Electron microscopies

High resolution TEM images can reveal important information about the asbestos bodies surface, which being at the interface with the biological tissue, is expected to play a crucial role in the carcinogenesis. Electron diffraction, also available on transmission electron microscopes, could give complementary information on the crystallographic structure of the embedded asbestos fibres, supporting and complementing µXRD data. Transmission electron microscopy has not been widely used in the past for the present subject due to the lack of a reliable procedure to prepare the samples. This part of the project will focus on the development and test of such a procedure and on the acquisition of high quality TEM micrographs. The key feature of the proposed method is to minimize the steps needed to isolate and fix the fibers on the TEM sample holder, avoiding possible damage or artefacts induced by strong chemical attacks or mechanical stress. This goal will be obtained starting from the protocol developed by Cook [1] and combining mild digestion using sodium hypochlorite with several centrifugation steps. The minimal centrifugation time required will be determined theoretically by calculating the sedimentation rate of the fibrous material through a mathematical model.

[1] P M Cook. Preparation of extrapulmonary tissues and body fluids for quantitative TEM analysis of asbestos. Ann. New York Acad. Sci. 330 (1979) 717.

Secondary electrons SEM image of asbestos bodies extracted from lung tissue by chemical digestion and filtration on porous membranes.

Scanning Electron Microscopy has been for decades the usual way to image asbestos bodies. It is an handy technique able to reveal their morphology with reasonable resolution, and also allows for semi-quantitative elemental quantification. Nevertheless, this latter information is limited to the surface (usually <1 micron), and, as for TEM, it is not possible (or very difficult) to image asbestos bodies embedded in the biological tissue. In this project SEM will be used to check the quality of the samples prepared for synchrotron radiation experiments, estimate the number of asbestos bodies per gram of dry tissue, and support SR-XRF quantification.

A new paper containing the latest results of combined nano-X-ray Diffraction (nXRD) and nano-X-ray Absorption Spectroscopy data has been published. The results of the new measurements, which were performed on single asbestos bodies using synchrotron radiation, demonstrated that these latter are mainly composed by a co-presence of two iron oxyhydroxides (ferrihydrite and goethite). The result is in line with the formation mechanism of the asbestos bodies: when the alveolar macrophages attempt fibers phagocytosis the start depositing ferritin, which contains ferrihydrite, on asbestos. Ferrhydrite is a metastable biomineral that can evolve to goethite at acidic pH (4 - 5). The phagolysosomes contained in the macrophages have pH values in the same range (4 - 5). Therefore, a reasonable scenario is that the acidic pH induces the transfrmation of ferrihydrite to goethite. More researches are needed to generalize the proposed mechanism.

April 5-11, 2018

A talk on advanced synchrotron-based tomography techniques applied to biological samples was given by the fellow at the International School of Nanomedicine (3rd Course: “Nanofluidics, Nanoimaging and Nanomanipulation”, held in Erice (Italy) the 5-11 of April. The course focuses on the state-of-the-art techniques and advanced biophysical methods related to Nanomedicin. Results of phase-contrast and fluorescence tomographic data acquired at the European synchrotron were presented as a case study during the talk. More at Dissemination.

An experiment was performed at the nano-imaging beamline (ID16A) at the European synchrotron in Grenoble (ESRF). This experiment was the continuation of an experiment done about one year before that was aimed to the acquisition of fluorescence and phase contrast tomographic data on asbestos bodies in human lung tissues. The continuation was necessary to acquire more data on a larger set of samples. The experiment drawn the attention of the press staff of the ESRF and the fellow and his collaborators have been interviewed during the experiment. The press release can be found at this link.

A lecture on advanced synchrotron-based tomography techniques was given by the fellow at the European Mineralogical Union (EMU) School that took place in Modena (Italy) in June 19-23. The school focused on the crystallographic and physical-chemical properties of mineral fibres, and on their biologocal interaction and toxicity. During the lecture, preliminary results of phase-contrast and fluorescence tomographic data acquired at the European synchrotron were presented. More at Dissemination.

Electron Microscopies