Imaging Protein Labels with Liquid STEM
Correlative fluorescence microscopy and liquid STEM
of QD-labeled cells. COS7 fibroblast cells were grown on microchips, and the cells were incubated for five minutes with EGF conjugated to QD (EGF-QD) and then fixed with glutaraldehyde [12]. Te cells were imaged on the microchip first by light microscopy, with the microchip placed upside- down (Figure 5) in a cell culture dish with phosphate-buffered saline. Figure 6A shows a fluorescence microscopy image of a window section partly covered with adhering cells. Te QD labels light up as bright spots on cells against the dark background of cell-free regions. Te cellular regions also contain faint fluorescence from the glutaraldehyde fixative. Te microchip with cells was assembled into a microfluidic chamber along with a chip containing a spacer layer for liquid STEM imaging. Figure 6B shows a STEM image recorded at the edge of the same cell shown in Figure 6A. Te lower two-thirds of the image contain bright spots of similar sizes, which we associate with the presence of QDs. Te separation line between regions with and without QDs is interpreted as
cell edge. Some debris leſt over from the microchip fabrication process is visible as well. Localizing the same region in both light and electron
microscopy images oſten requires dedicated procedures in correlative microscopy [19, 20]. For liquid STEM the localization was accomplished with a simple procedure. Te positions of features in the fluorescence image were measured with respect to the frame of the SiN window, faintly visible in Figure 6A as a thin line. With the sample in the STEM, the position of one corner of the SiN window was located first. Te stage position at this point was recorded and the scan rotation was aligned such that the scan direction of the electron beam ran parallel to the short side of the SiN window. During STEM imaging, the stage position of each image was recorded and correlated with the previously determined frame position of the SiN window. Te position of Figure 6B corresponds to the square in Figure 6A. Te position of the square in the fluorescent image is indeed at the edge of the cell.
Discussion Te spatial resolution achieved with liquid STEM of 4 nm
and better on labeled proteins is on the order of the size of individual proteins. Te high spatial resolution of liquid STEM obtained on sample volumes compatible with whole eukaryotic cells is not achievable with a liquid cell for TEM imaging [8] because the TEM contrast mechanism limits high-resolution imaging to a thickness of about 0.5 m. In the case of thin and weakly scattering samples, TEM yields a better resolution than STEM [21], but for the case of whole cells, STEM offers a particular advantage when imaging high-Z labels [22].
Imaging back-scattered
Figure 5: For imaging with light microscopy, prior to liquid STEM imaging, one of the microchips with the attached cells was placed upside down in a glass bottom culture dish and imaged using an oil-immersion lens. From [12].
electrons with a scanning electron microscope (SEM) from a specimen in liquid separated from the vacuum by a thin membrane [23, 24] provides a resolution of 8–20 nm on gold labels, but this is a surface technique that images only the top ~0.1 µm of the sample. Te resolution of liquid STEM also surpasses alternative approaches, such as the imaging of cooled cells in water vapor using an environmental TEM [25, 26], environmental SEM with a STEM detector [27], or X-ray microscopy [28]. For fixed samples, liquid STEM presents a novel alternative to fluorescence microscopy. Te resolution of
liquid STEM
Figure 6: Correlative light microscopy and liquid STEM of intact fixed eukaryotic cells in saline water. (A) Red fluorescence signals from COS7 cells with QD-labeled EGF receptors. Some unspecific fluorescence from the fixative is also visible. The thin line at the left indicates the location of the silicon nitride window. (B) Liquid STEM image of the region indicated with a square in (A). Individual QDs along the edge of the cell can be discerned as green spots on a blue background. Some debris is also visible. The magnification was M = 48,000. The signal intensity was color-coded to increase the visibility of the labels. Figure modified from [12].
2011 September •
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is a factor of 50 higher than that of confocal microscopy. Current ultra-high-resolution optical methods include stimulated emission depletion (STED) [6, 29], photo-activated localization microscopy (PALM) [7], and stochastical optical reconstruction microscopy (STORM) [30]. Tose techniques can obtain fluorescence images with a sub-diffraction spatial resolution of about 30 nm for practical imaging conditions of cells [31]. However, this length scale is still not sufficient to resolve the constituents of protein complexes, and the number of available orthogonal labels is limited. Te achieved spatial resolution of liquid STEM can be
used to study protein distributions in cells and is sufficient to discriminate nanoparticles differing in size, shape, and electron density for multi-label experiments. A high-resolution liquid STEM image can thus provide information about the constituents of protein complexes with the context of
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