High energy ionizing irradiation produces large amounts of low energy (<20 eV) secondary electrons (SEs). These electrons are produced via a cascade process following the ionization of a core (deeply bound) electron. Due to their low energy there is a high probability for the SEs to become trapped in antibonding orbitals, via resonant scattering, forming a temporary negative ion (TNI) resonance. If the lifetime of the TNI state is long enough, then bond rupture can occur by by a process known as dissociative electron attachment (DEA). There is vast literature on the role of TNI states and DEA in DNA related radiation chemistry.[1,2] Due to its high flux density, synchrotron radiation (SR) has often been used to induce and study radiation chemistry in numerous systems,[3] including DNA and related molecules. SR has also been used to probe the electronic structure and bonding of such molecules, primarily by probing the occupied states with X-ray photoelectron spectroscopy (XPS) and the unoccupied states with X-ray absorption (XAS) measurements. Bond overlap and localization can be revealed by XPS while XAS can determine the density of unoccupied states and the orientation of the orbitals. In this presentation we examine X-ray induced reactions of DNA adsorbed on a gold substrate when the DNA is either thiolated (tDNA) or when it is unthiolated (uDNA). By performing polarization-dependent XAS at the N K edge we determined that tDNA protrudes from the surface at ~45 degrees, in agreement with previous studies. We also found that the unthiolated molecules have a similar orientation. However, due to differences in charge transfer between the gold and the DNA in the two systems there is a higher density of unoccupied states in the N-C=N derived π* orbital for tDNA. We also found that the adsorbed tDNA has a significant higher cross section for radiation damage. The reason for this enhancement could arise from the greater probability of forming a TNI state for the tDNA due to the higher density of unoccupied π* states.

1. E. Alizadeh and L. Sanche, Chem. Rev.112, 5578 (2012).

2. R. Naaman and L. Sanche, Chem. Rev.107, 1553 (2007).

3. R. A. Rosenberg and S. P. Frigo, in Chemical Applications of Synchrotron Radiation, Part II: X-ray Applications, edited by T.K. Sham (World Scientific Publishing Co., Singapore, 2002), Vol. 12A, p. 462.

References

1 Nasse, M. J., Walsh, M. J., Mattson, E. C., Reininger, R., Kajdacsy-Balla, A., Macias, V., Bhargava, R., and Hirschmugl, C. J. (2011) Nat.Methods 8, 413-416

2 Martin, M. C., Dabat-Blondeau, C., Unger, M., Sedlmair, J., Parkinson, D. Y., Bechtel, H. A., Illman, B., Castro, J. M., Keiluweit, M., Buschke, D., Ogle, B., Nasse, M. J., and Hirschmugl, C. J. (2013) Nat.Methods 10, 861-864

Acknowledgements

This work was supported by the US NSF under awards CHE-0832298, CHE-1112433, and DMR-0619759, the Research Growth Initiative of the University of Wisconsin–Milwaukee, and is based on research conducted at the Synchrotron Radiation Center, University of Wisconsin–Madison which is supported by the University of Wisconsin– Milwaukee and University of Wisconsin–Madison.

The development of argon cluster sources has opened up new opportunities for ToF-SIMS depth profiling. These sources have enabled depth profiling of a wide range of materials that previously could not be accurately depth profiled. In addition, due to the low damage accumulation and sputtering efficiency of these sources, it is now possible to depth profile through microns of material. This in turn has opened up new opportunities for exploring the 3D chemical environments of a wide range of samples including drug eluting polymers, thick multilayer polymer films and porous tissue scaffolds. However, the ability to dig deeper into samples also results in significant challenges in 3D image reconstruction. For example, due to the fixed geometry of the analysis beam (at 45 deg from the surface normal in our instrument), sputtering away 1 micron of the surface will shift the analysis position by 1 micron. This means that if one were to depth profile 50 microns into a surface, the final image would be shifted by 50 microns. Traditional image registrations methods can be used to accommodate for these shifts, however when digging to depths larger than 10 microns, this requires significantly increasing the initial image size in order to end up with a usable image stack after the image shifting and cropping.

In this presentation we will summarize methods we have been developing to reconstruct deep depth profiles including adjusting the sample height during data acquisition and post acquisition image shifting. We will also show results from a new 3D image overlay tool that enables localization of different chemical environments in 3D and that can show areas of overlap between selected peak area images. These methods and tools will be demonstrated on data from control samples made from polymer beads on silicon and from data taken from polymer tissue scaffolds.

