Atomic Force Microscope Controlled Topographical Imaging and

Dec 30, 2013 - The basic instrumental setup and the general operation of the ... Amanda Rae Buchberger , Kellen DeLaney , Jillian Johnson , and Lingju...
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Atomic Force Microscope Controlled Topographical Imaging and Proximal Probe Thermal Desorption/Ionization Mass Spectrometry Imaging Olga S. Ovchinnikova,† Kevin Kjoller,‡ Gregory B. Hurst,† Dale A. Pelletier,§ and Gary J. Van Berkel†,* †

Organic and Biological Mass Spectrometry Group, Chemical Sciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831-6131 ‡ Anasys Instruments, Santa Barbara, California 93101 § Biological and Nanoscale Systems Group, Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831-6036 S Supporting Information *

ABSTRACT: This paper reports on the development of a hybrid atmospheric pressure atomic force microscopy/mass spectrometry imaging system utilizing nanothermal analysis probes for thermal desorption surface sampling with subsequent atmospheric pressure chemical ionization and mass analysis. The basic instrumental setup and the general operation of the system were discussed, and optimized performance metrics were presented. The ability to correlate topographic images of a surface with atomic force microscopy and a mass spectral chemical image of the same surface, utilizing the same probe without moving the sample from the system, was demonstrated. Co-registered mass spectral chemical images and atomic force microscopy topographical images were obtained from inked patterns on paper as well as from a living bacterial colony on an agar gel. Spatial resolution of the topography images based on pixel size (0.2 μm × 0.8 μm) was better than the resolution of the mass spectral images (2.5 μm × 2.0 μm), which were limited by current mass spectral data acquisition rate and system detection levels.

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combination of surface sampling process, ionization method, and mass analyzer used will determine the surface types and chemical species that can be analyzed and with what specific limits of detection and degree of chemical specificity. The coregistration or correlation of topography, physical and detailed chemical images of a surface, such as might be obtained from a hybrid AFM and MSI instrument platform, can be expected to provide a detailed understanding of the surface of interest.12,13 The combination of vacuum-based AFM and the elemental MSI technique, secondary ion mass spectrometry (SIMS), on one instrumental platform has been realized.14−16 Both elemental images (about 50 nm spatial resolution) and the corresponding topographical images of the surface are obtained from the sample within the same instrument platform. Incorporating AFM into a SIMS instrument provided the capability to produce topographically corrected 3D chemical images as demonstrated with Au/Cu and polystyrene/poly (methyl methacrylate) samples.14,15 For MS, surface sampling under ambient conditions, rather than at vacuum, eliminates the constraints on the size of the

tomic force microscopy (AFM) is one of the foremost tools for imaging, measuring, and manipulating matter at the nanoscale.1 With AFM, by measuring the deflection of a nanometer-sized mechanical probe tip connected to the free end of a microcantilever contacting the surface of a sample, one can deduce topography and viscoelastic properties of a surface with nanometer resolution. In addition, coating of probe tips with a conductive or a magnetic material has enabled nanoscale mapping of electric and magnetic properties, respectively, of surfaces.2 While topography and multiple physical property images of a surface can be obtained and correlated from a single sample, chemical identification is beyond the current capability of AFM.3 This has led to the use of a variety of optical spectroscopy techniques in combination with AFM to create hybrid tools that can obtain spatially resolved chemical signatures from a surface.4 Mass spectrometry imaging (MSI), with its ability to perform spatially resolved selective detection and identification within complex matrices, through either accurate mass measurements or through the use of tandem mass spectrometry, if combined with AFM, would provide complementary and more specific chemical information than optical spectroscopy.5−11 Mass spectrometry imaging can in theory be used to visualize the spatial distribution of any molecular or elemental species that can be sampled from a surface and converted into a gas-phase ion. The particular © 2013 American Chemical Society

Received: August 21, 2013 Accepted: December 23, 2013 Published: December 30, 2013 1083

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image pixel size of 2.5 μm × 2.0 μm, which was limited by mass spectral data acquisition rates and system detection levels.

