Advantages of Simultaneous Imaging
fluorescence image (Figure 7c) indicated a high degree of spatial correlation between the lipid raſts and the cyto- skeletal elements (correlation coefficient 0.85). Te 3D topo- graphical image (Figure 7d) indicates that the dimen- sions of the T24 cells are approximately 4 µm (Z) by 60 µm (X-Y).
Conclusions Many biological studies
Figure 5: AFM and fluorescence images of WI38 cells that were labeled with phalloidin—Alex568 to highlight the actin filaments. (a) The fluorescence image (image width = 300 µm) (b) 75 × 75 µm 3D contact-mode AFM topography image, which corresponds to the area indicated inside the yellow box in (a).
the cell nucleus. In addition, the cytoskeletal filaments were also stained with a fluorescent dye conjugate, phalloidin- rhodamine, which is a red fluorescent stain that binds to actin filaments. Several of the filaments appeared to be pointed directly from the nucleus to the cell border (Figure 7b). Overlaying the GFP fluorescence image with the rhodamine
require more capabilities than either a light microscope or a stand-alone AFM can provide. By combining the AFM with an inverted light microscope to form a hybrid ILM–AFM system, some inherent limi-
tations of AFM and light microscopy can be minimized without losing the favorable attributes of these methods. Open scanner architectures do not obstruct the optical path to the sample and permit
the use of off-the-shelf, high-NA
Figure 6: DIC, fluorescence, and AFM images of living T24 cells that were transfected to express GFP in their membranes. (a) DIC optical image of the T24 cells. (b) Lipid raft distribution measured by GFP fluorescence of the same area as in (a). (c) 3D AAC mode topography image (90 × 90 µm) of a GFP protein expressing cell interacting with other cells that do not over-express GFP. The cytoskeleton is clearly resolved and connections between individual cells are also visible. (d) Overlay of the topography and fluorescence images. Images and data courtesy of Institute for Biophysics, University of Linz, Austria.
28
Figure 7: Phalloidin-rhodamine stained T24 cells that also express GFP in lipid rafts. (a) The GFP fluorescence channel demonstrates the distribution of the lipid rafts in the cells. (b) The rhodamine fluorescence channel shows the location of actin filaments in the same cells. (c) A rhodamine/GFP fluorescence overlay image was used to correlate the positions of the lipid rafts in relation to the cytoskeleton. (d) 3D MAC mode topography image of the same cluster of cells (90 × 90 µm). Images and data courtesy of Institute for Biophysics, University of Linz, Austria.
www.microscopy-today.com • 2011 November
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