The developments in pharmaceutical delivery systems such as injectable drug eluting microparticles [1], topically applied medicines [2] and wound dressings [3] can be utilised in areas such as the treatment of HIV, basal cell carcinoma and microbial infections respectively. In this study, the characterisation of such systems has been performed using time of flight secondary ion mass spectrometry (ToF-SIMS), x-ray photoelectron spectroscopy (XPS) and atomic force microscopy (AFM).

Injectable controlled release formulations were produced by spray drying two biocompatible polymers, poly(lactic-co-glycolic acid) (PLGA) and polyvinylpyrrolidone (PVP). The samples were analysed using a range of techniques including ToF-SIMS, XPS and AFM showing that the samples were hollow microparticles with a surface PLGA rich phase and an underlying PVP phase [1]. Additionally, more complex ternary systems incorporating PLGA, PVP and a poorly soluble investigational drug compound were also analysed. These studies highlighted the influence of sample processing parameters and drug concentration upon factors such as surface composition which is influential in the drug release properties of the systems.

The permeation of an antibacterial drug, chlorhexidine, into skin tissue has been illustrated using ToF-SIMS chemical imaging of cross-sectioned treated skin samples [2]. This methodology has been further applied to investigate the topical delivery of imiquimod, a drug used in the treatment of basal cell carcinoma. This work demonstrates the ability of the ToF-SIMS technique to correlate chemical species specific to the drug with physiological features within tissue cross-sections. Further application of ToF-SIMS chemical mapping has also been used to successfully differentiate chemically dissimilar regions of anti-microbial films which could be developed as wound dressing materials. Observations made for these materials using a combination of ToF-SIMS and AFM analysis revealed the distribution of the active agents upon the surface which would be relevant to the the anti-microbial performance.

[1] Meeus, Scurr, Amssoms, Davies, Roberts, and Van den Mooter (2014) Molecular Pharmaceutics, 10 (8)

[2] Judd, Scurr, Heylings, Wan, and Moss (2013) Pharmaceutical Research, 30 (7)

[3] Liakos, Rizzello, Scurr, Pompa, Bayer and Athanassiou (2014) International Journal of Pharmacy, 463 (2)

**Sandia National Laboratories is a multi-program laboratory managed and operated by Sandia Corporation, a wholly owned subsidiary of Lockheed Martin Corporation, for the U.S. Department of Energy’s National Nuclear Security Administration under contract DE-AC04-94AL85000.

Mammalian cell analysis is of significant importance in providing detailed insights into biological system activities. Due to the complexity and heterogeneity of mammalian cell behavior and the technical challenge of spatially mapping chemical components in a hydrated environment, correlated chemical imaging from multiplexed measurement platforms is needed. Fluorescence structured illumination microscope (SIM), with super high resolution and visualization of proteins and sub-cellular structures in 3-D, provides more detailed information in cell imaging. Time-of-flight secondary ion mass spectrometry (ToF-SIMS) is a unique surface-sensitive analytical tool that provides molecular information and chemical mapping with a sub-micron lateral resolution. However, the understanding of how the spatial heterogeneity and structural difference affect the mammalian cell activities in an unperturbed, hydrated state by ToF-SIMS is severely limited due to the challenge to detect liquids with high volatility under high vacuum environment using surface sensitive technique like ToF-SIMS.

We recently developed a novel microfluidic reactor for C10 mouse lung epithelial cell growth for SIM imaging and direct probing of hydrated cell in vacuum using ToF-SIMS. C10 cells were inoculated into the microchannel, incubated at 37 ^oC for 24 hr., fed with 5 nM quantum dots, and then fixed with 4% paraformaldehyde before SIM imaging. In subsequent ToF-SIMS analysis, an aperture of 2 µm in diameter was drilled through SiN membrane to form the detection window to image biological surfaces directly; and surface tension is used for holding the liquid within the aperture.

SIM images show that C10 cells are successfully cultured on the SiN membrane, and quantum dots are uptaken by cells and dispersed in the cytoplasm. The ToF-SIMS m/z spectra showing characteristic fragments of dried cell sample, hydrated cells, and uninoculated medium in the microreactor will be presented. Moreover, 2D images of representative cell fragments and quantum dots ion mapping will be discussed. In addition, depth profiling will be used to provide time- and space-resolved imaging of the cells inside the microchannel. Furthermore, principal component analysis is conducted to evaluate the intrinsic similarities and discriminations among samples. Our results demonstrate feasibility for in situ imaging of cells in the hydrated state using ToF-SIMS for the first time. Correlative imaging using SIM and ToF-SIMS provides information across different space scales for investigating cell dynamics. This novel approach has great potential for studying intracellular processes in the future.