Exploiting the Semidestructive Nature of Gas Cluster Ion Beam Time-of-Flight Secondary Ion Mass Spectrometry Imaging for Simultaneous Localization and Confident Lipid Annotations.

Lipids have been recognized as key players in cell signaling and disease. Information on their location and distribution within a biological system, under varying conditions, is necessary to understand the contributions of different lipid species to an altered phenotype. Imaging mass spectrometry techniques, such as time-of-flight secondary ion mass spectrometry (ToF-SIMS) and matrix-assisted laser desorption/ionization (MALDI), are capable of revealing global lipid distributions in tissues in an untargeted fashion. However, to confidently identify the species present in a sample, orthogonal analyses like tandem MS (MS/MS) are often required. This can be accomplished by bulk sample analysis with liquid chromatography (LC)-MS/MS, which can provide confident lipid identifications, at the expense of losing location-specific information. Here, using planarian flatworms as a model system, we demonstrate that imaging gas cluster ion beam (GCIB)-ToF-SIMS has the unique capability to simultaneously detect, identify, and image lipid species with subcellular resolution in tissue sections. The parallel detection of both, intact lipids and their respective fragments, allows for unique identification of some species without the need of performing an additional orthogonal MS/MS analysis. This was accomplished by correlating intact lipid and associated fragment SIMS images. The lipid assignments, respective fragment identities, and locations gathered from ToF-SIMS data were confirmed via LC-MS/MS on lipid extracts and ultrahigh mass resolution MALDI-MS imaging. Together, these data show that the semidestructive nature of ToF-SIMS can be utilized advantageously to enable both confident molecular annotations and to determine the locations of species within a biological sample.

An unsupervised MVA method to compare specific regions in human breast tumor tissue samples using ToF-SIMS.

Imaging time-of-flight secondary ion mass spectrometry (ToF-SIMS) and principal component analysis (PCA) were used to investigate two sets of pre- and post-chemotherapy human breast tumor tissue sections to characterize lipids associated with tumor metabolic flexibility and response to treatment. The micron spatial resolution imaging capability of ToF-SIMS provides a powerful approach to attain spatially-resolved molecular and cellular data from cancerous tissues not available with conventional imaging techniques. Three ca. 1 mm(2) areas per tissue section were analyzed by stitching together 200 µm × 200 µm raster area scans. A method to isolate and analyze specific tissue regions of interest by utilizing PCA of ToF-SIMS images is presented, which allowed separation of cellularized areas from stromal areas. These PCA-generated regions of interest were then used as masks to reconstruct representative spectra from specifically stromal or cellular regions. The advantage of this unsupervised selection method is a reduction in scatter in the spectral PCA results when compared to analyzing all tissue areas or analyzing areas highlighted by a pathologist. Utilizing this method, stromal and cellular regions of breast tissue biopsies taken pre- versus post-chemotherapy demonstrate chemical separation using negatively-charged ion species. In this sample set, the cellular regions were predominantly all cancer cells. Fatty acids (i.e. palmitic, oleic, and stearic), monoacylglycerols, diacylglycerols and vitamin E profiles were distinctively different between the pre- and post-therapy tissues. These results validate a new unsupervised method to isolate and interpret biochemically distinct regions in cancer tissues using imaging ToF-SIMS data. In addition, the method developed here can provide a framework to compare a variety of tissue samples using imaging ToF-SIMS, especially where there is section-to-section variability that makes it difficult to use a serial hematoxylin and eosin (H&E) stained section to direct the SIMS analysis.

Hollow fiber membrane modification with functional zwitterionic macromolecules for improved thromboresistance in artificial lungs.

