Imaging Protein Labels with Liquid STEM
works for TEM imaging when a smaller spacer is used [16].
Results Liquid STEM imaging
of EGF-Au on COS7 cells. Liquid
STEM images with
Figure 2: The microfluidic chamber for liquid STEM. (A) Scanning electron microscopy image showing the backside of a microchip with the silicon nitride window in the middle. (B) Schematic of the top view of the slot in the tip of the specimen holder in which the microchips are placed. The microchips are aligned at their sides via alignment poles. The liquid flow path runs through the chips. In addition, a second flow path, serving as a bypass channel, runs along the sides of the microchips. (C) The closed tip of the specimen holder, enclosing two microchips. The light shining through the windows indicates that both microchips are aligned. (This is not visible in the figure.)
to allow liquid to flow between the microchips and to provide a specimen chamber with sufficient height to contain thin eukaryotic cells; for example, COS7 fibroblast cells. In addition, the spacer defined a flow channel between two microchips over their long side. Figure 2C shows the closed tip of the specimen holder with the microfluidic chamber loaded in the interior. Light shining through the window indicated that alignment was achieved. Te liquid-flow specimen holder sealed the microfluidic
chamber from the vacuum of the electron microscope via the use of O-rings. Plastic tubing connected the liquid specimen region to a syringe pump (Harvard Scientific, MA), which was outside of the electron microscope. Liquid was pumped from the syringe, through the microfluidic chamber in the tip of the holder, and back out of the microscope again. A photograph of the system is shown in Figure 3. Te liquid STEM system consists of only three elements (the microfluidic chamber, the specimen holder, and the pump), transforming a standard STEM into a system for imaging specimens in liquid. Note that the system can be used for in situ electron microscopy of specimens in gaseous environments at atmospheric pressure [15]. Te system also
4 nm resolution were obtained on gold nanoparticle tagged proteins in whole eukaryotic cells in liquid [9]. COS7 fibro- blast cells were labeled with 10 nm gold nanoparticles con- jugated to epidermal growth factor (EGF). Te cells were grown, labeled, and fixed with glutaradehyde directly on the microchips. Te STEM was
a 200 kV TEM/STEM (CM200, Philips/FEI, OR). Figure 4A shows the edge of a cell that was incubated for 5 minutes with EGF-Au. Gold labels are visible as green spots and the cellular material as light-blue matter over the dark-blue background. Te localization of the labels at the cell edges aſter 5 minutes of
label incubation is consistent with the physiological
distribution of the EGF receptor, which is randomly dispersed over the cell surface [17]. Te acquisition time for Figure 4A was 21 seconds for a 1024 × 1024 pixel image with a pixel-dwell time of t = 20 µs. Te imaging speed can be increased simply by recording smaller images. Te electron dose used for one image was 7 · 104 e−/nm2. Te achieved spatial resolution was consistent with calculations and with results obtained on test samples containing gold nanoparticles [18]. To observe molecular rearrangements of the EGF
receptors in the COS7 cells aſter ligand binding, a second batch of cells was incubated for 10 minutes with EGF-Au and then washed and incubated for an additional 15-minute period in buffer. Liquid STEM images of these cells are shown in Fig- ure 4B. Circular clusters of labels are visible, consistent with the clustering of the EGF receptor in internalized endosomes aſter receptor activation [17].
Figure 3: Photograph of
the liquid STEM system including the specimen
holder and a microfluidic syringe pump. Plastic tubing connects the liquid in the syringe with the microfluidic chamber at the tip of the specimen holder, which is placed in the vacuum chamber of the electron microscope.
18
Figure 4: Liquid STEM images of gold-labeled epidermal growth factor (EGF) receptors on COS7 fibroblast cells. (A) Image of the edge of a fixed COS7 cell after 5 minutes of incubation with EGF-Au. The gold labels are visible as bright green spots on the blue background. The background shows some detail of the edge of the cell. (B) Image of a COS7 cell incubated with EGF-Au for 10 minutes and then incubated in buffer for an additional 15 minutes. The signal intensity was color-coded to increase the visibility of the labels. Images modified from [9].
www.microscopy-today.com • 2011 September
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