A Great Challenge for Mass Spectrometry Imaging – DEFIMAGE
A great challenge for mass spectrometry imaging
Time-of-Flight Secondary Ion Mass Spectrometry (TOF-SIMS) imaging will be greatly improved by using massive argon clusters to sputter biological samples which are then analyzed by bismuth cluster ion beams. The dual beam depth analysis allows the acquisition of molecular images in the fields of medical research and cultural heritage.
Make secondary ion mass spectrometry imaging a method of choice for 3-D molecular analysis at a micrometric scale.
A new massive argon cluster ion source that will deliver clusters of thousands of argon atoms will be evaluated in three directions: depth analysis of thin tissue sections, improved sensitivity, and cleaning of the surface of samples by soft sputtering of the surface with the argon clusters. Particular attention will be given to the possible migration of lipids during defrosting of the samples and the possible matrix effects induced by certain very specific compounds such as cholesterol.<br />The first use of this new method will be to analyze the depth composition of matrix-covered tissue sections for Matrix-Assisted Laser Desorption-Ionization (MALDI) imaging, in order to improve the understanding of the desorption-ionization phenomena involved In MALDI, particularly in imaging.<br />Then two applications are devoted to very different domains. A first application in medical research will focus on the acquisition of ionic images of reshaped arteries, selected from complex samples of human explanted lungs of patients suffering from pulmonary arterial hypertension, compared to controls, as well as rats models, in order to help understand the physiopathology of this incurable disease.<br />Finally, in the field of cultural heritage and archeology, the second application will study pigments from samples of paintings dating from the Renaissance to the 20th century. The data acquired will help to characterize the use and the mode of preparation of the different pigments and binders, the origin of the minerals, the nature of the binders, the chemical synthesis of the compounds or the mixture of different organic molecules.
A first use of the newly developed method consists in analyzing, before and after the laser shots, the in-depth composition of matrix-coated tissue sections for Matrix-Assisted Laser Desorption-Ionization (MALDI) mass spectrometry to obtain a better understanding of the desorption-ionization phenomena involved in MALDI.
Next, studies will be devoted to very different fields of science, where an improvement in the capacity of TOF-SIMS imaging is expected with 3-D analyzes, or an improvement in the sensitivity or even possible cleaning of the surface of the sample without damaging it, when a sample is rare or unique.
In the field of biomedical research, a first study will focus on the acquisition of ionimages from selected remodeling artery samples in complex human explanted lung samples from patients with pulmonary arterial hypertension, as well as model rats, to help understand the physiopathology of this incurable disease.
Finally, in the field of cultural heritage and archeology, the latest study will focus on pigments from samples of ancient paintings from paintings by artists from the Renaissance to the twentieth century. The data will help to characterize the use and the method of preparation of different pigments and binders: for example, the origin of the minerals, the nature of the binders, the chemical synthesis of the compounds, the mixing of various organic molecules.
The first step of the project consisted of installing the massive argon cluster ion source on the existing TOF-SIMS mass spectrometer. The installation was carried out in March 2016.
The study of MALDI matrix deposition and lipid migration was performed. Principal component analysis has shown that diffusion is the process by which lipids migrate from the tissue to the matrix layer. Triacylglycerols and phospholipids have a delayed migratory tendency with respect to diacylglycerols and monoacylglycerols. The ability of a pure lipid to migrate in the matrix depends on its fluidity at room temperature. Cholesterol can only migrate in the presence of a lipid (fluid) and itself seems to fluidize the lipids, which could explain its co-location in the matrix with diacylglycerols and monoacylglycerols.
Leaf and fruit surfaces of Macaranga vedeliana, a endemic species to New Caledonia, were analyzed by depth profiling using the new AMCIS ion source. Vedelianin, an active metabolite of the prenyl stilbene family called schweinfurthines, is only localized in these glandular trichomes.
The desorption / ionization process under bombardment of massive argon clusters was studied by measuring the internal energy distributions of a series of benzylpyridinium ions. The internal energy distribution of the secondary ions increases with the speed of the cluster beam, reaching a plateau. The results were compared with those generated by the bismuth cluster impacts and the mean internal energies correspond to the plateau values for the argon clusters. However, the damage cross section is about 20 times lower with argon clusters than with bismuth ones, thus quantitatively showing the low damage effect of argon massive clusters.
The installation of a new argon cluster ion source will strengthen the laboratory in its position as an international leader in mass spectrometry imaging of biological samples. Many collaborations, some of which are already well established, will be generated by this new analytical capability. The recent improvement in MALDI-TOF MSI capabilities, which is much more popular than TOF-SIMS, particularly the spatial resolution which now reaches 5-10 µm, shows the necessary complementarity of these two molecular imaging methods. Strength is the determination of new diagnostic / prognostic biomarkers, in several chemical domains, including pathologies of identical morphology that can not be distinguished using established histopathological methods. The aim of this development is to make TOF-SIMS imaging the best chemical analysis method on a scale of 400 nm to 1 µm. It is truly hoped that this method finally becomes the method of choice for scientific communities that too often forget it.
The cost of diagnosing, monitoring and treating patients with pulmonary arterial hypertension in Europe is currently between 80 and 100,000 euros / patient / year. As life expectancy is about 3-5 years, with a possible lung transplant, this means that the cost of the complete management of life would reach 400 000 euros per patient. Indirect costs to society are also considerable because they are often unable to work.
