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.

Single gold-tagged epidermal growth factor (EGF) molecules bound to cellular EGF receptors of fixed fibroblast cells were imaged in liquid with a scanning transmission electron microscope (STEM). The cells were placed in buffer solution in a microfluidic device with electron transparent windows inside the vacuum of the electron microscope. A spatial resolution of 4 nm and a pixel dwell time of 20 s were obtained. The liquid layer was sufficiently thick to contain the cells with a thickness of 7 ؎ 1 m. The experimental findings are consistent with a theoretical calculation. Liquid STEM is a unique approach for imaging single molecules in whole cells with significantly improved resolution and imaging speed over existing methods. cellular imaging ͉ molecular labels U nderstanding cellular function at a molecular level requires imaging techniques capable of imaging whole cells with a resolution sufficient to image individually tagged proteins. Electron microscopy and X-ray diffraction are traditionally used to resolve the structures of individual proteins and to image proteins distributions in cells (1). Imaging with these techniques demands extensive sample preparation to obtain, e.g., proteins crystals, stained thin sections, or frozen samples. The cells are thus not in their native liquid state. Light microscopy is used to image protein distributions via fluorescent labels on fixed cells in liquid and in live cells to investigate cellular function (2). Superresolution techniques surpass the diffraction limit in optical microscopy (3-6), but despite recent advances, these methods are still restricted to spatial resolutions Ͼ10-20 nm. Further, their optimal performance requires extended imaging times, and significant data postprocessing. The speed can only be increased at the cost of resolution.Here, we describe a direct technique for imaging whole cells in liquid that offers nanometer spatial resolution and a high imaging speed. The principle is explained in Fig. 1. The eukaryotic cells in liquid are placed in a microfluidic flow cell with a thickness of up to 10 m contained between 2 ultrathin electron transparent windows. This flow cell is placed in the vacuum of a STEM, using a fluid specimen holder. The annular dark field (ADF) detector in the STEM is sensitive to scattered electrons, which are generated in proportion to the atomic number (Z) of the atoms in the specimen (7, 8), so-called Z contrast, where the contrast varies with ϷZ 2 . It is thus possible to image specific high-Z atoms, such as gold, inside a thick (several micrometer) layer of low-Z material, such as water, protein, or the embedding medium of a thin section (9). We used this approach to raster image single gold-tagged epidermal growth factor (EGF) molecules bound to cellular EGF receptors on fibroblast cells with a spatial resolution of 4 nm and a pixel dwell time of 20 s. ResultsCOS7 fibroblast cells were labeled with 10-nm gold nanoparticles conjugated with epidermal growth factor (EGF-Au). The cells were grown, labeled, and fixed directl...

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.

New microscopy method reveals local variations of functional state of membrane protein associated with cancer cell growth.

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 paper, 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.

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 µm. 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.

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Gold nanoparticles were observed to move at a liquid/solid interface 3 orders of magnitude slower than expected for the movement in a bulk liquid by Brownian motion. The nanoscale movement was studied with scanning transmission electron microscopy (STEM) using a liquid enclosure consisting of microchips with silicon nitride windows. The experiments involved a variation of the electron dose, the coating of the nanoparticles, the surface charge of the enclosing membrane, the viscosity, and the liquid thickness. The observed slow movement was not a result of hydrodynamic hindrance near a wall but instead explained by the presence of a layer of ordered liquid exhibiting a viscosity 5 orders of magnitude larger than a bulk liquid. The increased viscosity presumably led to a dramatic slowdown of the movement. The layer was formed as a result of the surface charge of the silicon nitride windows. The exceptionally slow motion is a crucial aspect of electron microscopy of specimens in liquid, enabling a direct observation of the movement and agglomeration of nanoscale objects in liquid.