SIMS2015 Session BI-ThP: Biological Imaging Poster Session

Amyloid fibril formation of Ab is a characteristic event closely related to Alzheimer’s disease. Lipid membranes play a role for a field where amyloidal beta peptides (Ab) aggregates through the interaction between Abs or between Ab and lipid membranes [1] . Imaging of Ab-adsorbed lipid membranes would give the better insight on the mechanism of fibril formation process. ToF-SIMS is a powerful tool for imaging of lipid membrane surfaces [2] because it has high special resolution below 100 nm and the analysis depth is below 2nm. These characters enable us to obtain the information of the surface of a giant molecular.

In this study, amyloidal beta with forty amino acid residues (Ab(1-40)) was firstly absorbed on lipid membranes formed on gold electrodes. The lipids used herein were 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and 1,2-dipalmitoyl- sn-glycero-3-phosphocholine (DPPC). These lipids have different transition temperatures. DOPC and DMPC are lipid crystal and DPPC is gel state at room temperatures.

Each sample was analyzed using ToF-SIMS with Bi₃⁺ primary ion source. Almost all of the peaks in ToF-SIMS spectra were chosen. All of the intensity of selected peaks on each pixel was analyzed by using principal component analysis ( PCA ) . The difference among the amino acids residues in Ab(1-40) on DOPC, DPPC, and DMPC was investigated in order to clarify the aggregation difference of Ab(1-40) depending on the lipid membranes condition. In addition, Ab(1-40)-related secondary ions of each sample were compared in order to evaluate the aggregation of Ab(1-40) on the lipid membranes. As a result, it is indicated that Ab (1-40) on DOPC rarely aggregates while that on DPPC aggregates. Moreover, the secondary ions of amino acid residues generated from the amyloid beta on DOPC are different from those from amyloid beta on DOPC or DMPC. It is suggested that the amino acid residues information is useful to understand the structural change of amyloid beta caused by aggregation.

References

[1] T. Shimanouchi, N. Kitaura, R. Onishi, H. Umakoshi, R. Kuboi, AIChE J., 57, 3625-3632 (2012).

[2] S. Aoyagi, T. Shimanouchi, T. Kawashima, H. Iwai, Anal. Bioanal. Chem., 407, 2859-2863 (2015).

TOF-SIMS provides detailed chemical structure information and high spetial resolution images. Therefore TOF-SIMS is useful for studying biological phenomena such as stroke. In this study, in order to evaluate biomolecular distributions, biological tissue samples were measured with TOF-SIMS. TOF-SIMS data were analyzed by means of multivariate analysis, in order to interpret complex samples containing unknown information and to obtain biomolecular distributions indicated by fragment ions from the target biomolecules.

Using conventional TOF-SIMS (primary ion source: Bi₃⁺), it is difficult to detect secondary ions beyond approximately 1000 Da. However, for the observation of biomolecular distributions in tissues, it is important to detect biological molecules such as peptides of molecular weight more than 1000 Da. Rat brain tissue samples were measured with TOF-SIMS (J105, Ionoptika Ltd, Hampshire), which has a continuous beam of Ar cluster as a primary ion source. TOF-SIMS with Ar clusters efficiently detects secondary ions more than 1000 Da. Molecules detected by TOF-SIMS were examined by analyzing TOF-SIMS data using multivariate analysis. Microspheres (45 μm) were injected into rat unilateral internal carotid artery (MS rat) to cause mimic cerebral infarction. The rat brain was sliced and then measured with TOF-SIMS.

The brain samples of a normal rat and the MS rat were examined to find specific secondary ions related to important biomolecules, and then the difference in them was investigated. Finally specific secondary ions were found around vessels incorporating spheres in the MS rat. The results suggest that important materials related to cerebral infarction would be detected by TOF-SIMS.

One of the oldest and most prolific communal ecosystems on Earth, bacterial biofilms are complex amalgamations of cells, extracellular DNA, proteins, polysaccharides, lipids, and various secondary metabolites. This structural and biochemical milieu affords biofilm inhabitants a substantial advantage over their planktonic counterparts, allowing for survival in harsh conditions, including on the outside of the international space station and the interior walls of hydrocarbon fuel tanks. Although many facets of the biofilm environment have been studied in great detail, intercellular chemical dynamics remain underexplored, leaving large gaps in our knowledge of these ecosystems. The inherent chemical and spatial complexity of a biofilm presents unique analytical challenges, necessitating both the application of existing analytical techniques and the advent of novel approaches. Here we compare and contrast two complementary methods for biomolecular mass spectrometry imaging (MSI) – namely Buckminsterfullerene time-of-flight secondary ion mass spectrometry (C₆₀-ToF-SIMS) and matrix-assisted laser desorption ionization (MALDI)-ToF-MS – for analysis of lipids, intracellular signaling molecules, and secondary metabolites in bacterial biofilms.