Respiratory assist devices seek optimized performance in terms of gas transfer efficiency and thromboresistance to minimize device size and reduce complications associated with inadequate blood biocompatibility. The exchange of gas with blood occurs at the surface of the hollow fiber membranes (HFMs) used in these devices. In this study, three zwitterionic macromolecules were attached to HFM surfaces to putatively improve thromboresistance: (1) carboxyl-functionalized zwitterionic phosphorylcholine (PC) and (2) sulfobetaine (SB) macromolecules (mPC or mSB-COOH) prepared by a simple thiol-ene radical polymerization and (3) a low-molecular weight sulfobetaine (SB)-co-methacrylic acid (MA) block copolymer (SBMAb-COOH) prepared by reversible addition-fragmentation chain transfer (RAFT) polymerization. Each macromolecule type was covalently immobilized on an aminated commercial HFM (Celg-A) by a condensation reaction, and HFM surface composition changes were analyzed by X-ray photoelectron spectroscopy. Thrombotic deposition on the HFMs was investigated after contact with ovine blood in vitro. The removal of CO2 by the HFMs was also evaluated using a model respiratory assistance device. The HFMs conjugated with zwitterionic macromolecules (Celg-mPC, Celg-mSB, and Celg-SBMAb) showed expected increases in phosphorus or sulfur surface content. Celg-mPC and Celg-SBMAb experienced rates of platelet deposition significantly lower than those of unmodified (Celg-A, >95% reduction) and heparin-coated (>88% reduction) control HFMs. Smaller reductions were seen with Celg-mSB. The CO2 removal rate for Celg-SBMAb HFMs remained comparable to that of Celg-A. In contrast, the rate of removal of CO2 for heparin-coated HFMs was significantly reduced. The results demonstrate a promising approach to modifying HFMs using zwitterionic macromolecules for artificial lung devices with improved thromboresistance without degradation of gas transfer.

Physico-chemical foundations underpinning microarray and next-generation sequencing experiments.

Hybridization of nucleic acids on solid surfaces is a key process involved in high-throughput technologies such as microarrays and, in some cases, next-generation sequencing (NGS). A physical understanding of the hybridization process helps to determine the accuracy of these technologies. The goal of a widespread research program is to develop reliable transformations between the raw signals reported by the technologies and individual molecular concentrations from an ensemble of nucleic acids. This research has inputs from many areas, from bioinformatics and biostatistics, to theoretical and experimental biochemistry and biophysics, to computer simulations. A group of leading researchers met in Ploen Germany in 2011 to discuss present knowledge and limitations of our physico-chemical understanding of high-throughput nucleic acid technologies. This meeting inspired us to write this summary, which provides an overview of the state-of-the-art approaches based on physico-chemical foundation to modeling of the nucleic acids hybridization process on solid surfaces. In addition, practical application of current knowledge is emphasized.

Surface modification of a biodegradable magnesium alloy with phosphorylcholine (PC) and sulfobetaine (SB) functional macromolecules for reduced thrombogenicity and acute corrosion resistance.

Siloxane functionalized phosphorylcholine (PC) or sulfobetaine (SB) macromolecules (PCSSi or SBSSi) were synthesized to act as surface modifying agents for degradable metallic surfaces to improve acute blood compatibility and slow initial corrosion rates. The macromolecules were synthesized using a thiol-ene radical photopolymerization technique and then utilized to modify magnesium (Mg) alloy (AZ31) surfaces via an anhydrous phase deposition of the silane functional groups. X-ray photoelectron spectroscopy surface analysis results indicated successful surface modification based on increased nitrogen and phosphorus or sulfur composition on the modified surfaces relative to unmodified AZ31. In vitro acute thrombogenicity assessment after ovine blood contact with the PCSSi and SBSSi modified surfaces showed a significant decrease in platelet deposition and bulk phase platelet activation compared with the control alloy surfaces. Potentiodynamic polarization and electrochemical impedance spectroscopy data obtained from electrochemical corrosion testing demonstrated increased corrosion resistance for PCSSi- and SBSSi-modified AZ31 versus unmodified surfaces. The developed coating technique using PCSSi or SBSSi showed promise in acutely reducing both the corrosion and thrombotic processes, which would be attractive for application to blood contacting devices, such as vascular stents, made from degradable Mg alloys.

Back to the basics of time-of-flight secondary ion mass spectrometry data analysis of bio-related samples. II. Data processing and display.

This is the second half of a two-part Tutorial on the basics of the time-of-flight secondary ion mass spectrometry (ToF-SIMS) analysis of bio-related samples. Part I of this Tutorial series covers planning for a ToF-SIMS experiment, preparing and shipping samples, and collecting ToF-SIMS data. This Tutorial aims at helping the ToF-SIMS user to process, display, and interpret ToF-SIMS data. ToF-SIMS provides detailed chemical information about surfaces but comes with a steep learning. The purpose of this Tutorial is to provide the reader with a solid foundation in the ToF-SIMS data analysis.

Back to the basics of time-of-flight secondary ion mass spectrometry of bio-related samples. I. Instrumentation and data collection.