surfaces, the volatility of the material and provides the possibility to analyze living and insulating materials. With the aim of better characterizing a wide range of material interfaces or surfaces under real world conditions, hybrid AFM/MS systems in which the AFM measurements and MS surface sampling process take place under atmospheric pressure (AP) conditions have been investigated by a few groups. In one example, Zenobi’s group combined scanning near-field optical microscopy (SNOM)-based laser desorption with an ion trap/ time-of-flight mass spectrometer using in vacuum electron ionization (EI).17 Material laser ablated from the surface was transported through an atmospheric pressure to vacuum interface, ionized in vacuum, and then mass analyzed. Molecular analysis of a pure crystal of anthracene in a spot sampling mode with a spot size of 5 μm (fwhm) was demonstrated. Topographical images of the same sample before or after laser desorption could be obtained in shear-force feedback mode with the same tip used for laser desorption. Reading and co-workers18−22 demonstrated the use of 5 μm tip diameter Wollaston wire heated AFM probes to perform both point thermal desorption (TD) and pyrolysis sampling of surfaces at AP, capturing the liberated vapor material then injecting it into a GC/MS for separation, in vacuum EI, and mass analysis. They also showed the ability to directly sample the vapors generated at AP into the vacuum-based EI source using a heated atmospheric-to-vacuum transfer capillary. Singlepoint sampling from polymer and plant tissue surfaces was demonstrated. The use of force-feedback mode with the AFM system allowed for the precise positioning of the Wollaston wire probes on the surface during the heating process as well as for subsequent topographic imaging of the thermal desorption craters. The smallest desorption craters achieved were from the pyrolysis of a poly(methylmethacrylate) sheet and were conical in shape, approximately 6 μm in diameter and 1.7 μm deep.20 More recently, we developed a combined AP-AFM/MS system utilizing nano thermal analysis (nano-TA) probes (30 nm tip diameter) for TD with subsequent electrospray ionization (ESI) and mass analysis.23 Thus, both the sampling from the surface by TD and the subsequent ionization process were achieved at AP. Ionization at AP using ESI, or another technique such as atmospheric pressure chemical ionization (APCI), ultimately broadens the range of compounds that can be ionized beyond those accessible with vacuum ionization techniques like EI.24 Our work showed automated sampling of a 5 × 2 array of spots, with 2 μm spacing between spots, and real-time selective detection of the desorbed caffeine using tandem mass spectrometry. Using a nano-TA probe temperature of 350 °C and a spot sampling time of 30 s, conical desorption craters 250 nm in diameter and 100 nm deep were created in a caffeine thin film as shown through subsequent topographical imaging of the surface within the same system. On the basis of the estimated crater volumes (∼2 × 106 nm3), only about 10 amol (2 fg) of caffeine was liberated from each TD spot. In the present work, we report on the advances in our system that enable the first report of combined AFM topography and mass spectral chemical images of a surface using AP surface sampling and ionization. The basic instrumental setup, the general operation of the system, and the optimization of performance metrics are discussed. The ability to coregister the mass spectral chemical image and topographical image is demonstrated using inked patterns on paper as well as a living bacterial colony on an agar gel. We achieved a mass spectral

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EXPERIMENTAL SECTION Samples. Uniform coatings of yellow ink were created by printing 1 mm wide lines of Epson yellow T0884 ink (Epson Inc., Tokyo, Japan) on ultra premium photo paper (Epson America Inc., Long Beach, CA) using an Epson Stylus NX110 inkjet printer (Epson, Inc.) controlled with in-house-developed software. Inked features ∼5−10 μm in size, spaced ∼5−10 μm apart were created by stamping Epson yellow T0884 printer ink on ultra premium photo paper using a 1500 mesh tunneling electron microscopy (TEM) grid with 11.5 μm wide square holes, 5 μm bars, and a 16.5 μm pitch (SPI Supplies, West Chester, PA). Pseudomonas species strain GM17 was isolated as an endophyte from roots of Populus deltoides trees, and its genome has been sequenced.25 For this work, GM17 was grown in Petri plates containing King’s B agar medium (SigmaAldrich, St. Louis, MO) and then stored under ambient conditions in a darkened environment for approximately 2.5 weeks prior to analysis. A 1 cm × 1 cm disk of the agar gel was cut out from the Petri dish and mounted on a metal puck for analysis. AFM Proximal Probe TD/APCI-MS Instrumentation. A Thermo LTQ XL mass spectrometer (Thermo Fisher Scientific Inc., Waltham, MA) equipped with an IonMax ion source and an APCI probe was used in this work. The IonMax ion source was operated in positive ion mode APCI with 450 °C nebulizer and 275 °C interface capillary temperatures. As shown in Figure 1, the IonMax source was modified by replacing the standard glass door window with an aluminum replica drilled and tapped for a 3/4 in. male pipe thread to 1/4 in. Swagelok connector (Swagelok, Solon, OH). A 10 cm long stainless steel sampling capillary (1/8 in. o.d. × 0.082 in. i.d., Supelco Analytical, Bellefonte, PA) was secured inside the connector using a 1/4 in. polyimide ferrule. Inside the source enclosure, the end of the

