Niels de Jonge

Electron microscopy of specimens in liquid

Imaging samples in liquids with electron microscopy can provide unique insights into biological systems, such as cells containing labelled proteins, and into processes of importance in materials science, such as nanoparticle synthesis and electrochemical deposition. Here we review recent progress in the use of electron microscopy in liquids and its applications. We examine the experimental challenges involved and the resolution that can be achieved with different forms of the technique. We conclude by assessing the potential role that electron microscopy of liquid samples can play in areas such as energy storage and bioimaging.

High brightness electron beam from a multi-walled carbon nanotube

Carbon nanotubes can act as electron sources with very rigid structures, making them particularly interesting for use as point electron sources in high-resolution electron-beam instruments. Promising results have been reported with respect to some important requirements for such applications: a stable emitted current and a long lifetime. Two parameters of an electron source affect the resolution of these instruments: the energy spread of the emitted electrons and a parameter called the reduced brightness, which depends on the angular current density and the virtual source size. Several authors have measured a low energy spread associated with electron emission. Here we measure the reduced brightness, and find a value that is more than a factor of ten larger than provided by state-of-the-art electron sources in electron microscopes. In addition, we show that an individual multi-walled carbon nanotube emits most current into a single narrow beam. On the basis of these results, we expect that carbon nanotube electron sources will lead to a significant improvement in the performance of high-resolution electron-beam instruments.

Carbon nanotube electron sources and applications

In this review we give an overview of the present status of research on carbon nanotube (CNT) field emitters and their applications. Several different construction principles of field–emission devices with CNTs are summarized. The emission mechanism is introduced and a detailed overview is given of the measured emission properties and related topics of CNT electron sources. We give also several examples of field–emission devices with CNT electron emitters that are presently being investigated in the academic world as well as in industry. Carbon nanotube electron sources clearly have interesting properties, such as low voltage operation, good stability, long lifetime and high brightness. The most promising applications are the field–emission display and high–resolution electron–beam instruments. But several hurdles remain, such as the manufacture of an electron source or an array of electron sources with exactly the desired properties in a reproducible manner.

Characterization of the field emission properties of individual thin carbon nanotubes

Electron emission measurements were conducted on individual carbon nanotubes. The nanotubes had a closed end and their surfaces were thoroughly cleaned. It is shown conclusively that individual carbon nanotube electron emitters indeed exhibit Fowler–Nordheim behavior and have a work function of 5.1±0.1eV for the nanotubes under investigation, which had diameters of 1.4 and 4.9nm.

Three-Dimensional Electron Microscopy Simulation with the CASINO Monte Carlo Software

Monte Carlo softwares are widely used to understand the capabilities of electron microscopes. To study more realistic applications with complex samples, 3D Monte Carlo softwares are needed. In this article, the development of the 3D version of CASINO is presented. The software feature a graphical user interface, an efficient (in relation to simulation time and memory use) 3D simulation model, accurate physic models for electron microscopy applications, and it is available freely to the scientific community at this website: www.gel.usherbrooke.ca/casino/index.html. It can be used to model backscattered, secondary, and transmitted electron signals as well as absorbed energy. The software features like scan points and shot noise allow the simulation and study of realistic experimental conditions. This software has an improved energy range for scanning electron microscopy and scanning transmission electron microscopy applications.

Visualizing Gold Nanoparticle Uptake in Live Cells with Liquid Scanning Transmission Electron Microscopy

The intracellular uptake of 30 nm diameter gold nanoparticles (Au-NPs) was studied at the nanoscale in pristine eukaryotic cells. Live COS-7 cells were maintained in a microfluidic chamber and imaged using scanning transmission electron microscopy. A quantitative image analysis showed that Au-NPs bound to the membranes of vesicles, possibly lysosomes, and occupied 67% of the available surface area. The vesicles accumulated to form a micrometer-sized cluster after 24 h of incubation. Two clusters were analyzed and found to consist of 117 ± 9 and 164 ± 4 NP-filled vesicles.

Nanometer-resolution electron microscopy through micrometers-thick water layers

Scanning transmission electron microscopy (STEM) was used to image gold nanoparticles on top of and below saline water layers of several micrometers thickness. The smallest gold nanoparticles studied had diameters of 1.4 nm and were visible for a liquid thickness of up to 3.3 microm. The imaging of gold nanoparticles below several micrometers of liquid was limited by broadening of the electron probe caused by scattering of the electron beam in the liquid. The experimental data corresponded to analytical models of the resolution and of the electron probe broadening as function of the liquid thickness. The results were also compared with Monte Carlo simulations of the STEM imaging on modeled specimens of similar geometry and composition as used for the experiments. Applications of STEM imaging in liquid can be found in cell biology, e.g., to study tagged proteins in whole eukaryotic cells in liquid and in materials science to study the interaction of solid:liquid interfaces at the nanoscale.

Microfluidic system for transmission electron microscopy.

We present a microfluidic system that maintains liquid flow in a specimen chamber for scanning transmission electron microscope (STEM) imaging. The specimen chamber consists of two ultrathin silicon nitride windows supported by silicon microchips. They are placed in a specimen holder that seals the sample from the vacuum in the electron microscope and incorporates tubing to and from the sample connected to a syringe pump outside the microscope. Using results obtained from fluorescence microscopy of microspheres flowing through the system, an equation to characterize the liquid flow through the system was calibrated. Gold nanoparticles of diameters of 30 and 100 nm moving in liquid were imaged with a 200 kV STEM. It was concluded that despite strong influences from Brownian motion, and sensitivity to small changes in the depth of the bypass channel, the electron microscopy flow data matched the calculated flow speed within an order of magnitude. The system allows for rapid (within a minute) liquid exchange, which can potentially be used, for example, to investigate the response of specimens, e.g., eukaryotic or bacterial cells, to certain stimuli.

Brightness of carbon nanotube electron sources

The virtual source sizes of individual multiwalled carbon nanotube electron emitters were investigated with a point projection microscope. The average radius of the virtual source size was found to be 2.6 nm, which does not correspond to the standard model of a field emitter. Instead, a model based on a flattened cap or an open cap seems to provide a more realistic description. The broadening effect of Coulomb interactions on the virtual source was calculated. The reduced angular current density was measured at the maximum current at which stable emission was obtained and arrived at an average of 30 nA sr−1 V−1. The reduced brightness values obtained for two emitters were (2.5±1)×109 and (1.3±0.5)×109 A m−2 sr−1 V−1, respectively. These values are an order of magnitude larger than the values of state-of-the-art commercial sources.

Correlative Fluorescence Microscopy and Scanning Transmission Electron Microscopy of Quantum Dot Labeled Proteins in Whole Cells in Liquid

Correlative fluorescence microscopy and transmission electron microscopy (TEM) is a state-of-the-art microscopy methodology to study cellular function, combining the functionality of light microscopy with the high resolution of electron microscopy. However, this technique involves complex sample preparation procedures due to its need for either thin sections or frozen samples for TEM imaging. Here, we introduce a novel correlative approach capable of imaging whole eukaryotic cells in liquid with fluorescence microscopy and with scanning transmission electron microscopy (STEM); there is no additional sample preparation necessary for the electron microscopy. Quantum dots (QDs) were bound to epidermal growth factor (EGF) receptors of COS7 fibroblast cells. Fixed whole cells in saline water were imaged with fluorescence microscopy and subsequently with STEM. The STEM images were correlated with fluorescence images of the same cellular regions. QDs of dimensions 7x12 nm were visible in a 5 microm thick layer of saline water, consistent with calculations. A spatial resolution of 3 nm was achieved on the QDs.