Synchrotron Radiation Center

Gelsomina DeStasio, Instituto de Struttura della Materia del CNR, has begun the application of spectromicroscopy to biological specimens at SRC. Her work uses the imaging XSEM (X-ray secondary electron-emission microscope) which Brian Tonner designed and developed. The microscope was designed for material science applications, and until five years ago, was not applied to biological experiments.

Biological samples pose some difficulties for study by traditional surface science techniques. These specimens by necessity must be prepared in complex liquid environments, and transferred into the microscope through air transfer. This results in a residue of a "contamination layer," which cannot be penetrated by a surface sensitive probe such as photoemission spectroscopy.

The advantage of XSEM for biological applications was evident since De Stasio’s first tests in 1991. With this microscope, the detected portion of the specimen is over a depth of 20 - 100 angstroms, well beyond the level of surface contamination. An additional capability of XSEM is the rapid production of element distribution maps. These maps may be obtained by collecting and digitally subtracting images of a sample area using photon energies above and below a specific absorption threshold of a given element.

Spectromicroscopy is applied to biological specimens in experiments by DeStasio using cultured neurons from rat cerebellum (prepared in Rome, in collaboration with Delio and Teresa Mercanti of the Institute of Neurobiology-CNR). These neuron cells are cultured on specially designed sample holders. An important consideration is that the network of cells be grown in a single layer, so each cell may be viewed from the top.

Neuron cultures are exposed to toxic elements (Fe, Co, Ni, Al, etc.) for a short time. These elements may be actively incorporated by cells, in specific areas of their structure. The cells are then carefully washed to remove of all contaminants which are not absorbed, dried and placed in the microscope. X-rays strike the sample, causing electrons to be emitted in an amount which corresponds to the number of absorbed rays and the chemical composition.

Among the many results of these experiments, the localization of Aluminum was the most significant. When neuron cultures were exposed to an Al ion solution, Al was strictly localized on a particular kind of neuron: Purkinje cells. Cross-checking this result on several other neuron cell-cultures has confirmed that Al is absorbed only by Purkinje neurons.

The relevance of this result lies in Aluminum’s involvement in pathologies such as Alzheimer’s and Parkinson’s diseases and Guam Complex. Many authors have reported the observation of aluminum in brain tissue samples taken (after death) from patients who had these diseases. Now, localization and observation of Aluminum’s selective uptake in Purkinje cell cultures signifies greater understanding of its suspected toxic role in the etiology of these diseases.

In a recent development, human brain tissue sections were observed with the XSEM. These samples were obtained from Alzheimer and Guam patients, and prepared in collaboration with Daniel P. Perl of the Mount Sinai School of Medicine. The preliminary tests were encouraging. Al spectra were successfully obtained, proving that XSEM is sensitive enough to detect trace concentrations of Al in natural cases. Nevertheless, technical problems are still being faced in the production of clear images of brain tissue sections. In the future it is hoped that XSEM may be established as a technique for human tissue analysis.

The chemical sensitivity and lateral resolution of XSEM are projected to be at the same level of other techniques such as PIXE (proton induced x-ray emission), SIMS (secondary ion mass spectroscopy) and LAMMA (laser-assisted mass microanalysis) which are now used for micro-chemical analysis of human brains. Based on the results from the past few years, an optimized design for XSEM with synchrotron radiation has been developed by Tonner, which if constructed, should reach spatial resolutions of below 50 nanometers.

The XSEM concept has begun to appear at a number of other synchrotron radation laboratories around the world, with projects in place or planned at ALS, Trieste, Taiwan and Bessy.