Each of the two ionization methods holds distinct advantages. MALDI is a soft ionization technique with unparalleled chemical coverage, but suffers from interference in the low mass range (m/z < 500) due to an increase in the noise profile caused by organic matrix ions. Cluster-ToF-SIMS offers a powerful alternative for small molecule imaging, with cluster beams such as C₆₀⁺, Ar₃₀₀₀⁺, and (H₂O)_n⁺ offering superior spatial resolution to MALDI and a softer form of ionization over traditional SIMS primary ion beams. C₆₀-ToF-SIMS in particular has become noteworthy for the high spatial resolution (a spot size of < 500 nm can be obtained with a few Pa beam), and a biomolecular coverage extending to about m/z 2000.

Our results show that while many target analytes are observable with both platforms, C₆₀-ToF-SIMS is particularly adept at imaging two important classes of intercellular signaling molecules, quinolones and phenazines, with limited chemical coverage for rhamnolipids surfactants, while MALDI, and the closely associated technique of metal enhanced-LDI, is better suited for detection and imaging of higher molecular weight rhamnolipids. Ongoing work focuses on depth profiling/imaging with SIMS, as well as methods for detection of a third class of important signaling molecules, namely homoserine lactones, which present a unique challenge due to their propensity for pH-induced degradation and a low ionization potential.

The use of nanoparticles is one of the most expanding fields in industrial as well as in medical applications, owing to their remarkable characteristics. Silver nanoparticles (AgNPs) are among the most-commercialized nanoparticles because of their antibacterial effects. Besides being employed as e.g. a coating material for sterile surfaces in household devices, the particles are also used in a broad range of medical applications. Their antibacterial properties make AgNPs especially useful for wound disinfection or as a coating material for prostheses and surgical instruments. Because of their optical characteristics, the particles are of increasing interest in biodetection as well.

Laser postionization secondary neutral mass spectrometry (Laser-SNMS) was used to investigate the effects of AgNPs on MDCK cells (epithelial cells of a dog kidney). The AgNPs had a size of about 20 nm and were synthesized by the addition of a peptide solution to a silver nitrate solution. MDCK cells grown on a silicon wafer were exposed to an AgNP concentration of 2.7 µg/ml with an exposure time of 24 h. To optimize the analysis of the MDCK cells, a special silicon wafer sandwich preparation technique was employed prior freeze-drying. 3D Laser-SNMS images were obtained using an Ar cluster ion beam for sputtering and Bi₃⁺ primary ions for the analysis. The 3D distribution of AgNPs in cells could be readily detected with very high efficiency, sensitivity and submicron lateral resolution. The Laser-SNMS images clearly showed that AgNPs are incorporated by MDCK cells and in part agglomerate to silver aggregates with a diameter of several microns at different distinct sites in the cell.

ToF-SIMS has been widely used to image small molecules of lipids and metabolites on tissue surfaces. Recently, various cluster ion beams (C₆₀, argon gas cluster, water cluster, etc.) were utilized to detect and identify large biomolecules from biological systems [1-2], despite the difficulties in detecting biomolecules higher than m/z 1,000 from tissue surfaces. For brain tissue imaging, electron-flooding has normally been used to carry out charge compensation during image acquisition, and all secondary ions were normally detected below m/z 1,000. It has been reported that electron-flooding would cause molecular fragmentation or damage as a function of electron energy and density [3].

In this study, we successfully obtained spectra and images from rat brain tissue surfaces without electron-flooding and were able to detect biomolecules higher than m/z 1,000. Several lipid molecules higher than m/z 1,000 were analyzed with and without electron-flooding to verify electron damage to these molecules. As a result, we conclude that TOF-SIMS imaging of tissue surfaces should be obtained without electron-flooding to get real and genuine chemical distributions of biomolecules on tissue surfaces.

Reference

[1] J. L. Denbigh, N. P. Lockyer, Materials Science and Technology, 31 (2015), 137-147.

[2] H. K. Shon, Y. Cho, C. S. Lim, J. S. Choi, S. J. Chung, T. G. Lee, Surface Interface and Analysis, 46 (2014) 189-192.

[3] I. S. Gilmore, M. P. Seah, Applied Surface Science, 187 (2002), 89-100.

One of the major problems of biological SIMS imaging is the lack of protein and peptide imaging. Most of biological story telling is mianly based on proteins. The biological implication of lipid SIMS imaging would be much higher if protein imaging is provided toghether. Utilizing high secondary ion yields of metals, proteins can be SIMS imaged with metal tagged proteins. Rare earth metals such as Yb, Sm, Pr, Tm, Eu were used for imaing NeuN and HUC proteins in neuron cells in hypothalamus mouse tissues with a spatial resolution of ~2 μm using a TOF-SIMS. Lipids and neurotransmitters images such as cholesterol, sterol lipids, glycerophospholipids, GABA, glumate, depamine, and acetylcholine obtained simultaneously with protein images were overlayed for more deeper understanding of neurobiology, which is not allowed by any other bioimaging technqiues. The protein images from TOF-SIMS were compared with confocal fluorescence miscroscopy and nanoSIMS images.

A new sample preparation method for imaging single cell membranes in a tissue using the vibrotome technique to prepare a tissue slice without any fixation and freeze drying will be also presented briefly for hypothalamus tissues.

