SIMS2015 Session BI2-FrM: Biological Imaging

Friday, September 18, 2015 8:40 AM in Grand Ballroom I
Friday Morning

Time Period FrM Sessions | Abstract Timeline | Topic BI Sessions | Time Periods | Topics | SIMS2015 Schedule

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8:40 AM BI2-FrM-1 TOF-SIMS Imaging Analysis of Rats with Focal Cerebral Ischemia by Using Ar Cluster Ion Beams
Tae Geol Lee (Korea Research Institute of Standards and Science (KRISS), Republic of Korea); Ji-Won Park (Institute for Basic Science, Republic of Korea); Hyun Kyong Shon (Korea Research Institute of Standards and Science (KRISS), Republic of Korea); Min-hyeoung Kim (POSTECH, Republic of Korea); JaeWon Lee (Kyung Hee University); Jeong Hee Moon (Korea Research Institute of Bioscience and Biotechnology); ChanYoung Shin (Konkuk University); Kwang Pyo Kim (Kyung Hee University); Daehee Hwang (Institute for Basic Science)

Focal cerebral ischemia occurs when a blood clot has blocked a cerebral vessel. Focal cerebral ischemia reduces blood flow to a particular brain region, increasing the risk of cell death to that area. Ischemia altered brain metabolism, reduced metabolic rates, and caused energy crises. Recently, the matrix-assisted laser desorption and ionization time-of-flight (MALDI-TOF) imaging technique was used to analyze the lateral distributions of lipid molecules on normal and ischemic rat brain tissue surfaces [1].

In this study, we analyzed the surfaces of ischemic rat brains using TOF-SIMS with Ar gas cluster ion beams. Results of the TOF-SIMS spectrum compared with MALDI-TOF spectrum indicate that TOF-SIMS provides complementary information to MALDI-TOF for lipid compositions from the tissue surface by detecting shared and distinct new ions with different intensities. The TOF-SIMS spectra and images in conjunction with multivariate statistical analyses such as PCA and NMF were used for comparison between normal and ischemic areas. The results of our statistical analysis indicated that several unique peaks showed different distributions between normal and ischemic areas of the brain surface. By analyzing lipids extracted from the ischemic area with the ESI-MS/MS technique, we successfully identified several distinct new lipid molecules, which were more abundant in ischemic areas but were not detected in the MALDI-TOF data. Our results show that the TOF-SIMS imaging technique with Ar cluster ion beams can be a very powerful technique to discover new biomarkers related to focal cerebral ischemia.

[1] Kim et al., J Lip Res. 2012, 53, 1823-1831

9:00 AM BI2-FrM-2 Imaging of Lipids and Proteins in Alzheimer’s Disease Transgenic Mouse Brains Using Immunoliposomes and ToF-SIMS
Louise Carlred (SP Technical Research Institute of Sweden); Björn Johansson, Vladana Vukojević, Martin Schalling (Karolinska Institutet); Fredrik Höök (Chalmers University of Technology); Peter Sjövall (SP Technical Research Institute of Sweden)

Time-of-flight secondary ion mass spectrometry (ToF-SIMS) has been proven to successfully image different kinds of molecules, especially a variety of lipids, in biological samples. Proteins, however, are difficult to detect as specific entities with this method due to extensive fragmentation. To circumvent this issue, we are developing a method for detection of proteins using antibody-conjugated liposomes, so called immunoliposomes, which are able to bind to the specific protein of interest. Using specifically designed liposomes that are easily recognized with ToF-SIMS, the immunoliposomes can reveal the spatial distribution of the protein of interest in the sample. Moreover, the antibodies and lipid composition in the immunoliposomes can be altered to create different kinds of immunoliposomes for detection of many different proteins at the same time. In combination with the capability of ToF-SIMS to detect native lipids in tissue samples, this method opens up the opportunity to analyze many different biomolecules, both lipids and proteins, at the same time, with high spatial resolution.