Time-of-flight secondary ion mass spectrometry (ToF-SIMS) is used widely throughout industrial and academic research due to the high information content of the chemically specific data it produces. Modern ToF-SIMS instruments can generate high mass resolution data that can be displayed as spectra and images (2D and 3D). This enables determining the distribution of molecules across and into a surface and provides access to information not obtainable from other methods. With this detailed chemical information comes a steep learning curve in how to properly acquire and interpret the data. This Tutorial is aimed at helping ToF-SIMS users to plan for and collect ToF-SIMS data. The second Tutorial in this series will cover how to process, display, and interpret ToF-SIMS data.

Quantitative evaluation of perfluorinated alkanethiol molecular order on gold surfaces.

Self-assembled monolayers (SAMs) of perfluoroalkanethiols [CF3(CF2)xCH2CH2SH (x = 3, 5, 7, and 9)] on gold were characterized by x-ray photoelectron spectroscopy (XPS), near edge x-ray absorption fine structure (NEXAFS), and static time-of-flight secondary ion mass spectrometry (ToF-SIMS). Perfluoroalkanethiols of several chain lengths were synthesized using a known hydride reduction method for transforming commercially available perfluoroalkyliodides to corresponding perfluoroalkanethiols. This strategy provides improved product yields compared to other known routes based on hydrolysis from the common thioacetyl perfluoroalkyl intermediate. Angle-dependent XPS analysis revealed that CF3(CF2)xCH2CH2SH (x = 5, 7, and 9; F6, F8, and F10, respectively) SAMs on gold exhibited significant enrichment of the terminal CF3 group at the outer monolayer surface with the sulfur present as a metal-bound thiolate located at the monolayer-gold interface. XPS of the CF3(CF2)3CH2CH2SH (F4) monolayer revealed a thin film with a significant (>50%) amount of hydrocarbon contamination consistent with poorly organized monolayers, while the longest thiol (F10) showed XPS signals attributed to substantial ordering and anisotropy. ToF-SIMS spectra from all four SAMs contained molecular ions representative of the particular perfluorinated thiol used to prepare the monolayer. NEXAFS methods were used to determine degrees of ordering and average tilt for molecules comprising monolayers. The SAMs prepared from the longest (F10) thiols exhibited the highest degree of ordering with the molecular axis nearly perpendicular to the gold surface. The degree of ordering decreased significantly with decreasing length of the perfluorocarbon tail.

High-resolution epifluorescence and time-of-flight secondary ion mass spectrometry chemical imaging comparisons of single DNA microarray spots.

DNA microarray assay performance is commonly compromised by spot-spot probe and signal variations as well as heterogeneity within printed microspots. Accurate metrics for captured DNA target signal rely upon uniform spot distribution of both probe and target DNA to yield reliable hybridized signal. While often presumed, this is neither easily achieved nor often proven experimentally. High-resolution imaging techniques were used to determine spot heterogeneity in identical DNA array microspots comprising varied ratios of unlabeled and dye-labeled DNA probes contact-printed onto commercial arraying surfaces. Epifluorescence imaging data for individual array microspots were correlated with time-of-flight secondary ion mass spectrometry (TOF-SIMS) chemical state imaging of the same spots. Epifluorescence imaging intensity distinguished varying DNA density distributed both within a given spot and from spot to spot. TOF-SIMS chemical analysis confirmed these heterogeneous printed DNA distributions by tracking bound Cy3 dye, DNA base, and phosphate specific ion fragments often correlating to fluorescence patterns within identical spots. TOF-SIMS ion fragments originating from probe DNA and Cy3 dye are enriched in microspot centers, correlating with high fluorescence intensity regions. Both TOF-SIMS and epifluorescence support Marangoni flow effects on spot drying, with high-density DNA-Cy3 located in spot centers and nonhomogeneous DNA distribution within printed spots. Microspot image dimensional analysis results for DNA droplet spreading show differing DNA densities across printed spots. The study directly supports different DNA probe chemical and spatial microenvironments within spots that yield spot-spot signal variations known to affect DNA target hybridization efficiencies and kinetics. These variations critically affect probe-target duplex formation and DNA array signal generation.

Probing the orientation of electrostatically immobilized Protein G B1 by time-of-flight secondary ion spectrometry, sum frequency generation, and near-edge X-ray adsorption fine structure spectroscopy.

