5. Structure and Function
Although structural details of the lamellipodium can be revealed by electron microscopy there are limitations that must be considered. Since samples in the electron microscope must be viewed in a vacuum we are unable to directly observe living cells by this technique (but see also cryo-electron microscopy, section ...). We must therefore immobilise cells by chemical fixation before electron microscopy. In addition, if we wish to view the cytoskeleton, the cell membrane must be removed to expose it. This is achieved by adding a detergent to the fixation mixture.
An example of using this technique is presented in Koestler, et al. 2008, Urban et al., 2010 and Vinzenz et al., 2012 and summarized in the following sections (The pushing network and Lamellipodium in 3D).
In order to correlate structure with function in a moving cell it is essential to know the state of movement at the time of fixation. This can be achieved by performing live cell imaging and electron microscopy on the same cell (Auinger and Small, 2008). Briefly, cells are grown on a thin plastic film cast on a glass coverslip. The cells can be imaged live in the fluorescence microscope and fixed directly on the microscope stage during a video sequence. Thereafter, the plastic film is peeled off the coverslip, transferred to an EM grid and negatively stained for electron microscopy. Re-location of the cell observed in the light microscope in the EM is facilitated by embossing an indexed grid pattern in gold onto the plastic film (Fig.5-1)
Figure 5-1. shows a schematic illustration of how the coverslip-film combination used for cell culture and imaging is prepared. A formvar film is produced on a glass slide and floated onto a water surface (1-4). Prewashed coverslips are coated on the rim with concentrated formvar (5). The coverslips are added to the floating film and removed with parafilm (6-7). A grid pattern is then applied to the film by shadowing gold through a custom-made mask in an evaporation unit (8). After sterilization under UV the film is coated with any desired matrix molecule and used for cell culture.
Figure 5-2. Schematic illustration of how the plastic film is retrieved from the coverslip and transferred to an EM grid for negative staining. With the coverslip immersed in buffer in a Petri dish (1), the film is loosed on the edges and flipped onto the buffer surface, with the cells down (2-3). Under a dissecting microscope, the film is maneuvered onto a ring and the grid placed on the film using forceps mounted in a micromanipulator (4). Parafilm is layed over the floating grid and the parafilm and formvar film pressed together at the periphery with forceps (5). The parafilm-grid combination is then removed, rinsed with several drops of stain and blotted on the edge with filter paper (6-8).From Auinger and Small (2008).
Figure 5-3. Maneuvering the grid onto the film. A. Overview of the mounting set-up. B. Forceps in the modified dual pipette holder, mounted on the micromanipulator. C. Application of the grid with the film immobilised on the support ring. D. Close-up view of the 50 mesh hexagonal grid over the finder grid pattern on the film. From Auinger and Small (2008).
Figure 5-4. Images of cells on the plastic film embossed with the finder grid pattern. A, example of a field of cells observed in the light microscope; B, example of EM image of film with finder grid pattern after transfer to EM grid.