Electron microscopy of whole cells in liquid with nanometer resolution

Abstract

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...

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