The method has been applied for identification of biologically relevant proteins in Alzheimer’s disease (AD), such as amyloid-β, in transgenic mouse brain tissue. To ensure specific binding, the immunoliposome binding was studied on a model surface using quartz crystal microbalance with dissipation monitoring (QCM-D). The immunoliposome binding was also investigated with fluorescence microscopy, by including fluorescent lipids for detection of the immunoliposomes, and compared with conventional immunohistochemistry.

The results show specific binding of the immunoliposomes to amyloid-β, both in QCM-D, fluorescence microscopy and ToF-SIMS. Using ToF-SIMS imaging, several lipids could also be investigated in the vicinity of the amyloid-β deposits, which is an advantage compared to fluorescence microscopy. Expanding this method by including other important protein targets in AD, such as Tau, can potentially provide further information about lipid-protein interactions, which is important to understand the mechanisms of neurodegeneration in AD.

9:20 AM BI2-FrM-3 Lipid Imaging of Invertebrate Model Systems by Secondary Ion Mass Spectrometry
Nhu Phan (University of Gothenburg, Sweden); Andrew G. Ewing, John S. Fletcher (Chalmers University of Technology, Sweden)

Drosophila melanogaster and C. elegans (flies and worms respectively) are common biological model systems, which have relatively simple nervous systems but possess high order brain functions similar to humans. We have applied time of flight secondary ion mass spectrometry (ToF-SIMS) to study the lipid structure of these invertebrate organisms. From this, we were able to investigate the effects of the stimulant drug methylphenidate on the fly brain structure and the stress response of the worm caused by temperature on its lipid compositions.

Different distributions of various molecules, particularly eye pigments, fatty acids, diacyl-glycerides, and phospholipids have been found across Drosophila brain. The lipid structures, particularly diacylglycerides (DAG), phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylinositiol (PI), are shown to dramatically alter following the administration of methylphenidate in our previous study.[1] Here, we successfully use tandem MS on ToF-SIMS with the high energy 40 keV Ar4000 cluster gun to elucidate the structures of the biomolecules affected by the drug.

For C. Elegans, the entire freeze-dried worm and worm sections were imaged using a 3-dimensional imaging approach with interlaced C60 and Ar4000 cluster guns. Lipids and lipid-related compounds such as fatty acids, DAG, PC and triacylglycerides (TAG) distribute in specific regions of the worm. The correlation of ion images between the entire and sectioned worms provides reliable structural information of the worm following a change of temperature, a typical stress used to study this organism. ToF-SIMS imaging shows great potential to elucidate chemical distributions in small invertebrate systems in relation to endogenous and exogenous effects.

Reference:

[1] Nhu T. N. Phan, et al. Anal.Chem., 2015, 87 (8), 4063–4071. DOI: 10.1021/acs.analchem.5b00555.

9:40 AM BI2-FrM-4 High Resolution ToF-SIMS Tandem Imaging MS of Lipid Species in Infected Thin Tissue Sections
Anne L. Bruinen (Maastricht University, Netherlands); Gregory L. Fisher (Physical Electronics); Astrid van der Sar (VU University Medical Center); Nina Ogrinc Potočnik, Ron M.A. Heeren (Maastricht University, Netherlands)
In mass spectrometry imaging based biological tissue analysis, one has to contend with a high level of complexity concerning the molecules that are found on the surface. In many cases, it is predictable and generally accepted which compounds should be detected in specific samples. However, tandem mass spectrometry is considered the bench mark technique to identify and elucidate structures of unknown molecular species of which there are a multiplicity.

A PHI NanoTOF II mass spectrometer (Physical Electronics, USA) was equipped with a second time of flight spectrometer and developed for simultaneous ToF-SIMS and tandem MS imaging experiments. A precursor ion can be selected from the MS1 mass spectrum and subsequently fragmented by collision induced dissociation and the fragments detected by the MS2 detector.

MALDI is often the ionization technique of choice for tandem MS imaging in biological tissues; however, the described ToF-SIMS instrument is capable of 2D mapping of relevant molecules within biological tissues with a much higher lateral resolution, less sample preparation, and with minimal surface damage. The strength of this instrument is the possibility of collecting MS1 and MS2 data simultaneously, analysis of unlabeled samples without an applied ionization matrix and the reuse of samples for many analyses.

