SIMS2015 Session SP1-TuM: Sample Prep & Analysis of Biological Samples

ToF-SIMS provides chemically specific images that can increase our understanding of chemical changes in cells and tissues as a function of an applied stress or as a result of disease, and enable tracking the spatial distribution of metabolites and lipids. However, there are currently still many challenges with tissue analysis using ToF-SIMS. In this presentation, challenges with sample preparation for the ToF-SIMS environment including working with formalin fixation followed by paraffin embedding (FFPE) processed samples will be discussed. The advantage of combining ToF-SIMS images with informational images using other techniques allows researchers to visualize a molecular map that correlates with specific biological features or functions. The challenge of aligning tissue images from different instruments will be presented along with solutions for processing large amount of data (including multivariate analysis of the ToF-SIMS image data).

Endophytes are microorganisms (either bacteria or fungi) that live within plants, most of them without causing any symptom of disease. They have attracted a growing worldwide interest especially because of their enormous biological diversity as well as their ability to biosynthesize secondary metabolites. However, the precise role of endophytes in plant microbiome as well as ecological significance of their metabolites remains underexplored [1].

In this context, we initiated a program based on the cultivable endophytic microbial diversity associated with the conifer Cephalotaxus harringtonia from which more than 640 fungal and bacterial isolates were obtained and identified via the determination of their Rdna sequences [2]. Among them, the endophytic fungus Paraconiothyrium variabile and the bacterium Bacillus subtilis displayed a strong and mutual antibiosis never observed between other partners of the C. harringtonia microbiome.

We used mass spectrometry imaging to decipher the chemical communication between the two partners. Indeed, mass spectrometry imaging is proved to be a complementary technique to metabolomic studies which are generally done by liquid chromatography, to allow precise mapping of the chemical communication by comparing ion images of different metabolites involved in the competition [3].

In this study, both Matrix Assisted Laser Desorption / Ionisation Time-Of-Flight tandem mass spectrometry (UltrafleXtreme, Bruker Daltonics, Wissembourg, France) and Time–Of–Flight Secondary Ion Mass Spectrometry (TOF-SIMS IV, ION-TOF GmbH, Münster, Germany) have been used to compare the localization of metabolites from P. variabile and B. subtilis which have grown together in competition on a Petri dish. Specific sample preparation methods have been developed and adapted for both instruments. Hydrolyzed surfactines have been detected in the competition area using both MALDI-TOF and TOF-SIMS imaging technics. That means the fungus is able to hydrolyze surfactines produced by the bacterium before these compounds inhibit its growth. Although MALDI imaging of microbial competitions is not new [4], we propose a sample preparation to study microbial competition by TOF-SIMS imaging.

[1]Prado S. et al. Stud. Nat. Prod. Chem. 2012, 36:249-296.

[2] Langenfeld, A. et al. Fungal Biol. 2012, 117:124-136.

[3] Hoefler, B.C. et al. Proc. Natl. Acad. Sci. U.S.A. 2012, 109: 13082-13087.

[4] Yang, Y-L. et al. Nat. Chem. Biol. 2009, 5:885-887.

Imaging time-of-flight secondary ion mass spectrometry (ToF-SIMS) has the unique capability to detect and map a great number of compounds on a tissues surface simultaneously, without the need of labelling specific molecules. The development of cluster and polyatomic ion beams have improved the ability of ToF-SIMS to detect higher mass molecular species, therefore addressing biological questions with ToF-SIMS, such as drug localisation in cells/tissues and disease studies, was facilitated.^[1] Large gas cluster ion beams (GCIBs), commonly Ar_n⁺ (where n = 500-5000), have shown to provide further improvements as the energy is partitioned over a large number of constituent atoms but with drawbacks such as less spatial resolution and lower ionisation efficiency compared to conventional ion beams.

At the National Centre for Imaging Mass Spectrometry (NCIMS) in Gothenburg we are using a 40 keV GCIB mounted on a J105-3D Chemical Imager, (Ionoptika Ltd). We showed that the benefits of this system are higher ionisation efficiency and beam spot sizes of below 3 µm that allow the imaging of larger molecular species at high resolution. This brings ToF-SIMS closer to the MALDI regime but without the need of a matrix and with generally better spatial resolution.^[2] Those improvements still leave issues such as greatly varying ionisation efficiencies for different molecules, matrix effects and high surface sensitivity unaddressed.

The latter has been recognized as a problem in traditional tissue sample preparation as it emphasizes signals of molecules which migrate to the tissues surface (e.g. cholesterol) while other molecules are being covered up and/or suppressed. We have overcome some of those issues by exposing rodent brain sections to reactive vapour compounds e.g. trifluoracetic acid (TFA).^[3] This treatment led to increased high mass signal and showed signal from previously unobserved species in the mass spectrum. Vapour sample pre-treatment allowed new chemical signatures of different brain regions to be distinguished, therefore expanding the chemical coverage and the potential application of ToF-SIMS imaging in neurological studies.

[1] D. Touboul, A. Brunelle, F. Halgand, S. De La Porte, O. Laprevote, J. Lipid Res. 2005, 46, 1388-1395.

[2] T. B. Angerer, P. Blenkinsopp, J. S. Fletcher, Int. J. Mass Spectrom. 2015, 377, 591-598.

[3] T. B. Angerer, M. Dowlatshahi Pour, P. Malmberg, J. S. Fletcher, Anal. Chem. 2015, 87, 4305-4313.

