ID-1-IN-1629 Imaging Labeled Protein Complex Subunits in Whole Eukaryotic Cells in their Native Aqueous Environment

We have used scanning transmission electron microscopy (STEM) of cells in liquid [1, 2], so-called Liquid STEM, to study protein complex subunits. Live eukaryotic cells were grown on silicon microchips with silicon nitride (SiN) membrane windows, and incubated with specific protein labels consisting of gold nanoparticles or fluorescence nanoparticles, quantum dots (QDs). The samples were imaged with STEM. On account of the atomic number (Z) contrast of the annular dark field (ADF) detector of STEM, the nanoparticles of high-Z material can be detected within the background signal produced by the low-Z material of the cell and surrounding liquid. The highest resolution was obtained using STEM at 200 keV electron beam energy, for which the cells were placed in a microfluidic chamber with electron transparent windows (Fig. 1A). The flat parts of cells can also be imaged in a thin layer of water with environmental scanning electron microscopy (ESEM) at 30 keV with the STEM detector (Fig. 1B).

Liquid STEM was used to study several different proteins and protein complexes on intact cells in liquid, such as the epidermal growth factor receptor, and the closely related ErbB2 receptor. The particular distribution of monomers, homodimers, and heterodimers of these receptors is of relevance for basic research as well as for the analysis of drug mechanisms. We used COS7 fibroblast cells and SKBR3 breast cancer cells. The receptors were specifically labeled with nanoparticles via small, specific ligands, much smaller than antibodies used for immunogold labeling. It was observed that the expression levels and the distribution of the receptors differed significantly from cell to cell. Therefore, we used correlative fluorescence microscopy to localize cells and cellular regions where a certain type of receptor was present (see Fig. 2A). The sample was transferred into the ESEM chamber and an overview image was recorded at the same location (Fig. 2B). ESEM-STEM images were then recorded at a higher magnification to localize the individual QDs (Fig. 2C). The regions of stronger fluorescence correlated with a higher density of the QDs. Since the extensive sample preparation common for biological electron microscopy is mostly avoided, the Liquid STEM method is as simple as fluorescence microscopy. It is readily possible to acquire data of thousands of labels on dozens of cells. This advantage was used to study the distribution of EGFR monomers, dimers and clusters using a total of 1411 obtained from images of 15 cells [3].

References:

[1] N de Jonge et al., Proc Natl Acad Sci 106, 2159-2164, 2009.

[2] N de Jonge & FM Ross, Nature Nanotechnology 6, 695-704, 2011.

[3] DB Peckys et al, Scientific reports 3, 2626, 2013.