To fully develop techniques that provide an accurate description of protein structure at a surface, we must start with a relatively simple model system before moving to increasingly complex systems. In this study, X-ray photoelectron spectroscopy (XPS), sum frequency generation spectroscopy (SFG), near-edge X-ray adsorption fine structure (NEXAFS) spectroscopy, and time-of-flight secondary ion mass spectrometry (ToF-SIMS) were used to probe the orientation of Protein G B1 (6 kDa) immobilized onto both amine (NH(3)(+)) and carboxyl (COO(-)) functionalized gold. Previously, we have shown that we could successfully control orientation of a similar Protein G fragment via a cysteine-maleimide bond. In this investigation, to induce opposite end-on orientations, a charge distribution was created within the Protein G B1 fragment by first substituting specific negatively charged amino acids with neutral amino acids and then immobilizing the protein onto two oppositely charged self-assembled monolayer (SAM) surfaces (NH(3)(+) and COO(-)). Protein coverage, on both surfaces, was monitored by the change in the atomic % N, as determined by XPS. Spectral features within the SFG spectra, acquired for the protein adsorbed onto a NH(3)(+)-SAM surface, indicates that this electrostatic interaction does induce the protein to form an oriented monolayer on the SAM substrate. This corresponded to the polarization dependence of the spectral feature related to the NEXAFS N(1s)-to-π* transition of the ß-sheet peptide bonds within the protein layer. ToF-SIMS data demonstrated a clear separation between the two samples based on the intensity differences of secondary ions stemming from amino acids located asymmetrically within Protein G B1 (methionine: 62 and 105 m/z; tyrosine: 107 and 137 m/z; leucine: 86 m/z). For a more quantitative examination of orientation, we developed a ratio comparing the sum of the intensities of secondary-ions stemming from the amino acid residues at either end of the protein. The 2-fold increase in this ratio, observed between the protein covered NH(3)(+) and COO(-) SAMs, indicates opposite orientations of the Protein G B1 fragment on the two different surfaces.

Chemical patterning of ultrathin polymer films by direct-write multiphoton lithography.

We applied 2-photon laser ablation to write subdiffraction nanoscale chemical patterns into ultrathin polymer films under ambient conditions. Poly(ethylene glycol) methacrylate brush layers were prepared on quartz substrates via surface-initiated atom-transfer radical polymerization and ablated to expose the underlying substrate using the nonlinear 2-photon absorbance of a frequency-doubled Ti:sapphire femtosecond laser. Single-shot ablation thresholds of polymer films were ~1.5 times smaller than that of a quartz substrate, which allowed patterning of nanoscale features without damage to the underlying substrate. At a 1/e(2) laser spot diameter of 0.86 µm, the features of exposed substrate approached ~80 nm, well below the diffraction limit for 400 nm light. Ablated features were chemically distinct and amenable to chemical modification.

Surfactants influence polymer nanoparticle fate within the brain.

Drug delivery to the brain is limited by poor penetration of pharmaceutical agents across the blood-brain barrier (BBB), within the brain parenchyma, and into specific cells of interest. Nanotechnology can overcome these barriers, but its ability to do so is dependent on nanoparticle physicochemical properties including surface chemistry. Surface chemistry can be determined by a number of factors, including by the presence of stabilizing surfactant molecules introduced during the formulation process. Nanoparticles coated with poloxamer 188 (F68), poloxamer 407 (F127), and polysorbate 80 (P80) have demonstrated uptake in BBB endothelial cells and enhanced accumulation within the brain. However, the impact of surfactants on nanoparticle fate, and specifically on brain extracellular diffusion or intracellular targeting, must be better understood to design nanotherapeutics to efficiently overcome drug delivery barriers in the brain. Here, we evaluated the effect of the biocompatible and commonly used surfactants cholic acid (CHA), F68, F127, P80, and poly (vinyl alcohol) (PVA) on poly (lactic-co-glycolic acid)-poly (ethylene glycol) (PLGA-PEG) nanoparticle transport to and within the brain. The inclusion of these surfactant molecules decreases diffusive ability through brain tissue, reflecting the surfactant's role in encouraging cellular interaction at short length and time scales. After in vivo administration, PLGA-PEG/P80 nanoparticles demonstrated enhanced penetration across the BBB and subsequent internalization within neurons and microglia. Surfactants incorporated into the formulation of PLGA-PEG nanoparticles therefore represent an important design parameter for controlling nanoparticle fate within the brain.