A huge change in lifestyle and eating habits, dramatic increase in risk of gastrointestinal diseases every year. Early detection and treatment can reduce mortality caused by gastrointestinal diseases. Fecal occult blood is a small amount of blood in the stool which is invisible. If there is blood detection reaction, the cause may be gastrointestinal disorders, such as colorectal polyps, colorectal cancer, etc. Fecal occult blood test (FOBT) is easy operation, rapid judgment and non-invasive.

There are two types of fecal occult blood test: The chemical method used heme from hemoglobin, which has a similar activity as catalase, to performed redox reactions. It is applicable to both upper and lower gastrointestinal bleeding, and is simple and inexpensive. The disadvantage is possible false positive influence caused by food. Immunization method is the use of human hemoglobin antigen-antibody binding reaction. The advantages of high specificity, more sensitive to the lower gastrointestinal tract, less susceptible to the impact of food, the disadvantage is that the detection limit is strict and higher prices.

Secondary ion mass spectrometry analyzes the molecular composition of the surface. The advantage is a simple pre-treatment or not, maintaining the original state analysis. This study is a direct or simple treatment stool samples using TOF-SIMS analysis, the difference between whether or not containing blood in stool. Preliminary findings at m / z = 86 and m / z = 184, there are significant differences. We intend to use a different pre-treatment to achieve consistency and accuracy.

1. M. Dannoura et al. (2011) New Phytol. 190, 181-192

2. D. Epron et al. (2012) Tree Physiol. 32, 776-798

3. Takabe et al. (1984) Mokuzai Gakkaishi 30, 103-109

The interest in using plant-based products as raw material in industrial processes, replacing oil-based or geological products, or as renewable energy source is increasing. With the current scientific, economical and societal interest in sustainable development, industrial products wherein synthetic fibers are replaced by fibers originating from plant cell walls are no longer niche products but a real industrial product .

Important influences of the environment on the productivity of alfalfa have been described. However fairly little is known about the influence of mineral imbalances, deficiency or excess, on the production of biofibers in alfalfa or in plants in general [1]. This is found to be essential for the extracellular polymerization of lignin monomers. Alfalfa species can accumulate relatively high concentrations of heavy metals and minerals in their aboveground tissues.To investigate on plant cell wall responses, the study was focussed on copper distribution.

In the present study, a set of plants grew in a hydroponic system for 3 months with a solution concentration of copper from a deficiency to an excess, in the range of 3 to 3000 mg/mL. For stabilizing biological plant, the strategy chosen was the ultra-rapid cryofixation [2]. First the sodium and calcium ions of the samples were imaged by SIMS in positive mode with a modified ims6f equipped with a gallium source [3-4]. As the specimen preparation had apparently been quite good in preserving the cellular morphology of the stem, the copper localization in the fiber plant material was revealed by NanoSIMS in secondary negative mode. Higher copper signal intensity is observed in the sclerenchyma cells of the stem. According to the concentration of original solution, the elemental distribution was qualitatively evaluated and compared with measurements performed by ICP-MS. The role and the quantity of the copper mapped will be discussed on the base of the growth of the alfalfa.

[1] Moura JC et al., J Integr Plant Biol.;52(4):360-76 (2010).

[2] Smart et al. The Plant Journal 63, 870–879 (2010).

[3] Migeon et al., Surface and Interface Analysis 43 (1-2), 367-369 (2011).

[4] Frache et al., Surface and Interface Analysis 43 (1-2), 639-642 (2011).

SIMS is showing significant promise for cellular and sub-cellular resolution imaging of pharmaceutical compounds¹. A recent important review has highlighted that “the distribution of drugs into and within individual cells is arguably one of the most neglected areas of pharmacology, pharmacokinetics and therapeutics”² and that “knowledge of the drug concentration at its site of action within cells is becoming increasingly important in pharmacology and therapeutics”². That same review alsostates that “To predict the pharmacological effect, there must be data concerning the concentration at the target, and for intracellular targets, plasma concentrations may poorly represent what is happening inside the cell”. It is well-known that matrix effects in SIMS can entirely suppress analyte signal in tissue as well as promote it through, for example, cationisation. It is therefore critical to obtain a better understanding of matrix effects on pharmaceutical compound signal intensity in biological matrices.

In this study, we measure the matrix effect of two well-studied drugs by the pharmaceutical industry: (1) amiodarone, a class III antiarrhythmic agent used for various types of cardiac dysrhythmias and (2) chloroquine, which is used in the treatment or prevention of malaria. The drugs contain a halogenated moiety, iodine and chlorine, respectively. This is useful for quantification by XPS as well as comparison with the molecular ion signal matrix effect. We measure the matrix effect in three systems of increasing complexity; (1) a molecular multilayer of drug-cholesterol-drug, (2) a concentration gradient of drug on a lipid layer and (3) drug quantitatively doped into a tissue paste reference material. These measurements will be discussed and presented in terms of data for drug in a tissue model system.

References

1. M. K. Passarelli et al., Analytical Chemistry (in press), 2015.

2. C. T. Dollery, Clinical Pharmacology & Therapeutics, (2013); 93, 263–266.