Advantages of Simultaneous Imaging
Figure 3: Normal human lung (WI38) cell. These cells display a fibroblast-like morphology that is clearly evident in the AFM and DIC optical images. (a) 3D contact-mode AFM topography image (90 × 90 µm). (b) Corresponding 40× DIC optical image of the same cell (image width = 120 µm).
in the optical view using DIC along with fluorescence microscopy, and then it was positioned precisely under the tip of the AFM probe using “Point and Shoot.” Te cell was then imaged in contact mode as shown in figure 5. Te fluorescence image of the cell is shown in figure 5a. Te yellow box indicates the area from which the AFM 75 × 75 µm contact-mode AFM image was acquired. Te fluorescence image was acquired using a Lumen Dynamics X-CITE 120Q fluo- rescence light source, an LD Plan-Neofluar 40× objective, and a 63 HE mRFP shiſt-free filter set. Lipid raſts serve as
Figure 4: 3D contact-mode AFM images of a MyEND cell. (a) Contact-mode AFM topography image (70 × 70 µm). In (b), a feature of interest that was found in image (a) was repositioned under the AFM probe and imaged at higher resolution (14 × 14 µm). Inside the blue squares, small humps may be indicative of the presence of WPBs. The pore-like indentations in the red squares may indicate fusion events between the plasma membrane and WPBs leading to exocytosis of VWF (see text for further explanation).
exocytosis of VWF following hypoxic stimulation or vascular injury. In the high-magnification contact-mode AFM image shown in Figure 4b, both humps (blue squares) and pores (red squares) can clearly be observed. Because the WPBs are on the order of several hundred nanometers in diameter, the humps and the pores are too small to be easily resolved by standard light microscopy techniques. Using an ILM-AFM outfitted with fluorescence microscopy
capabilities permits combined AFM-fluorescence microscopy studies [3]. Te AFM can oſten resolve small structures that cannot be easily or efficiently labeled with fluorescent probes. Figure 5 shows a specific example of a combined AFM-fluorescence microscopy application using WI38 cells. In this case, the cells were grown on fibronectin-coated glass cover slips and fluorescently labeled with Alexa568 conjugated to a phallotoxin (phalloidin—Alexa568), which tightly and specifically binds to actin filaments [14]. A cell was located
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organizing centers for the assembly of signaling molecules. Tey also influence membrane fluidity, receptor trafficking, as well as other protein trafficking events. Te ILM-AFM permitted investigation of the morphology of live T24 (human bladder carcinoma) cells and the distribution of the lipid raſts. T24 cells were transfected to express green fluorescent protein (GFP) in the lipid raſts [15]. Te “Point and Shoot” capability of the soſtware and the motorized sample stage allowed for rapid screening of the sample surface for suitable cells. Te cell positions were located using DIC imaging (Figure 6a). GFP
expression levels were determined by fluorescence intensity (Figure 6b). Te brightest areas indicate locations where GFP was over-expressed. Te distribution of the lipid raſts was determined by changing the focal plane of the fluorescence images. For topographical imaging, a protein-expressing cell that was surrounded by non-fluorescent cells was selected (red square in Figure 6a) and imaged in AAC mode. Te filaments of the cytoskeleton are clearly resolved, as are lamellipodia. Several small connections between individual cells can also be observed. An overlay of the fluorescence and the topography image (Figure 6d) allowed the position of GFP-labeled lipid raſts to be correlated with topographical features such as cytoskeleton filaments and lamellipodia. In Figure 7, the locations of lipid raſts and cytoskeletal
elements in T24 cells were correlated using fluorescence microscopy. Te GFP fluorescence image (Figure 7a) represents the distribution of lipid raſts, most of which were found near
www.microscopy-today.com • 2011 November
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