Human Neocortex Layer Features Evaluated by PIXE, STIM, and STXM Techniques.

The human neocortex has a cytoarchitecture composed of six layers with an intrinsic organization that relates to afferent and efferent pathways for a high functional specialization. Various histological, neurochemical, and connectional techniques have been used to study these cortical layers. Here, we explore the additional possibilities of swift ion beam and synchrotron radiation techniques to distinguish cellular layers based on the elemental distributions and areal density pattern in the human neocortex. Temporal cortex samples were obtained from two neurologically normal adult men (postmortem interval: 6-12 h). A cortical area of 500 × 500 µm2 was scanned by a 3 MeV proton beam for elemental composition and areal density measurements using particle induced x-ray emission (PIXE) and scanning transmission ion microscopy (STIM), respectively. Zinc showed higher values in cortical layers II and V, which needs a critical discussion. Furthermore, the areal density decreased in regions with a higher density of pyramidal neurons in layers III and V. Scanning transmission X-ray microscopy (STXM) revealed the cellular density with higher lateral resolution than STIM, but not enough to distinguish each cortical lamination border. Our data describe the practical results of these approaches employing both X-ray and ion-beam based techniques for the human cerebral cortex and its heterogeneous layers. These results add to the potential approaches and knowledge of the human neocortical gray matter in normal tissue to develop improvements and address further studies on pathological conditions.

Micro-ATR FTIR, SEM-EDS and X-ray micro-CT: an innovative multi-technique approach to investigate bone affected by peri-implantitis.

PURPOSE: The aim of this work was to prove the synergic complementarity of attenuated total reflection Fourier transform infrared microspectroscopy (micro-ATR FTIR), scanning electron microscopy (SEM) coupled with energy-dispersive x-ray spectroscopy (EDS) and x-ray computed microtomography (micro-CT) by studying implant samples with bone affected by peri-implantitis. MATERIALS AND METHODS: Six samples of implanted bone affected by peri-implantitis and one control healthy bone were analyzed. Thick bone sections included in epoxy-resin and removed implants were analyzed by micro-ATR FTIR, SEM-EDS, and micro-CT. RESULTS: Micro-ATR FTIR revealed the complex nature of the bone composition. Vibrational bands characteristic of both mineral bone phase (acidic phosphates, CO32- groups) and organic bone phase (mostly collagen) could be recognized, and their proportion could be seen to change accordingly with the bone degradation. Similarly, SEM-EDS clearly revealed the cortical nature of the control mandible and its homogenous mineral composition. On the contrary, EDS analyses performed over relevant portions of pathologic samples revealed that defective areas were almost Ca and P free. Micro-CT data showed that the morphology of the interface was smooth and linear in the physiologic periimplant bone, while in the pathologic samples, an altered morphology was evident. CONCLUSION: This study demonstrated that morphologic, elemental, and biochemical modifications of periimplant bone can be studied using micro-ATR FTIR, SEM-EDS, and micro-CT. The complement of these techniques can be considered a new multipurpose approach to investigate bone affected by peri-implantitis.

Contribution of Ribonucleic Acid (RNA) to the Fourier Transform Infrared (FTIR) Spectrum of Eukaryotic Cells.

We report on an optimized protocol for the digestion of cellular RNA, which minimally affects the cell membrane integrity, maintaining substantially unaltered the vibrational contributions of the other cellular macromolecules. The design of this protocol allowed us to collect the first Fourier transform infrared (FTIR) spectra of intact hydrated B16 mouse melanoma cells deprived of RNA and to highlight the in-cell diagnostic spectral features of it. Complementing the cellular results with the FTIR analysis of extracted RNA, ds-DNA, ss-cDNA and isolated nuclei, we verified that the spectral component centered at ∼1220 cm-1 is a good qualitative and semiquantitative marker of cellular DNA, since it is minimally affected by cellular RNA removal. Conversely, the band centered at ∼1240 cm-1, conventionally attributed to RNA, is only a qualitative marker of it, since its intensity is majorly influenced by other macromolecules containing diverse phosphate groups, such as phospholipids and phosphorylated proteins. On the other hand, we proved that the spectral contribution centered at ∼1120 cm-1 is the most reliable indicator of variations in cellular RNA levels, that better correlates with cellular metabolic activity. The achievement of these results have been made possible also by the implementation of new methods for baseline correction and automated peak fitting, presented in this paper.