Here, zebrafish (Danio rerio) are used as a model for tuberculosis, a granulomatous inflammatory disease. In an earlier study, lipid changes were observed in tuberculosis granulomas found in thin tissue sections of an infected adult zebrafish. These structures were imaged by high resolution SIMS analysis, but the identification of lipid species was dependent on additional MALDI tandem MS analysis.

Whole body sections were prepared from snap frozen and embedded diseased and healthy zebrafish. Without further preparation, the samples were analyzed in both positive and negative ion mode using a PHI NanoTOF II mass spectrometer equipped with a liquid metal ion gun (Bi3+), a gas collision cell, and a second ToF detector. ToF-SIMS tandem imaging MS was used to localize and identify both the absence and presence of diacylglycerol species and fatty acids as well as host membrane phospholipids that are believed to play an important role in the bacterial inflammation cascade.

10:00 AM BI2-FrM-5 Analysis of Pharmaceutically Modified Biomaterials from In Vitro to In Vivo – and its Own Special Challenges
Anja Henss, Marcus Rohnke, Julia Kokesch-Himmelreich, Peter Schmitz (Justus-Liebig University Giessen, Germany); Matthias Schumacher (Technical University of Dresden, Germany); Seemun Ray (Justus-Liebig University Giessen, Germany); Thomas Hanke (Technical University of Dresden, Germany); Volker Alt (Justus-Liebig University Giessen, Germany); Michael Gelinsky (Technical University of Dresden, Germany); Jürgen Janek (Justus-Liebig University Giessen, Germany)

Time of flight secondary ion mass spectrometry (ToF-SIMS) is applied to detect pharmaceutical agents and their distribution within new developed implant materials, in cells cultured on these materials and to elucidate the release kinetics of the drugs in an animal experiment.

We concentrate on strontium (Sr) and bortezomib (BZ) enriched biomaterials since Sr is known to be an effective antiosteoporotic drug whereas BZ as proteasome inhibitor is used for the treatment of multiple myeloma. (1-3)

Based on the good ionization properties of inorganic Sr, the analysis of human osteoblast-like cells cultured on the Sr-modified cements and their mineralized extracellular matrix (Mecm) prove clearly, that there is an uptake of Sr into the cells as well as Sr is incorporated into the Mecm.(4)

Cell experiments with myeloma cells prove also the uptake of BZ into the cells but the unambiguous detection of BZ is quite more challenging. Because of its instability and its low ionization probability the detection limit is hardly reached. This is aggravated by the fact that BZ is only applied in low dose due to its cell toxic effects (in vitro) or inflammatory response (in vivo). Additionally the high solubility and less stability of the molecule require optimized sample preparation protocols and special measurement procedures. Therefore a pretreating protocol for samples prepared as cut sections after explantation (in vivo) is developed using Ar1500.

In the animal experiments the biomaterials were used to fill defects in the femur of rats. We imaged the fragments of the inorganic and organic compounds of the bone as well as the distribution of the particular drugs. Using ToF-SIMS the Sr release in the surrounding tissue is investigated and it is proven that Sr is localized in regions of newly formed bone but also within the pre-existing tissue.(5) The BZ samples had to be cleaned prior analysis using Ar-clusters. The samples showed a nearly uniform distribution of the B+/BO+ in the entire tissue and implant area. No concentration gradient of boron from the implant to the neighbored tissue was detected but rather an equal distribution with less intensity for the complete section. These findings prove the good solubility and diffusion properties of BZ but also support the assumption of a too fast degradation process of the molecule.