Structure and order of phosphonic acid-based self-assembled monolayers on Si(100).

Organophosphonic acid self-assembled monolayers (SAMs) on oxide surfaces have recently seen increased use in electrical and biological sensor applications. The reliability and reproducibility of these sensors require good molecular organization in these SAMs. In this regard, packing, order, and alignment in the SAMs is important, as it influences the electron transport measurements. In this study, we examine the order of hydroxyl- and methyl-terminated phosphonate films deposited onto silicon oxide surfaces by the tethering by aggregation and growth method using complementary, state-of-art surface characterization tools. Near-edge X-ray absorption fine structure (NEXAFS) spectroscopy and in situ sum frequency generation (SFG) spectroscopy are used to study the order of the phosphonate SAMs in vacuum and under aqueous conditions, respectively. X-ray photoelectron spectroscopy and time-of-flight secondary ion mass spectrometry results show that these samples form chemically intact monolayer phosphonate films. NEXAFS and SFG spectroscopy showed that molecular order exists in the octadecylphosphonic acid and 11-hydroxyundecylphosphonic acid SAMs. The chain tilt angles in these SAMs were approximately 37° and 45°, respectively.

XPS and ToF-SIMS investigation of alpha-helical and beta-strand peptide adsorption onto SAMs.

14-mer alpha-helix and 15-mer beta-strand oligopeptides composed of leucine (L) and lysine (K) were used to investigate peptide adsorption and orientation onto well-defined methyl and carboxylic acid-terminated self-assembled monolayer (SAM) surfaces with X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (ToF-SIMS). XPS showed that both peptides reached monolayer thickness on both SAMs, but significantly higher solution concentrations were required to reach this coverage on the methyl SAMs. This shows that the peptides adsorb more strongly onto the carboxyl-terminated SAMs. The excess oxygen detected by XPS and the H(3)O(+) signal detected by ToF-SIMS for the SAMs with adsorbed peptides indicated that water molecules are associated with the adsorbed peptides, even under ultrahigh-vacuum conditions. Changes in the number of L and K fragments detected by ToF-SIMS indicate that the beta-strand oriented differently on the two SAMs. The L side chains were preferentially associated with the methyl-terminated SAM, and the K side chains were preferentially associated with the carboxyl SAM. In contrast, little change in the ToF-SIMS K/L ratio was observed for the alpha-helix peptide absorbed on the two SAMs, indicating that ToF-SIMS was not as sensitive to the orientation of the alpha-helix peptide.

Probing the orientation and conformation of alpha-helix and beta-strand model peptides on self-assembled monolayers using sum frequency generation and NEXAFS spectroscopy.

The structure and orientation of amphiphilic alpha-helix and beta-strand model peptide films on self-assembled monolayers (SAMs) have been studied with sum frequency generation (SFG) vibrational spectroscopy and near-edge X-ray absorption fine structure (NEXAFS) spectroscopy. The alpha-helix peptide is a 14-mer, and the beta-strand is a 15-mer of hydrophilic lysine and hydrophobic leucine residues with hydrophobic periodicities of 3.5 and 2, respectively. These periodicities result in the leucine side chains located on one side of the peptides and the lysine side chains on the other side. The SAMs were prepared from the assembly of either carboxylic acid- or methyl-terminated alkyl thiols onto gold surfaces. For SFG studies, the deuterated analog of the methyl SAM was used. SFG vibrational spectra in the C-H region of air-dried peptides films on both SAMs exhibit strong peaks near 2965, 2940, and 2875 cm(-1) related to ordered leucine side chains. The orientation of the leucine side chains was determined from the phase of these features relative to the nonresonant gold background. The relative phase for both the alpha-helix and beta-strand peptides showed that the leucine side chains were oriented away from the carboxylic acid SAM surface and oriented toward the methyl SAM surface. Amide I peaks observed near 1656 cm(-1) for the alpha-helix peptide confirm that the secondary structure is preserved on both SAMs. Strong linear dichroism related to the amide pi* orbital at 400.8 eV was observed in the nitrogen K-edge NEXAFS spectra for the adsorbed beta-strand peptides, suggesting that the peptide backbones are oriented parallel to the SAM surface with the side chains pointing toward or away from the interface. For the alpha-helix the dichroism of the amide pi* is significantly weaker, probably because of the broad distribution of amide bond orientations in the alpha-helix secondary structure.

