nanoparticles, and buckyferrocene could serve as catalysts for single-walled carbon nanotube (SWNT) forest growth when supported on AlO(x) and annealed in hydrogen. This observation was explained by subsurface diffusion of Fe atoms into the AlO(x) support induced by hydrogen annealing where most of the deposited Fe left the surface and the remaining Fe atoms reconfigured into small nanoparticles suitable for SWNT growth. Interestingly, the average diameters of the SWNTs grown from all iron compounds studied were nearly identical (2.8-3.1 nm). We interpret that the offsetting effects of Ostwald ripening and subsurface diffusion resulted in the ability to grow SWNT forests with similar average diameters regardless of the initial Fe catalyst.

Review of recent advances in spectrum imaging and its extension to reciprocal space.

Using examples from various domains of science, this review covers some recent developments in spectrum imaging (SI) using scanning transmission electron microscopy (STEM) and electron energy loss spectroscopy (EELS). Advanced multi-dimensional acquisition methods allow the acquisition of STEM-EELS data with other complementary data such as energy dispersive X-ray spectroscopy (EDS), cathodoluminescence and even combining them with reciprocal space analysis through a new method called diffraction imaging. This method allows real and reciprocal space information to be mixed to get a more complete description of the electron-sample interaction. The developments in SI data analysis such as multiple linear least-squares fitting, non-linear least-squares fitting and multivariate analysis allow for a robust extraction not only of each elemental distribution but also of each chemical phase in a sample through an intuitive computer-assisted method.

Revealing the secret of water-assisted carbon nanotube synthesis by microscopic observation of the interaction of water on the catalysts.

We elucidated the secret of water-assisted chemical vapor deposition (CVD) by elucidating the influence of water on the catalysts, through ex situ microscopic and spectroscopic analysis. We unambiguously showed that catalyst deactivation readily occurs due to carbon coating and that water acted to remove this coating and revive catalysts activity. This represents the central point of water-assisted CVD.

Photoinduced electron transfer on aqueous carbon nanohorn-pyrene-tetrathiafulvalene architectures.

Water-soluble carbon-nanohorn-tetrathiafulvalene (CNH-TTF) nanoensembles were prepared by utilizing positively charged pyrene as an assembly medium and characterized by spectroscopy and electron microscopy. Electronic interactions within the nanoensemble were probed by optical spectroscopy, indicating electron transfer between the TTF units and CNHs after light illumination.

Soluble functionalized carbon nanohorns.

Carbon nanahorns (CNH) were functionalized following the methodology of 1,3-dipolar cycloaddition of azomethine ylides and found to form stable solutions in either organic solvents or water. The number of added functional units, in the form of pyrrolidine moieties, was calculated when a pyrene chromophore was utilized in the modification scheme. Moreover, complementary theoretical calculations revealed that reactivity enhancement is expected at locations near the conical-shaped tip of CNH, where the highest curvature and strain exist. Finally, additional organic transformation of already modified CNH was exploited by covalently linked ferrocene units.

Optimum hole-opening condition for Cisplatin incorporation in single-wall carbon nanohorns and its release.

We enclosed cisplatin (CDDP), an anticancer drug, inside single-wall carbon nanohorns (SWNH) with holes opened by being heated from room temperature to a target temperature (475-580 degrees C) in flowing dry air, with an increase rate of 1 degrees C/min. The optimum target temperature was found to be 500 degrees C, in terms of the least amount of CDDP deposited outside the SWNH, when the quantity of CDDP encapsulated inside the SWNH was 12 wt %. The incorporated CDDP was slowly released from the SWNH in phosphate buffer saline, and the released quantity was 80%, which was greatly improved from the previous value of 15%. This indicated that a CDDP-containing SWNH could become more potentially useful for biological applications.

Effect of functional groups at hole edges on cisplatin release from inside single-wall carbon nanohorns.

We incorporated cisplatin inside single-wall carbon nanohorns (NHs) and revealed that 70% of the cisplatin was released from NHs having holes with hydrogen-terminated edges when they were immersed in phosphate-buffered saline (PBS). However, only 15% was released from NHs having holes with oxygen-containing functional groups at the hole edges (NHox). Elemental analysis indicated that -COOH and -OH groups at the hole edges of NHox changed mainly to -COONa and -ONa groups by immersion in PBS. These groups decreased the practical hole diameters, which resulted in hindering the cisplatin release from NHox. This means that the release of the material from inside NHox would be controlled by chemically modifying the functional groups attached to the hole edges of NHox; thus the potential applicability of NHox to a material carrier would be enhanced.

Carbon nanohorns as anticancer drug carriers.

We demonstrate that oxidized single-wall carbon nanohorns (SWNHs), a type of single-wall nanotube, entrap cisplatin, an anticancer agent. We found that the cisplatin structure was maintained inside the SWNHs and that the cisplatin was slowly released from the SWNHs in aqueous environments. The released cisplatin was effective in terminating the growth of human lung-cancer cells, while the SWNHs themselves had no such effect. Cisplatin-incorporated oxidized SWNHs are thus a potential drug delivery system.