1. Schumacher M, et al.,Acta Biomater. 2013;9(7):7536-44.

2. Marie PJ, et al., Calcif Tissue Int. 2001;69(3):121-9.

3. Kortum KM, et al., Internist. 2013;54(8):963-75.

4. Kokesch-Himmelreich J, et al., Biointerphases. 2013;8.

5. Rohnke M, et al., Analytical and Bioanalytical Chemistry. 2013.

10:40 AM BI2-FrM-7 C60-SIMS Imaging of Nanoparticles Within Mammalian Cells
Anna Bloom, Hua Tian, Nicholas Winograd (The Pennsylvania State University)

Targeted drug delivery systems have become increasingly prevalent in the pharmaceutical industry due to more successful treatment outcomes and reduced side effects when compared to traditional treatment methods. The high biocompatibility and reduced toxicity of gold nanoparticles, coupled with their ability to accumulate in areas of interest, and tunable stability, have made them a promising delivery platform.1 Research efforts continue to focus on developing materials which target specific tissues and organs through the incorporation of a variety of biomolecules to the surface of gold nanoparticles.2 Traditional analysis methods for nanoparticles within biological systems, such as transmission electron microscopy (TEM), require complicated sample preparation methods and do not offer chemical specificity. This makes it difficult to obtain information regarding the distribution of nanoparticle-bound pharmaceutical compounds, as well as the distribution of the compounds themselves after uptake into the biological system. TOF-SIMS is a tool well-suited to providing not only the location of the nanoparticles, but also offering insight into the chemical environment in the system being studied, and has previously been used to image TiO2 nanoparticles in a biological system.3

Gold nanoparticles have been chemically imaged within macrophage-like RAW cells utilizing a C60+ primary ion beam on the J105 3D Chemical Imager (Ionoptika LTD).4 The ability to locate the nanoparticle laterally, as well as a function of depth, without sacrificing information regarding the surrounding areas of the cell, makes it possible to acquire greater understanding of the drug pathways and mechanisms of action in treatments utilizing nanoparticles. As with any technique, sample preparation is crucial in order to maintain native biological environments, while optimizing the capabilities of the instrument. Existing cellular sample preparation techniques for SIMS5-6 have been adapted to ensure successful incorporation of nanoparticles, without altering the biological balance present within the cell.

[1]E. Connor, J. Mwamuka, A. Gole, C. Murphy, M. Wyatt, Small 2005, 1, 325.

[2]D. Giljohann, D. Seferos, W. Daniel, M. Massich, P. Patel, C. Mirkin, Angew. Chem. Int. Ed.2010, 49, 3280.

[3]T. Angerer, J. Fletcher, Surf. Interface Anal.2014, 46, 198.

[4]J. Fletcher, S. Rabbani, A. Henderson, P. Blenkinsopp, S. Thompson, N. Lockyer, J. Vickerman, Anal. Chem.2008, 80, 9058.

[5]A. Piwowar, S. Keskin, M. O. Delgado, K. Shen, J. Hue, I. Lanekoff, A. Ewing, N. Winograd, Surf. Interface Anal.2013, 45, 302.

[6]S. Rabbani, J. Fletcher, N. Lockyer, J. Vickerman, Surf. Interface Anal.2011, 43, 380.

11:00 AM BI2-FrM-8 Detection of Organic Nanoparticles in Human Bone Marrow Stromal Cells using ToF-SIMS and PCA
Julia Kokesch-Himmelreich (Justus-Liebig-University of Giessen, Germany); Beatrice Woltmann, Bernhard Torger (Dresden University of Technology); Marcus Rohnke (Justus-Liebig-University of Giessen, Germany); Stefan Arnhold (Justus-Liebig-Universitity of Giessen); Ute Hempel, Martin Müller (Dresden University of Technology); Jürgen Janek (Justus-Liebig-Universitity of Giessen, Germany)

The medical application of nanoparticles relies on the understanding of the interaction between cells and nanoparticles. Therefore it is very important to know whether and how the nanoparticles are taken up by the cells. Optical methods like fluorescence microscopy are commonly used to visualize nanoparticles inside cells. This method requires chemical linking of a fluorescence marker to the nanoparticles. Instead here we used the label free mass spectrometric method "Time of Flight Secondary Ion Mass Spectrometry" (ToF-SIMS) for the detection of organic nanoparticles.