Principal components analysis (PCA) revealed spectral differences between treatment groups. Several characteristic negative ion peaks were observed in the scaffolds exposed to SDS and deoxycholate. These peaks could be assigned to characteristic molecular fragments seen in spectra from the respective pure detergent indicating the presence of residual detergent in the UBM. Peaks indicative of phospholipid membranes and residual nuclear material, suggesting incomplete decellularization, were observed in all samples, but to a greater extent with scaffolds not exposed to detergent. We further probed these data sets to investigate how detergent selection impacts proteinaceous ECM components. Using a peak list of known amino acid fragments, PCA distinguished native tissue from decellularized UBM and highlighted spectral differences between treatment groups. Notably, the BMC surface of UBM prepared with ionic detergents SDS and deoxycholate yielded less intense characteristic peaks from hydrophobic amino acids than UBM treated with charge-neutral detergents CHAPS and Triton X-100. Harsher detergents may denature protein structure and break protein-protein interactions through binding of their hydrophobic tail to hydrophobic amino acid residues. We further examined cell-matrix interactions of human urothelial cells seeded on the BMC of UBM, investigating how detergent exposure affected cell proliferation and permeability of the cell monolayer. An understanding of the effects of detergent exposure on the structure, composition and surface molecular functionality of decellularized scaffolds will facilitate a rational strategy for successful recellularization and subsequent positive clinical outcomes.

In the present study two powerful techniques imaging mass spectrometry techniques, Time of Flight Secondary Ion mass spectrometry (ToF-SIMS) and Matrix Assisted Laser Desorption Ionization (MALDI) were used for lipid profiling of intestine tissue sections from rats fed specially processed cereals (SPC-fed group) and rats fed ordinary feed as a control. Intake of such cereals increase active antisecretory factor (AF) in plasma, an endogenous protein with proven regulatory function on inflammation and fluid secretion. This endogenous increase counteracts the clinical intestinal symptoms in patients suffering from inflammatory bowel disease (IBD), endocrine diarrhea and Meniere’s disease and has also been shown to provide protection against raised intracranial pressure induced by experimental head trauma. Although, the exact mechanism for the activation process of AF at the cellular level remains unclear. Here, multimodal Imaging mass spectrometry has been used to examine changes in lipid distribution in intestine tissue samples and to gain insight into the possible mechanisms involved.

An IONTOF V instrument equipped with a Bi cluster ion gun was used to analyze the tissue sections. Data from 15 intestine sections from control SPC-fed rats were recorded using the stage scan macro raster with a lateral resolution of 5 μm. Data were subsequently exported to MatLab and subjected to PLS-DA. MALDI imaging was performed using an Ultraflextreme IV (Bruker Daltonics, Bremen, Germany) equipped with a Smartbeam laser Nd:YAG/355nm as a complimentary technique for higher mass lipid species. Data were collected from one tissue section from each group. The MALDI data were analyzed in SCiLS Lab 2014b.

The data clearly show changes of certain lipids in induced by the SPC-feed. Scores plots showed a well-defined separation between two groups. The corresponding loadings plots revealed that the groups separated mainly due to changes the c18:2, c18:1, c16:0 and other short chain monocarboxylic fatty acids. This study used ToF-SIMS to track food induced changes in lipid content in intestinal tissue sections and we have successfully shown that lipid content changes in rats fed SPC-Flakes® and this is consistent with recent studies on the effect of this diet supplementation. The changes in lipids found might give insight into the working mechanisms of AF in the body and this has successfully been used to understand the working mechanism of SPC-induced AF activation in intestine.

Poultry meat is considered to be one of the most widespread in the world and the amount of chicken meat in human diets is still growing in many societies. This trend has been related with the occurrence of a number of diseases, for example coronary heart disease or arteriosclerosis. At the same time, the presence of the meat in people’s diets has an positive impact on human health as well. Generally poultry meat is an invaluable source of proteins, nutrients such as iron, zinc, vitamins B3, B6, and B12 and has a relatively low fat content. The quality of chicken meat has been the subject of research for many years and there are several ways to improve the quality of the meat. One of the possibilities is to change the contents of some certain components, as omega-3 and -6 or reducing the level of cholesterol by enriching chickens feed by elements of different types, such as vegetable oils. So far there has been used various biochemical and biophysical methods to study quality of different types of meat, especially broilers meat. Here we demonstrate the application of high resolution ToF-SIMS mass spectrometry to determine how changes in the animal nutrition influence the level of specific lipids, such as cholesterol and vitamin E.

In the present experiments, there were four different chicken groups with various dietary treatments. The first group was supplemented with 50% soy oil, second one with 50% linseed oil, in the third group there was combination of these oils in the proportion of 44%:56% and as reference - group fed from beef tallow. In each group four animals were selected and for each one positive and negative ion spectra were generated from the left carcass side from the pectoralis superficialis muscle tissue. Using TOF-SIMS and based on some of the characteristic peaks for fatty acids, glycerolipids, glycerophospholipids, sphingolipids, sterol lipids and prenol lipids we have illustrated that there are significant changes in the levels of individual lipids in meat tissue due to the different nutrition programs, the dependence of these lipids from the feeding program is different for fat and muscle fibre, and that ToF-SIMS mass spectrometry in a very good way reflects these changes.

Thanks to the ToF-SIMS measurements and the proposed methodology for analyzing spectra we have obtained relationship between applied nutrition program for the each group and the level of some essential lipids in animals organisms. Presented results show that SIMS imaging is a useful approach for assessing changes in lipid content meat tissue from animals treated with different nutritional plans and gives a very good prospects for future research in this field .