Confocal laser scanning microscopy of fixed and stained cells

Petri P. Lehenkari, Guillaume T. Charras, Stephen A. Nesbitt and Mike A. Horton

Figure 4. Confocal laser scanning microscopy of fixed and stained cells. A computer-produced image reconstruction is shown of fixed and immuno-stained osteoclasts (bone-resorbing cells) that were cultured on dentine. Three-dimensional and volumetric data have been derived from a series of confocal laser scanning microscope images that were produced using a Leica TCS-NT system. The surface features shown in this figure can be compared with those of the atomic force microscope topographical image of a living osteoclast [see Fig. 1b; fig001mhu]. Osteoclasts resorb mineralised tissues following a series of cellular polarisation events. Cytoskeletal rearrangement in osteoclasts creates a structure that is rich in F-actin, which forms a tight seal that encloses a specialised secretory membrane called the ruffled border. Protons and proteases cross the ruffled border and degrade bone matrix by extracellular demineralisation and proteolytic activity. Subsequently, a resorption pit is formed beneath the osteoclast, as released bone matrix is transported through the cell before being expelled into the extracellular space (Refs 61, 62). (a1–a4) An image, taken in a single confocal plane, of two osteoclasts, one in an early resorption state (top right), the other merely adhering to the dentine surface. (a1) The vitronectin receptors of the osteoclasts have been stained with fluorescein-labelled antibodies (and appear green); (a2) F-actin has been stained using rhodamine-phalloidin (and appears red); (a3) the dentine surface has been biotinylated and stained with Cy5-labelled streptavidin (and appears blue). The images were taken at the surface of the dentine, where the F-actin ‘ring’ structure (a tight seal at the resorption area) is maximal [as shown by an arrowhead on image (a2)]. (a4) This image has been produced by merging the previous three images. (b) A pseudo three-dimensional projection of a stack of single confocal slices viewed from above; the arrow indicates the view direction for the images shown in parts (c) and (d). (c) This image shows an isosurface reconstruction of the data-set for all three colours [i.e. from (a1–a3)], which has been tilted 30° downwards and rotated through 90° clockwise relative to the image shown in (b). (d) This shows the same view as that shown in (c), but the intensity of the ‘green signal’ (i.e. of the vitronectin receptor) has been reduced to visualise the internal structure of the osteoclasts (particularly the F-actin ring, which is indicated by the arrowhead) (fig004mhu).