The collagenic structure of human digital skin seen by scanning electron microscopy after Ohtani maceration technique.

We performed a morphological scanning electron microscope (SEM) study to describe the fine structure and disposition of collagenous tissue in the human toe. After therapeutic amputation of a human right Leg, we applied the Othani maceration technique to the skin of three toes surgically explanted from the foot. We distinguished eight cutaneous regions and focused on some specialized collagenous structures differing in the thickness of the skin. The eight areas investigated were: the dorsal skin, the eponychium, the perionychium, the hyponychium, the region under the visible nail, the nail root, the plantar skin and finally the toe tip. Each of these areas is characterized by a distinctive collagenous surface disposition, with some peculiar features mostly related to dermal. papillae. At high magnification, we observed the spatial arrangement of the cottagen fibers constituting the top of the dermal, papillae that represents the attachment site of the proliferative basal layer of the epidermis. We also noted an impressive density of collagen fibers throughout the thickness of the dermal layer, organized in specialized structures and constituting the skeleton of dermal, thermoreceptorial corpuscles or sweat glands. A combination of SEM and Ohtani technique disclosed the three-dimensional architecture of the collagenous matrix of tarsal skin under physiologic conditions, giving a detailed description of the most reactive tissue during pathologic processes

The quest for the third dimension: from the Electron Microscope to the 3D printer

Conventional light microscopy (LM) and transmission electron microscopy (TEM) are meant to image planar sections, i.e. bidimensional specimens, and are therefore constrained into a bidimensional world. In contrast, the scanning probe microscopy (SPM) and scanning electron microscopy (SEM) are able to image surfaces, i.e. three-dimensional subjects. Of these techniques, SPM has the additional advantage of directly obtaining three-dimensional datasets from three-dimensional specimens, although this ability is seldom exploited. The SEM is per se limited to 2D pictures of 3D subjects, but its flexibility and performance make possible to re-obtain the third dimension indirectly. A first, simple, time-proven approach is stereophotography. This makes possible an immediate visual appreciation of depth and volume but does not allow quantitative measurements. A subsequent approach is represented by shape-from-stereo reconstruction, which builds a quantitative computer model of the specimen. This is now a consolidated technique and several solutions, both hardware- and software-based, are readily available. Although limited to the development of 2 ½ dimensions, rather than real 3D, this technique is simple and effective and for several years the authors have used a proprietary package [1] featured in a number of published papers. More recently a new generation of shape-from-motion or shape-from-video photogrammetric software [2] makes possible the full recovery of the third dimension, complete with undercuts and texture mapping. All these techniques are now complemented and extended by the availability of inexpensive three-dimensional printers. Going beyond visual appreciation and beyond computer graphics, this technique makes possible to obtain a tangible, material model of the specimen. 3D printing is already in use for educational purposes but can be effectively deployed also in morphological research, making possible to obtain highly magnified, accurate copies of microscopic structures such as molecules, cells and interfaces, adding to the visual appreciation the immediacy of the tactile experience. A few examples are shown

Ultrastructural aspects of mineralization-induced modifications in turkey tendon

In all tendons the collagen fascicles follow a wavy course (actually a flattened lefthanded helix) forming visible crimps. Each crimp corresponds to a sharp bend and/ or an axial twisting of individual collagen fibrils (Raspanti et al., 2005; Franchi et al., 2010), and even once the fibril are straightened out a permanent local deformation remains visible, still revealing the original crimp location (Raspanti et al., 2005). The tendons of some birds represent a special case as they undergo a physiological process of gradual mineralization involving heavy modifications of the tissue architecture. In the present research, turkey tendons appeared to be more finely subdivided into thinner fascicles than most tendons; they contained a greater amount of cell-rich endotenon tissue as well as occasional nodules of cartilage-like matrix. The most striking finding, however, was the complete disappearance of the crimps in the calcified portions of the tendon, while they were present with the usual morphology in the non-mineralized portion. The mineralized fibrils ran perfectly straight, but the electron microscopy revealed traces of pre-existing crimps locked in the extended position by the mineralization process. The inorganic phase itself appeared composed of two different types of fine particles, respectively growing inside or around the collagen fibrils and looking as tightly packed fine needles or as larger platelets regularly arranged in relation with the D-period. The perifibrillar mineral could play a critical role in the mechanical coupling of adjoining fascicles and in the transmission of tensile loads along the tendon itself