For our experiments polyelectrolyte complex nanoparticles (NP) made of polyethylenimine (PEI) and cellulose sulfate (CS) were chosen. These polymer-based nanoparticles were developed as potential drug carrier and also as coatings for implant materials [1]. For this reason human bone-marrow derived stromal cells (hBMSC) were used for the cell experiments.

One of the key questions of the current study was whether it is possible to identify specific masses for these PEI/CS-NP using ToF-SIMS. Since cells contain various sugars and peptides, their fragment patterns look quite similar to the fragment mass spectra of the PEI/CS-NP. In this study PCA was used to identify characteristic masses of the PEI/CS nanoparticles. Therefore mass spectra of the nanoparticles were compared with mass spectra of the hBMSC.

To investigate whether the PEI/CS-NP were taken up by the hBMSC, the cells were cultured with PEI/CS nanoparticles for 8 days. On the one hand, cross sections of the cells were prepared and SIMS images were compared with optical images. On the other hand, hBMSC were fixed with PFA, air dried and depth profiled by ToF-SIMS.

Mass fragments originating from the NP compounds especially from cellulose sulfate could be used to unequivocally detect and image the PEI/CS-NP inside the hBMSC. The findings were confirmed by light and transmission electron microscopy [2].

[1] M. Muller, B. Kessler, Journal of Pharmaceutical and Biomedical Analysis 2012, 66, 183-190.

[2] J. Kokesch-Himmelreich, B. Woltmann, B. Torger, M. Rohnke, S. Arnhold, U. Hempel, M. Müller, J. Janek, Analytical and Bioanalytical Chemistry 2015.

11:20 AM BI2-FrM-9 Combined ToF-SIMS and Fluorescence Microscopy Imaging for Visualization of Metal Anticancer Complexes in Single Cells
Fuyi Wang, Suyan Liu, Kui Wu, Zhaoying Wang (Institute of Chemistry, Chinese Academy of Sciences, China)
The elucidation of the cellular uptake and subcellular distribution of drugs and drug candidates is helpful for better understanding on mechanism of action of the drugs. With the virtues of labeling-free, chemical specificity and high spatial resolution, secondary ion mass spectrometry (SIMS) imaging has emerged to be a powerful tool to visualize the distribution of drugs in cells. In the present work, we develop a combined ToF-SIMS and confocal fluorescence imaging strategy for the visualization of metal anticancer complexes in single cells cultured on house-made addressable silicon wafers which assists to localize one or a few single cells for sequentially confocal fluorescence imaging and SIMS imaging. The fluorescence imaging visualizes the subcellular organs, e.g. membrane and nucleus, and the SIMS imaging localize the tested metal complexes inside the same single cells. Thus, we can elucidate the subcellular distribution of the studied complexes according to the morphological and molecular imaging results. The developed method has been successfully applied to visualize the cellular distribution of novel organometallic ruthenium and platinum anticancer complexes synthesized in our laboratory, demonstrating that these organometallic complexes co-localizes with both membrane and nucleus dyers, verifying the dual-targeting property of the complexes.
11:40 AM BI2-FrM-10 TOF-SIMS Analysis of Pharmacological Active Components in Cordyceps Sinensis
Zhanping Li (Tsinghua University, China); Noriaki Sanada (ULVAC-PHI); Suqin Sun (Tsinghua University)

