Photon emission and changes in fluorescent properties of bone after laser irradiation.

Laser scalpels used in medical surgery concentrate light energy, heating the tissues. Recently, we reported thermoluminescence emission from laser-treated soft tissues. Here we investigated the thermo-optical effects caused by a laser operating at 808 nm on animal bones (beef ribs) through luminescence and fluorescence imaging, thermal imaging and scanning electron microscopy. Laser-induced artificial lesions emitted luminescence peaking around 650 nm, with a half-life of almost 1 hour. As concerns fluorescence, 24 hours after laser treatment we observed an increase of the emission and a shift from 500 (untreated) to 580 nm (treated). Recrystallization observed by SEM indicates that the temperature in the artificial lesions is over 600°C. We can conclude that laser treatment induces specific luminescent and fluorescent emissions due to heating of the bone and modification of its components. Monitoring these emissions could help prevent tissue overheating and its potential damages during laser-assisted medical procedures.

The synovial surface of the articular cartilage.

The articular cartilage has been the subject of a huge amount of research carried out with a wide array of different techniques. Most of the existing morphological and ultrastructural data on the this tissue, however, were obtained either by light microscopy or by transmission electron microscopy. Both techniques rely on thin sections and neither allows a direct, face-on visualization of the free cartilage surface (synovial surface), which is the only portion subject to frictional as well as compressive forces. In the present research, high resolution visualization by scanning electron microscopy and by atomic force microscopy revealed that the collagen fibrils of the articular surface are exclusively represented by thin, uniform, parallel fibrils evocative of the heterotypic type IX-type II fibrils reported by other authors, immersed in an abundant matrix of glycoconjugates, in part regularly arranged in phase with the D-period of collagen. Electrophoresis of fluorophore-labeled saccharides confirmed that the superficial and the deeper layers are quite different in their glycoconjugate content as well, the deeper ones containing more sulfated, more acidic small proteoglycans bound to thicker, more heterogenous collagen fibrils. The differences found between the synovial surface and the deeper layers are consistent with the different mechanical stresses they must withstand.

Weak biophoton emission after laser surgery application in soft tissues: Analysis of the optical features.

Nowadays, laser scalpels are commonly used in surgery, replacing the traditional surgical scalpels for several applications involving cutting or ablating living biological tissue. Laser scalpels are generally used to concentrate light energy in a very small-sized area; light energy is then converted in heat by the tissues. In other cases, the fiber glass tip of the laser scalpel is heated to high temperature and used to cut the tissues. Depending on the temperature reached in the irradiated area, different effects are visible in the tissues. In this study, we report the discovery and characterization of the light emitted by soft mammalian biological tissues from seconds to hours after laser surgery application. A laser diode (with hot fiber glass tip) working at 808 nm and commercially available for medical and dentistry applications was used. The irradiated tissues (red meat, chicken breast and fat) showed light emission in the visible range, well detectable with a commercial charge coupled device (CCD) camera. The time decay of the light emission, the laser power effects and the spectral features in the range 500 to 840 nm in the different tissues are here reported.

Visualizing the Supramolecular Assembly of Collagen.

Among biological macromolecules, collagen enjoys quite a peculiar status. Making up as much as a third of the protein fraction of the body it is the main responsible for the functional properties of the extracellular matrix, which can be efficiently tuned and tailored by modifying the length, volume fraction, and spatial layout of its collagen content. The supramolecular aggregates of collagen are therefore subject to be investigation by several viewpoints and at different scales, from the finest interactions of individual collagen molecules to the spatial layout of fibril bundles. As a consequence, no treatise can pretend to be exhaustive about the several techniques that can be useful in different moments and/or for different purposes. So, in this chapter, we focus only on some applications of the transmission electron microscope (TEM), of the scanning electron microscope (SEM), and of the atomic force microscope (AFM).

Multiple Organ Dysfunction Syndrome (MODS) induced by Candida krusei in an Aldabra giant tortoise (Aldabrachelys gigantea) and confirmed by electron microscopy analysis.

A young female Aldabra giant tortoise (Adabrachelys gigantea) was presented with anorexia, ataxia, severe constipation and bloating. Analysis revealed liver disease and collected biopsy diagnosed Candida krusei infection. Despite Itraconazole treatment, the tortoise got worse and died. Full necropsy was performed; microbiology showed Candida krusei presence in liver, but histopathology didn't confirm fungal presence with special stains, so scanning electron microscopy was essential to prove a detailed diagnosis of extensive mycosis.

Not only tendons: The other architecture of collagen fibrils.

For many decades the fibrillar collagens have been the subject of a remarkable body of ultrastructural research. The vast majority of the studies, however, were carried out on tendon or on tendon-derived material. For many reasons this reflects an obvious choice but at the same time it also is an unfortunate circumstance, because this flooding of tendon-related data can easily encourage the false confidence that all connective tissues are similar. The reality is quite the opposite, and a different fibrillar structure has been long time observed on collagen fibrils from different tissues, the most notable example being offered by corneal fibrils. The same architecture can be found in a number of disparate tissues and may actually be the prevalent one on a whole-body scale. Although these fibrils diverge from those of tendon in their architecture, size, D-period, composition, cross-linking and fibrillogenesis mechanism, their structure was the subject of rather sparse ultrastructural studies and even today their mere existence is often overlooked or ignored. This paper summarizes the main aspects of the structural biology of these forgotten fibrils.

Multilayer Microstructure of Idiopathic Epiretinal Macular Membranes.