Probing the orientation of surface-immobilized protein G B1 using ToF-SIMS, sum frequency generation, and NEXAFS spectroscopy.

The ability to orient active proteins on surfaces is a critical aspect of many medical technologies. An important related challenge is characterizing protein orientation in these surface films. This study uses a combination of time-of-flight secondary ion mass spectrometry (ToF-SIMS), sum frequency generation (SFG) vibrational spectroscopy, and near-edge X-ray absorption fine structure (NEXAFS) spectroscopy to characterize the orientation of surface-immobilized Protein G B1, a rigid 6 kDa domain that binds the Fc fragment of IgG. Two Protein G B1 variants with a single cysteine introduced at either end were immobilized via the cysteine thiol onto maleimide-oligo(ethylene glycol)-functionalized gold and bare gold substrates. X-ray photoelectron spectroscopy was used to measure the amount of immobilized protein, and ToF-SIMS was used to measure the amino acid composition of the exposed surface of the protein films and to confirm covalent attachment of protein thiol to the substrate maleimide groups. SFG and NEXAFS were used to characterize the ordering and orientation of peptide or side chain bonds. On both substrates and for both cysteine positions, ToF-SIMS data showed enrichment of mass peaks from amino acids located at the end of the protein opposite to the cysteine surface position as compared with nonspecifically immobilized protein, indicating end-on protein orientations. Orientation on the maleimide substrate was enhanced by increasing pH (7.0-9.5) and salt concentration (0-1.5 M NaCl). SFG spectral peaks characteristic of ordered α-helix and ß-sheet elements were observed for both variants but not for cysteine-free wild type protein on the maleimide surface. The phase of the α-helix and ß-sheet peaks indicated a predominantly upright orientation for both variants, consistent with an end-on protein binding configuration. Polarization dependence of the NEXAFS signal from the N 1s to π* transition of ß-sheet peptide bonds also indicated protein ordering, with an estimated tilt angle of inner ß-strands of 40-50° for both variants (one variant more tilted than the other), consistent with SFG results. The combined results demonstrate the power of using complementary techniques to probe protein orientation on surfaces.

Amine Terminated SAMs: Investigating Why Oxygen is Present in these Films.

Self-assembled monolayers (SAMs) on gold prepared from amine-terminated alkanethiols have long been employed as model positively charged surfaces. Yet in previous studies significant amounts of unexpected oxygen containing species are always detected in amine terminated SAMs. Thus, the goal of this investigation was to determine the source of these oxygen species and minimize their presence in the SAM. The surface composition, structure, and order of amine-terminated SAMs on Au were characterized by X-ray photoelectron spectroscopy (XPS), time-of-flight secondary ion mass spectroscopy (ToF-SIMS), sum frequency generation (SFG) and near edge X-ray absorption fine structure (NEXAFS) spectroscopy. XPS determined compositions of amine-terminated SAMs in the current study exhibited oxygen concentrations of 2.4 ± 0.4 atomic %, a substantially lower amount of oxygen than reported in previously published studies. High-resolution XPS results from the S(2p), C(1s) and N(1s) regions did not detect any oxidized species. Angle-resolved XPS indicated that the small amount of oxygen detected was located at or near the amine head group. Small amounts of oxidized nitrogen, carbon and sulfur secondary ions, as well as ions attributed to water, were detected in the ToF-SIMS data due to the higher sensitivity of ToF-SIMS. The lack of N-O, S-O, and C-O stretches in the SFG spectra are consistent with the XPS and ToF-SIMS results and together show that oxidation of the amine-terminated thiols alone can only account for, at most, a small fraction of the oxygen detected by XPS. Both the SFG and angle-dependent NEXAFS indicated the presence of gauche defects in the amine SAMs. However, the SFG spectral features near 2865 cm(-1), assigned to the stretch of the methylene group next to the terminal amine unit, demonstrate the SAM is reasonably ordered. The SFG results also show another broad feature near 3200 cm(-1) related to hydrogen-bonded water. From this multi-technique investigation it is clear that the majority of the oxygen detected within these amine-terminated SAMs arises from the presence of oxygen containing adsorbates such as tightly bound water.

Time-of-flight secondary ion mass spectrometry three-dimensional imaging of surface modifications in poly(caprolactone) scaffold pores.