Cordyceps sinensis is a well-known traditional Chinese medicine, is also called DongChongXiaCao (winter worm summer grass) in Chinese. The nucleoside, sterols and mannitol of Cordyceps sinensis, which are important pharmacological active components, were studied with TOF-SIMS analysis. The chemical information correspond to the fragment ions of m/z 251 and 181 is the key point of the study for mannitol and cordycepin. Base on the advantage of high mass resolution (M/ΔM) of TOF-SIMS, The positive fragment ion detected and identified by TOF-SIMS at m/z 251 and 252 might not be the molecular ion M+ and [M+H]+ of cordycepin C10H13N5O3 (251amu), which is a sanitarian and pharmacological active component. At the same time, the negative fragment ion of m/z 181 (near molecular ion of mannitol (C6H14O6): [M-H]-) was made detail interpretation. This confirms the fragment ion of m/z 181 is a reliable basis for the identification of mannitol in Cordyceps sinensis. The positive fragment ions of m/z 121, 152, 205, 411, 412, 413, 575, 577, 578 and the negative fragment ions of m/z 181, 573, which arise from pharmacological active component, can be used as the characteristic ions for the identification of Cordyceps sinensis. The study also found that, there are some “splicing” ions, which are formed between pharmacological active compounds and weakly polar compounds (stearic acid, palmitic acid, oleic acid, linoleic acid, seventeen acid, eighteen acid, nineteen alkanes, twenty dilute acid etc.) of Cordyceps sinensis, and/or between two kind of the pharmacological active compounds themselves, and/or between two kind of the weakly polar compounds themselves, appear in the TOF-SIMS mass spectrum of Cordyceps sinensis. These “splicing” ions also can be used as the characteristic ions for the identification of Cordyceps sinensis. The study also proves that, if TOF-SIMS is used as a tool to detect, analyze, research and identify Cordyceps sinensis, the mass resolution(M/ΔM) at least needs to be over 4000, and the error of mass number (ΔM) needs to be less than several mamu. This study shows that TOF-SIMS is an effective mean to analysis, research and identify Cordyceps sinensis.

Reference

[1] Committee of National Pharmacopoeia, Pharmacopoeia of People’s Republic of China, China Medicine Science and Technology Press, Beijing, 2012: p.106.

[2] Li S P., Yang F Q., Karl W.K., Tsim, Journal of Pharmaceutical and Biomedical Analysis, 2006, 41: 1571–1584

[3] Guo F Q., Li A., Huanga L F., Liang Y Z., Chen B M., Journal of Pharmaceutical and Biomedical Analysis,2006, 40: 623–630

12:00 PM BI2-FrM-11 Compositional Gradients in Wax Coatings across Various Plant Epidermis Cell Types Captured by Time-of-Flight Secondary Ion Mass Spectrometric Imaging
Radu Racovita, John Kim, Reinhard Jetter (University of British Columbia, Canada)

The epidermis of almost all above-ground organs of land plants is covered by a cuticle, i.e. a lipid coating composed of a polyester and waxes embedded in and also lying atop of this polymer. Using wet chemistry techniques, such as solvent extraction followed by chromatography, the wax mixtures were found to differ widely between plant species, and between organs and even tissues of the same species. Indirect evidence suggested that further compositional gradients existed on the cellular scale, with significant implications for materials properties and biological function. However, solvent extraction has the inherent disadvantage of homogenizing the complex wax mixtures, thus making it impossible to map wax composition on a cellular scale. In contrast, ToF-SIMS imaging can provide sub-micron resolution, as illustrated in a previous pilot study focusing on select compounds in a single plant species (Jetter and Sodhi, 2011). Here, we now performed comprehensive ToF-SIMS analyses first of authentic standards covering the entire range of wax compounds, both in positive and negative mode. All homologs and isomers tested were found to have either unique fragments or, in some cases, isobaric ions in characteristic abundance ratios. ToF-SIMS imaging revealed micro-relief features on various surfaces of the model plant species, Arabidopsis thaliana, comparable to those previously described by SEM and interpreted as epidermal cell patterns. Three different cell types could be distinguished in total ion images, most notably trichomes (hairs), guard cells (forming pores) and pavement cells (forming relatively smooth areas). All three cell types were covered with waxes comprising similar compounds in complex mixtures, and all major compounds were identified by comparison with the spectra of authentic standards. Most importantly, the relative amounts of these compounds were found to differ between the wax surfaces of the different cell types. Thus, the previously suspected lateral gradients between epidermis cells lining the plant surface were here confirmed directly. The finding of chemical gradients on the cellular level has implications for the cell-autonomous wax formation, thus enabling further studies into the biosynthetic machinery, and for the surface properties such as super-hydrophobicity.

References:

Jetter, R.; Sodhi, R. Surf. Interface Anal.2011, 43, 326–330.

Time Period FrM Sessions | Abstract Timeline | Topic BI Sessions | Time Periods | Topics | SIMS2015 Schedule