Blanc SIMI 8 - Sciences de l'information, de la matière et de l'ingénierie : Chimie du solide, colloïdes, physicochimie

New methods in mass spectrometry imaging for the study of the Alzheimer's disease – MASS-IMAGE

Mass spectrometry imaging: development and application to Alzheimer's disease

New methods in mass spectrometry imaging to study Alzheimer's disease

Methodological developments in biological mass spectrometry imaging for in situ study of lipids involved in Alzheimer's disease

TOF-SIMS (Time-of-Flight Secondary Ion Mass Spectrometry) mass spectrometry imaging provides access to the chemical composition of the surface of a sample with an accuracy of one micron, without any a priori on the mapped species.<br />The main objective of this project was to greatly improve with analytical chemistry the capabilities of biomedical mass spectrometry imaging, to bring this method to clinical research, and to make it a method of choice for routine human histopathology. First three tasks of the project aimed to make it compatible with histology, to achieve in situ structural identification (directly on tissue sections), or to build lipid databases, and finally testing a method called G-SIMS. In a fourth and last task, the new methods had to be tested for the investigation of samples from patients who died of Alzheimer's disease.<br />

At first, the compatibility between histological staining and mass spectrometry imaging has been tested and methods to optimize the compatibility have been developed. Then, since the TOF / TOF analyzer that we intended to device for structural analysis of lipids by SIMS could not be built, a database of lipids was established and validated by complementary analyses made by MALDI-TOF. We have tested new sources of massive argon clusters recently arrived on the market of instruments: we have adapted the method of «dual beam depth profiling«, through 3-D analyses of rat brain sections. We have also shown that this method can be used to greatly increase the sensitivity of imaging mass spectrometry. Finally, the reproducibility and repeatability of TOF-SIMS imaging have been characterized on rat brain sections, and proved excellent.

In addition to the method developments already summarized in the previous paragraph, and which made up three-quarters of the work load of the project, sections from frozen brains of patients who died of Alzheimer’s disease were analyzed, showing an overload of cholesterol in the brain cortex. Finally, after having developed a method to analyze samples from brains fixed with formaldehyde, the last experiments showed a potential reduction of sulfatide amount in senile plaques, size of which is only a few tens of microns.

From the point of view of analytical developments, new sample preparation methods that can combine histology and mass spectrometry imaging have been developed. A database of lipid spectra was produced. A new source of massive argon clusters has been tested, which opens interesting perspectives in the future to make 3-D analysis of biological samples and to increase the sensitivity of TOF-SIMS imaging. Finally concerning the analysis of brain sections of patients who died from Alzheimer's disease, changes of the composition in cholesterol and sulfatides were detected in situ. For this, a method for the analysis of fixed samples has been developed and a repeatability study was conducted.

This project resulted in 9 publications in international peer-reviewed journals, one book chapter, 8 conferences, 8 oral communications, and 4 poster communications.

Mass spectrometry Imaging using TOF-SIMS (Time-of-Flight Secondary Ion Mass Spectrometry) give access to the chemical composition of a sample surface with a micrometric precision and without any a priori on the species which are mapped.
The main goal of the program is to enhance the capabilities of biomedical Mass Spectrometry Imaging, making it a “molecular histology” method fully compatible with protocols of histology and immunohistochemistry, by working on the same samples. Our wish is to bring this method closer to the clinical research, to make it a method of choice for histopathology in hospitals, very near to the operating room.
Although several biological and biomedical applications of mass spectrometry imaging have already been published in different fields (Duchenne Muscular Dystrophy, Non-Alcoholic Fatty Liver Disease, Fabry Disease, atherosclerotic plaque), the necessity of in situ identification of the molecular species is always facing the results. Imaging ions at the surface of a sample without identifying them cannot be a good and robust analytical method. To the contrary the combination of imaging and identification of species, with tandem mass spectrometry and/or databases of standard spectra will be an invaluable strength for mass spectrometry imaging. A database of standard TOF-SIMS spectra of lipids is to the best of our knowledge not existing.
A SIMS-TOF-TOF analyzer will be completely new. We believe this will make TOF-SIMS imaging really emerging, since this will cancel its actual major drawback when compared to other methods. Nevertheless, although the technology is now ready and existing for such an instrumental improvement, manufacturers need to design it from scratch. This represents a risk which cannot be neglected, and that is the reason why an alternative solution is presented with the study of in-source decay, together with a database of TOF-SIMS lipid spectra. Although it must be kept in mind that this alternative way will never be able to replace a true tandem mass spectrometry method, this will undoubtedly make a great progress for TOF-SIMS mass spectrometry imaging.
G-SIMS (Gentle-SIMS) is a very promising analytical improvement which deserves to be evaluated with biological samples. This method has been introduced a few years ago by Gilmore and coworkers. It aims to take advantage of the different fragmentations when using as projectile a single light atomic ion or a single heavy ion, or even a polyatomic ion. A new tip of bismuth emitter has recently been developed by Gilmore in collaboration with ION-TOF, which is made of a bismuth-manganese alloy. Using the same primary ion column, therefore having the same ion optics, beams of manganese and of bismuth clusters can alternatively irradiate the sample and then generate G-SIMS spectra. Such kind of spectra have for the moment only been recorded onto polymer films. The spectra generated are much more informative than traditional SIMS spectra, with a higher signal to noise ratio of the molecular ion and also characteristic fragments which are not swamped by a huge number of irrelevant fragment peaks. This method will be applied to lipids, standards as well as tissue sections, and is expected to greatly help the identification of these lipids.
These new methods will finally be utilized for the study of Alzheimer’s disease samples. We plan to use samples chosen according to topography determining the type of lesions and severity. The plaques will be shown by Aß immunohistochemistry using antibodies directed against the C-terminal of Aß42 and Aß40. The neurofibrillary alterations will be visualized with the AT8 anti-tau antibody. A series of control samples from normal young cases, old patients with minimal alterations (considered normal for the age) and from cases with other degenerative diseases (Parkinson's disease, fronto-temporal dementia, amyotrophic lateral sclerosis, multiple sclerosis for white matter lesions) will be used.

Project coordination


The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.



Help of the ANR 285,000 euros
Beginning and duration of the scientific project: - 36 Months

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