Scaffolds composed of synthetic polymers such as poly(caprolactone) (PCL) are widely used for the support and repair of tissues in biomedicine. Pores are common features in scaffolds as they facilitate cell penetration. Various surface modifications can be performed to promote key biological responses to these scaffolds. However, verifying the chemistry of these materials post surface modification is problematic due to the combination of three-dimensional (3D) topography and surface sensitivity. Time-of-flight secondary ion mass spectrometry (ToF-SIMS) is commonly used to correlate surface chemistry with cell response. In this study, 3D imaging mass spectrometry analysis of surface modified synthetic polymer scaffolds is demonstrated using PCL porous scaffold, a pore filling polymer sample preparation, and 3D imaging ToF-SIMS. We apply a simple sample preparation procedure, filling the scaffold pores with a poly(vinyl alcohol)/glycerol mixture to remove topographic influence on image quality. This filling method allows the scaffold (PCL) and filler secondary ions to be reconstructed into a 3D chemical image of the pore. Furthermore, we show that surface modifications in the pores of synthetic polymer scaffolds can be mapped in 3D. Imaging of "dry" and "wet" surface modifications is demonstrated as well as a comparison of surface modifications with relatively strong ToF-SIMS peaks (fluorocarbon films [FC]) and to more biologically relevant surface modification of a protein (bovine serum albumin [BSA]). We demonstrate that surface modifications can be imaged in 3D showing that characteristic secondary ions associated with FC and BSA are associated with C3 F8 plasma treatment and BSA, respectively within the pore.

"Chip-on-a-Transwell" Devices for User-Friendly Control of the Microenvironment of Cultured Cells.

We present microfabricated poly(dimethylsiloxane) templates assembled atop Transwell membranes ("Chip-on-a-Transwell") for focal delivery of soluble reagents to cells. This modified Transwell operates based on area-selective diffusion of soluble species to the basal side of cells. The device was capable of addressing small groups of cells, with a maximum spatial resolution of 20 µm, while sustaining focal signal for up to 48 hours. Furthermore, we developed a fluorosilane-based cell micropatterning method that complements the Transwell system, creating well-defined arrays of muscle cell cultures and enabling automated microscopy and image analysis protocols. The micropatterning method is based on plasma-etching hydrophilic cell-adhesive tracks against a hydrophobic fluorosilane cell-repellent background. We applied the platform as a model of the neuromuscular junction (NMJ), in which we effectively substituted motor neurons with apertures delivering agrin stimuli to subcellular regions of micropatterned myotubes. With the Transwell system, we demonstrated that focal agrin application to subcellular sections of myotubes induced localized acetylcholine receptor microclustering, mimicking the in vivo NMJ. We also demonstrated spatially selective transfection of a monolayer of cells using Lipofectamine carrying the plasmid DNA for pMax GFP, which is ubiquitously expressed in transfected cells. In summary, we present a user-friendly cell culture tool that provides spatiotemporal control of the fluidic and substrate microenvironments with potential applications to developmental biology and tissue engineering.

Insights into the histology of planarian flatworm Phagocata gracilis based on location specific, intact lipid information provided by GCIB-ToF-SIMS imaging.

Planarian flatworms are known as the masters of regeneration, re-growing an entire organism from as little as 1/279th part of their body. While the proteomics of these processes has been studied extensively, the planarian lipodome remains relatively unknown. In this study we investigate the lipid profile of planarian tissue sections with imaging Time-of-Flight - Secondary-Ion-Mass-Spectrometry (ToF-SIMS). ToF-SIMS is a label-free technique capable of gathering intact, location specific lipid information on a cellular scale. Lipid identities are confirmed using LC-MS/MS. Our data shows that different organ structures within planarians have unique lipid profiles. The 22-carbon atom poly unsaturated fatty acids (PUFAs) which occur in unusually high amounts in planarians are found to be mainly located in the testes. Additionally, we observe that planarians contain various odd numbered fatty acid species, that are usually found in bacteria, localized in the reproductive and ectodermal structures of the planarian. An abundance of poorly understood ether fatty acids and ether lipids were found in unique areas in planarians as well as a new, yet unidentified class of potential lipids in planarian intestines. Identifying the location of these lipids in the planarian body provides insights into their bodily functions and, in combination with knowledge about their diet and their genome, enables drawing conclusions about planarian fatty acid processing.