PURPOSE: To identify the ultramicroscopic structure of idiopathic epiretinal macular membranes (iEMMs) by scanning electron microscopy (SEM). METHODS: We examined 28 iEMMs surgically removed from 28 eyes of 28 patients. All specimens, previously observed at stereomicroscope, were treated with an osmium maceration technique. Fine resolution of iEMMs' 3D architecture and their interaction with the retina were studied using a Philips SEM-FEG XL-30 microscope. RESULTS: The specimens appeared as laminar connective structures partially or completely adherent to the inner limiting membrane (ILM). We identified 4 types of structures: ( 1 ) distinct layers of thin sheets of woven fibers; ( 2 ) folded layers of inhomogeneous thickness of fibrous material more consistent; ( 3 ) thicker and more rigid layers recognizable as collagen fibrils with typical 64-nm period, collagen fibrils isolated or intermingled between them; ( 4 ) lacunar structures with inflammatory and/or necrotic material. The first 3 types of structures appear to thicken towards a centripetal direction from the ILM to the vitreous in order from 1 to 3. The interface of ILM-iEMM tissue shows particular small bridges of connection. Cells are rarely found, especially in the tissue near the ILM. CONCLUSIONS: Layers of various materials follow one another in iEMMs. Cells are rarely found. The interface ILM-iEMM tissue shows particular small bridges of connection. The dynamic modeling of bended layers begins in soft tissue.

The application of heat-deproteinization to the morphological study of cortical bone: A contribution to the knowledge of the osteonal structure.

Observation of heat-deproteinized cortical bone specimens in incident light enabled the high definition documentation of the osteonal pattern of diaphyseal Haversian bone. This prompted a study to compare these images with those revealed by polarized light microscopy, carried out either on decalcified or thin, undecalcified, resin-embedded sections. Different bone processing methods can reveal structural aspects of the intercellular matrix, depending on the light diffraction mode: birefringency in decalcified sections can be ascribed to the collagen fibrils orientation alone; in undecalcified sections, to both the ordered layout of collagen and the inorganic phase; in the heat-deproteinized samples, exclusively to the hydroxyapatite crystals aggregation mode. The elemental chemical analysis documented low content of carbon and hydrogen, no detectable levels of nitrogen and significantly higher content of calcium and phosphorus in heat-deproteinized samples, as compared with dehydrated controls. In both samples, the X-ray diffraction (XRD) pattern did not show any significant difference in pattern of hydroxyapatite, with no peaks of any possible decomposition phases. Scanning electron microscopic (SEM) morphology of heat-deproteinized samples could be documented with the fracturing technique facilitated by the bone brittleness. The structure of crystal aggregates, oriented in parallel and with marks of time periods, was documented. Comparative study of deproteinized and undecalcified samples showed that the matrix inorganic phase did not undergo a coarse grain thermal conversion until it reached 500°C, maintaining the original crystals structure and orientation. Incident light stereomicroscopy, combined with SEM analysis of deproteinized bone fractured surfaces, is a new enforceable technique which can be used in morphometric studies to improve the understanding of the osteonal dynamics. Microsc. Res. Tech. 79:691-699, 2016. © 2016 Wiley Periodicals, Inc.

Chemical etching in processing cortical bone specimens for scanning electron microscopy.

Transverse and longitudinal sectioning of undecalcified cortical bone is a commonly employed technique for investigating the lamellar structure of the osteons. Since a flat surface is required, the specimen has to be grinded and then polished. Whereas the smear of debris and inorganic/organic deposits left by these treatments cannot be removed by ultrasonication alone, a chemical treatment of the specimen surface with either a basic or an acid etching solution is currently employed. A further effect of the latter can be the enhancement of the lamellar bone pattern. The kind of etching solution, its pH, the concentration of etchants, and the contact time significantly affect the sectioned surface when it is observed with scanning electron microscopy (SEM). The etching procedures can severely influence the obtained images. Homogeneous cortical bone specimens were sampled from the first metatarsal of two fresh human subjects. One or two cut surfaces were exposed to different acid and basic solutions in bonded conditions. Considering the type of chemical agents, the solution pH, and the exposure time of the specimens, the effects of several etching media have been investigated and compared. Strong etching, either acid or basic produced surface decalcification and severe damage of the collagen matrix, compromising any morphological or morphometric analysis. Weak acid etching (for example citric and acetic acid), even though causing distinctive alteration of the sample, enhanced the visibility of the lamellar pattern, while the polyphosphate treatment of the surface decalcified a thin layer matrix, ensuring a good visibility of fibrils and avoiding rough distortions.

Colonization of a Central Venous Catheter by the Hyaline Fungus Fusarium solani Species Complex: A Case Report and SEM Imaging.

The incidence of opportunistic infections by filamentous fungi is increasing partly due to the widespread use of central venous catheters (CVC), indwelling medical devices, and antineoplastic/immunosuppressive drugs. The case of a 13-year-old boy under treatment for acute lymphoblastic leukemia is presented. The boy was readmitted to the Pediatric Ward for intermittent fever of unknown origin. Results of blood cultures drawn from peripheral venous sites or through the CVC were compared. CVC-derived bottles (but not those from peripheral veins) yielded hyaline fungi that, based on morphology, were identified as belonging to the Fusarium solani species complex. Gene amplification and direct sequencing of the fungal ITS1 rRNA region and the EF-1alpha gene confirmed the isolate as belonging to the Fusarium solani species complex. Portions of the CVC were analyzed by scanning electron microscopy. Fungi mycelia with long protruding hyphae were seen into the lumen. The firm adhesion of the fungal formation to the inner surface of the catheter was evident. In the absence of systemic infection, catheter removal and prophylactic voriconazole therapy were followed by disappearance of febrile events and recovery. Thus, indwelling catheters are prone to contamination by environmental fungi.