Three-dimensional digital elevation models reconstructed from stereoscopic image of platinum replica in sheared open...

Computer-generated microscopic images can be valuable tools for analyzing cell structure. We have used a computerized surface topography technique to convert platinum replica images into measurable 3D digital elevation model reconstructiondata. The commercially available Alicona MeX software can be successfully applied to the 3D reconstruction images of the platinum replicas, resulting in a series of digital elevation models in grayscale and coloured elevation maps in RGB mode of the selected area of interest. Here, we present accessible methods to analyze cell structures in sheared-open osteoclasts in 3D and at nanometre resolution, focusing on the podosome cytoskeleton, membrane-bound clathrin lattices, and surface topography. These structures on the surface of the ventral membrane appear to be highly characterized for their specific cellular functions. Extraction data from these reconstructed digital elevation models lead to the presentation of 3D information on some ultrastructural architectures on the ventral membrane, including the height of podosomes, the thickness of clathrin-coated structures and the non-coplanar surface of the flat clathrin lattices. In particular, we found that flat clathrin lattices appear on the curved surface of the basal part of the cell protrusions, or the non-coplanarity of their surface topography further indicates their morphological diversity. This new analytical approach provided a fast and easy way to reveal the ventral membrane surface structures in sheared open osteoclasts using high quality 3D reconstructed images.