Figure 1. Experimental setup. Schematic illustration showing the IonMax source enclosure configured for APCI that was modified by inclusion of a metal sampling capillary in the back window and a venturi pump on the drain connection. The AFM was positioned so that the sampling capillary was within about 0.5 mm of the AFM probe. Inset shows an SEM image of the AFM probe tip (nano-TA probe). 1084

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quadrupole ion trap via collision-induced dissociation at a normalized collision energy of 35; product ions were mass analyzed in FTMS mode.

sampling capillary was positioned slightly above and about 8 mm back from the entrance to the interface capillary. The sampling capillary extended outside the source housing to transfer thermally desorbed material from the AFM tip area to the ion source. A variable leak valve (Granville-Phillips, Boulder, CO) with a gas pressure regulator (Parker, Mayfield Heights, OH) connected to a Vaccon CDF 200H venturi pump (Vaccon, Medway, MA) was placed at the drain opening on the source to enable gas flow into the ion source through the sampling capillary. The gas flow rate into the source enclosure through the sampling capillary was measured using an Aalborg GFM37 mass flow meter (Aalborg, Orangeburg, NY). A custom-designed atomic force microscope (Anasys Instruments, Santa Barbara, CA) equipped with a closed-loop stage was used to obtain topographical images and to control the temperature profile for thermal desorption for both spot sampling and lane scanning (imaging). Material was thermally desorbed from the surface using AN2−300 nanothermal analysis (nano-TA) AFM probes (Anasys Instruments). Temperature calibration of the nano-TA AFM probes was carried out using the measured melting point transitions of the polymeric samples polycarbolactone (PCL), high-density polyethylene (HDPE), and polyethylene terephthalate (PET))26,27 and fitting the values to a quadratic regression, a technique first introduced by Lee et al.28 Data Collection and Image Creation. For measurements in spot-sampling mode, the AFM was controlled using custom software that allowed varying of both time spent and AFM tip temperature on the surface. For lane scanning and imaging (multiple lane scans), custom software was used that provided a TTL signal at the start of each lane scan in the forward direction. Mass spectral data for each lane scan was collected into individual files. Movement of the AFM was synchronized with the corresponding mass spectral data by triggering the start of the data collection at the beginning of a lane scan. Spatial distribution of the analyte of interest obtained from the mass spectral data was visualized as a chemical image using the image visualization module of the HandsFree Surface Analysis© software package.29 HPLC-MS. A 10 mg piece of agar medium containing the portion of a colony with abundant visible yellow-green, needleshaped crystals was extracted with 10 mL of solvent (water/ acetonitrile/formic acid (65/35/0.1 v/v/v)). The extract was centrifuged for 10 min at 10 000g to remove particulates and then diluted 100 times prior to analysis. A 50 μL aliquot was injected onto a reverse-phase HPLC column (Agilent Eclipse XDB-C18, 5 μm, 4.6 × 150 mm) and separated using a gradient from 100% solvent A (5% acetonitrile, 0.1% formic acid in water) to 100% solvent B (70% acetonitrile, 0.1% formic acid in water) over 40 min at a flow rate of 0.5 mL/min. The eluent was directed to the ESI source of an LTQ-XL Orbitrap mass spectrometer (ThermoScientific), operating in FTMS mode at resolution 60 000. External m/z calibration was employed, and instrument calibration was performed on the same day as the measurements of the extract. Predicted m/z values and m/z measurement errors were obtained using the Elemental Composition and Isotope Simulation Calculator in XCalibur (version 2.1.0.1140, ThermoScientific). Three scan types were performed in succession throughout the chromatographic separation: full-scan, MS/MS (precursor ion m/z 224), and MS/MS/MS (m/z 224 → m/z 207 → ○). Precursor ions for the tandem mass spectral scans were dissociated in the