Finally, the new developments will provide new data on artistic materials, on the conditions and processes of artistic creation, but also to preserve our cultural heritage. The role of materials science and interdisciplinary research is now very important in this field and recognized internationally by several programs in Europe and in the United States.
1. High mass and spatial resolution mass spectrometry imaging of Nicolas Poussin painting cross-section by cluster-TOF-SIMS, M. Noun, E. Van Elslande, D. Touboul, H. Glanville, S. Bucklow, P. Walter, A. Brunelle, J. Mass Spectrom. 51 (2016) 1196-1210, dx.doi.org/10.1002/jms.3885
2. Dual Beam Depth Profiling and Imaging with Argon and Bismuth Clusters of Prenylated Stilbenes on Glandular Trichomes of Macaranga vedeliana, T. Péresse, N. Elie, D. Touboul, V.C. Pham, V. Dumontet, F. Roussi, M. Litaudon, A. Brunelle, Anal. Chem. 89 (2017) 9247-9252, dx.doi.org/10.1021/acs.analchem.7b02020
3. Insights into the MALDI Process after Matrix Deposition by Sublimation using 3D ToF-SIMS Imaging, S. Van Nuffel, N. Elie, E. Yang, J. Nouet, D. Touboul, P. Chaurand, A. Brunelle, Anal. Chem. 90 (2018) 1907-1914, dx.doi.org/10.1021/acs.analchem.7b03993
4. Radial distribution of wood extractives in European larch Larix decidua by TOF-SIMS imaging, T. Fu, N. Elie, A. Brunelle, Phytochemistry 150 (2018) 31-39, dx.doi.org/10.1016/j.phytochem.2018.02.017
TOF-SIMS (Time-of-Flight Secondary Ion Mass Spectrometry) mass spectrometry imaging can be greatly improved by using massive argon clusters to sputter biological samples, which are further analyzed by a beam of bismuth clusters. Recent preliminary results showed that this dual beam depth profiling method provides for organic molecules such as lipids, 3-D ion images, and a significant increase in sensitivity, as well as cleaning without damage of the surface of precious samples.
The DEFIMAGE project is multi- and interdisciplinary and aims to make a big breakthrough in TOF-SIMS imaging, thus opening the door for new original applications, in health science, and chemical analysis of cultural heritage artifacts.
After the installation on the existing TOF-SIMS mass spectrometer of a new Argon Massive Cluster Ion Source (AMCIS), delivering clusters of hundreds to thousands of argon atoms at a few keV of energy, different experimental conditions for dual beam depth profiling onto tissue samples will be assessed: size and energy of the argon clusters used for sputtering, as well as size and energy of the bismuth cluster used for analyzing, and relative doses of each of the beams, will be optimized with rat brain tissue sections, used as a working model. This will be done according to three directions: 3-D analysis, or in depth analysis of thin tissue sections, enhancement of the sensitivity, by summing the signal of the whole sample depth during the sputtering (a large increase of more than 100 is expected), and cleaning of sample surface by soft sputtering with the argon clusters. Particular attention will be paid to possible migration of lipids during in-vacuum defrosting of samples, and to possible matrix effects induced by specific compounds like cholesterol.
A first use of the newly developed method will be to scan, before and after the laser shots, the in-depth composition of tissue sections coated by a matrix for MALDI (Matrix-Assisted Laser Desorption-Ionisation) mass spectrometry imaging, in order to gain a better understanding of desorption-ionization phenomena involved in MALDI, particularly on tissue in the context of imaging.
Then two work-packages are devoted to very different fields of science, in which an improvement of the capability of TOF-SIMS imaging is expected with 3-D analyses, or enhancement of the sensitivity, or even the possible cleaning of the sample surface without damaging it, when a sample is rare or unique. For each of these work-packages, informal collaborations have already been started since several years, and preliminary results or proof-of-concept have also been obtained.
In the field of healthcare research, a first work-package will focus on the acquisition of ion images from samples of remodeled arteries selected in complex samples of explanted human lungs from patients suffering from pulmonary arterial hypertension, compared to controls, as well as from model rats, to help understanding of the pathophysiology of this incurable condition.
Finally in the field of cultural heritage and archaeology, the last work package will study pigments from ancient painting samples, taken from paintings of artists dating from the Renaissance period to the twentieth century. The data will help to characterize the use and the mode of preparation of different pigments and binders: for example, the origin of the minerals, the nature of binders, the chemical synthesis of compounds, the mixing of different organic molecules.
In each of these two applications, limitations of TOF-SIMS imaging have already been pointed, such as the intrinsic limitation of surface analysis, which will be lifted by the in-depth analysis, or the difficulty of obtaining clean surfaces, or the limited sensitivity which will now be increased.
The final goal will be to make TOF-SIMS imaging a method of choice for micrometric localizedchemical analysis.
Project coordination
Alain Brunelle (Centre National de la Recherche Scientifique)
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.
Partner
CNRS-UPMC Laboratoire d'Archéologie Moléculaire et Structurale
INSERM UMR S_999 Hypertension Artérielle Pulmonaire: Physiopathologie et Innovation Thérapeutique
CNRS-ICSN Centre National de la Recherche Scientifique
Help of the ANR 467,000 euros
Beginning and duration of the scientific project:
December 2015
- 48 Months