3D imaging of the osteon lacunar-canalicular system: an improved corrosion-casting method

The osteocyte lacunar-canalicular system has been the subject of several studies either by serial sectioning of embedded specimens, or by confocal laser scanning microscopy, or with FIB/SEM imaging. With all these techniques the persistence of the bone matrix hampers the visualization of the finer details of the canalicular network. In the present research the methyl-metacrylate casting technique was applied to human cortical bone in order to evidentiate the 3-D organization of the osteons lacunar-canalicular system. The MMA monomer infiltration into the vascular canals, and hence into the lacunar-canalicular system was driven by capillarity, helped by evaporation and the resulting negative pressure. The resin penetration was complete in the Haversian canal but limited to a depth of 4 – 5 layers of lacunae around the canal itself, suggesting that each secondary osteon is a close system limited by an impenetrable cementing line. The casts duplicated the shape, position and connections of the lacunae, and allowed an unrestricted exploration of lacunar-canalicular network without manipulations such as cutting or sawing. Two systems of canalicula could be distinguished: the equatorial, which connects osteocytes lying on the same concentrical level, and the radial, which interconnects different levels. The equatorial canalicula radiate from the lacunar border on a planar surface around the lacuna, forming straight and basically parallel canals; the radial have a predominant direction perpendicular to the equatorial plane. The mean length of the radial canalicula was 38.4 +/- 7.35 um in human osteons, while their mean diameter was 195.7 +/- 79.58 nm. The equatorial canalicula had an average diameter of 249.7 +/- 73.78 nm, significantly larger (p < 0.001) than the radial. The architecture of lacunar-canalicular network obtained by the MMA casts was compared with theoretical models. Our data suggests that the intracanalicular pericellular fluid is static and that the supply of ions, oxygen and metabolites to the osteocytes can be assured by the diffusion of these molecules into the static volume of the solution without the need to hypothesize a fluid flow within the canalicular system

Scanning Electron Microscopy in forensic investigations: More views from more applications

The purpose of this presentation is to expand and highlight the range of applications of scanning electron microscopy to forensic science, following the overview which was shown last year at the LXX SIAI Congress. All examples shown of forensic uses of SEM were carried out over the last few years at the Human Morphology Laboratory of the University of Insubria. These studies include: - The identification and characterization of different toolmarks found on human bones. Some toolmarks have a distinctive morphology and allow a reliable identification of the weapon or instrument used. For this purpose, we will illustrate a few examples of dismemberment with different types of saw and will show the peculiar bone patterns left by different cutting edges (knives, axes, cutters ...); - The examination of human tissues and of medical devices (catheters etc.) for the early detection and identification of slow-growing microorganisms (e.g. some fungi). The diagnosis of these microorganisms would have otherwise required molecular biology techniques, which are not only expensive but also not always available or applicable in the field of forensics (for instance, when the specimen is inadequate for external contaminations or is into a state of conservation far from optimal), or conventional cultures in vitro, which require much longer times and may be easily spoiled by inopportune drug administration; - The use of scanning electron microscopy and of X-ray spectroscopy as auxiliary and “creative” tools to discover mystifications and frauds against insurance companies

Atherosclerotic alterations in human carotid observed by scanning electron microscopy

Atherosclerosis involves all the layers of the artery wall, but the events involving the intimal portion are fundamental to understand the evolution and gravity of lesions. This study shows that scanning microscopy is instrumental for better understanding the physiopathology of this disease