Advantages of Simultaneous Imaging
giſts from professor Tatiana P. Ugarova and Ivan Yermolenko of Arizona State University. Te cells were grown at 37°C at 5% CO2, on bare glass cover slips or protein coated cover slips in cell-specific culture media. In some cases, the cells were lightly fixed by adding low concentrations of glutaraldehyde to the medium. Fluorescently labeled normal human cells were stained with phalloidin—Alexa568. All AFM experiments were performed using an Agilent 6000ILM AFM on a Zeiss Axio Observer that was equipped with phase contrast, DIC, and fluorescence as well as a 0.55 NA condenser. Cover slips containing the cells were mounted onto a perfusion cell sample holder or a standard liquid cell. Te laser was aligned on the AFM probes manually via soſtware control. Te sample holder was filled with PBS buffer, and the photo detector was aligned using the automated alignment feature in PicoView. For MAC, and AAC mode imaging, MAC Levers were tuned to resonance using the auto tune feature. Te cells were imaged at room temperature in either contact, AAC or MAC mode as described in the figure legends. Te “Point and Shoot” feature was used to position areas of interest under the tip of AFM probes to enable precise AFM imaging at the predefined locations indentified by the light microscope (bright field, DIC, phase contrast, and/ or fluorescence). Te overlay feature was used to overlay the fluorescent images onto the AFM images. Figure 2a shows a 40× DIC optical image of living cells
derived from human cervical cancer cells (HeLa) [9]. Te red box indicates the area from which an AFM image is to be acquired using “Point and Shoot.” In Figure 2b, the AFM contact-mode deflection image was overlaid on the optical image from 2a in real time. Figure 2c is a 3D contact-mode AFM image of cells in (b). Te deflection image was overlaid on the 3D topography image using PicoImage soſtware in (c) to show both the depth and finer details of the cells. Te cells were grown on collagen-coated cover slips and imaged in HEPES buffer containing BSA.
Applications Differential interference contrast and phase contrast light
microscopy techniques [10] permit even semi-transparent samples to be visualized and located so that they can be investigated with the AFM. An example of DIC combined with AFM imaging is presented in Figure 3. A normal cell line (WI38) originating from human lung tissue [11] can be seen in the optical image as well as in the AFM image; they have a fibroblast-like morphology. In this case, a WI38 cell was identified by DIC (Figure 3b) so the sample could be positioned directly under the tip of the AFM probe. Te cell was then imaged in PBS buffer using contact-mode AFM (Figure 3a). Mouse vascular endothelial (MyEND) cells [12] and
other vascular cells contain particular membrane-bound structures, called Weibel-Palade bodies (WPB). WPBs are storage organelles for von Willebrand factor (VWF), which is a glycoprotein that mediates the adherence of platelets to one another and to sites of vascular damage [13]. In this manner VWF promotes blood clot formation leading to wound healing. Bumps on the vascular endothelial cell surface observed in high-resolution AFM images may be indicative of the presence of intact WPBs located under the plasma membrane. WPB secretion pores can also form on the surface of vascular endothelial cells. Te pores oſten indicate fusion events between WPBs and the plasma membrane, leading to
24
Figure 2: (a) 40× DIC optical image of living cells derived from human cervical cancer cells (HeLa). The red box indicates the area from which the AFM image is to be acquired using “Point and Shoot.” (b) Contact-mode deflection image (60 × 60 µm) overlaid on the area in the red box indicated in 2a. (c) 3D contact-mode topography image combined with the deflection image of the cells in (b) (60 × 60 µm).
www.microscopy-today.com • 2011 November
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