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RESULTS AND DISCUSSION General Operation and Optimized Performance Metrics. The schematic in Figure 1 shows the AFM proximal probe TD/APCI-MS setup. The AFM probe could be used in a conventional nondestructive fashion to obtain AFM topographical images of a surface, or it could be heated to enable AFM controlled TD sampling of material from a surface. The neutral, thermally desorbed material was drawn into an ion source enclosure through a straight line transfer capillary for ionization by positive ion mode corona discharge APCI. The ions created were transferred through a heated atmospheric pressure to vacuum capillary inlet into the mass spectrometer and detected. Mass spectral data obtained were linked with the spatial position of the AFM probe during the desorption event and coregistered with the topographic information that was obtained from the surface by the AFM immediately before the thermal desorption interrogation. The topography and mass spectral data could be acquired in succession without the need to change the AFM probe tip or move the sample manually. To optimize parameters affecting thermal desorption of material from a surface and the gas phase transport, ionization, and detection of this material, Epson yellow ink printed or stamped on photo paper was used as the main test analyte/ substrate combination. This ink contains pigment yellow 74 (386 Da),30 a monoazo dye, which we found amenable to intact thermal desorption, and is also amenable to ionization by APCI.31 The full scan mass spectrum obtained by thermal desorption of a printed ink spot (1.0 s heating, 350 °C), which resulted in a 1 μm wide × 0.6 μm deep hole in the ink, is shown in Supplemental Figure S1a. The base peak in the spectrum was that expected for the protonated molecule at m/z 387. The major product ion from m/z 387 was m/z 264 (normalized collision energy (CE) = 25%), which is consistent with previous reports (Supplemental Figure S1b). During optimization studies described below, selective detection of this compound was achieved by monitoring the MS/MS product ion transition of m/z 387 → m/z 264. The magnitude of the signal from a thermal desorption event is dependent strongly on the distance between the extraction capillary and the heated AFM tip. Through a series of positioning experiments (data not shown) it was found that placing the capillary near to the surface and as close as possible to the AFM probe tip maximized signal levels. For all the subsequent experiments discussed here the sampling edge of the capillary was placed approximately 0.5 mm from the probe. While positioning the extraction capillary even closer to the AFM would probably improve on the collection efficiency, at distances closer than 0.5 mm, the capillary blocked the deflection laser signal that was required for the AFM force feedback operation. Heating of the sampling capillary external to the source enclosure could also improve signals in certain cases. However, when placed in close proximity to the surface, radiant heat from the capillary did thermal damage to the surface. Therefore, the sampling capillary external to the source enclosure was not heated for the experiments reported here. Figure 2a shows the relative mass spectral peak area obtained in a series of thermal desorption spot-sampling experiments (AFM tip temperature of 366 °C) as a function of gas flow rate into the source enclosure through the sampling capillary. The 1085

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19% relative standard deviation (RSD) in the measured volume of the craters and a 19% RSD in the mass spectral signals for pigment yellow 74. In general, though, there was good correlation between crater volume and mass spectral signal at each probe temperature. It was not possible to determine definitively from the present data set whether the variation observed between hole size and mass spectral signal were the result of actual surface composition variation or the result of experimental variability in the gas-phase sampling, ionization, and mass spectral detection. However, these results are similar to those we reported for spot sampling with a caffeine thin film and an alternative interface design.23 While spot sampling can be used for single location profiling of a material or for imaging of a surface by point-by-point sampling, line scan arrays can be a preferred alternative for imaging, especially when considering total speed of analysis. The effect of line scan speed on mass spectral signal and on pixel size was investigated by scanning across a printed yellow ink surface at scan speeds ranging from 5 to 20 μm/s at a fixed probe temperature (350 °C). Without oversampling, the pixel size in spot sampling is equivalent to the diameter for the desorption crater.32 The pixel size in line scanning is influenced by the probe temperature and line scan speed, which determine the desorption line width, and by the mass spectral data acquisition rate. Mass spectral signal was observed to increase with surface scan rate because more material was sampled during the mass spectral data acquisition period as surface scan rate increased (open squares, Figure 3a). Desorption line width decreased with increasing scan speed due to less heat transfer from the heated probe to the surface at any particular point. This resulted in a smaller pixel size perpendicular to the scan direction with increasing surface scan speed (filled triangles, Figure 3a). However, because of the fixed minimum mass spectral data acquisition rate with the instrumentation in use (approximately 250 ms/spectrum in MS/MS mode), the pixel size in the scan direction linearly increased with surface scan rate (filled circles, Figure 3a). Comparable pixel size in both directions (∼2.5 μm) was achieved at a surface scan speed of 10 μm/s, which was the scan speed used for all subsequent line scan and imaging experiments. Figure 3b shows an AFM surface topography image of the remaining yellow ink surface when three lanes, spaced 8 μm apart, were scanned at a scan speed of 10 μm/s. The cross section profile in Figure 3c was taken from the topography image in Figure 3b at the location indicated by the dashed line showing desorption crater widths of ∼2.5 μm and a depth of approximately 1 μm. Chemical Imaging of Inked Patterns. The quality of the chemical images and achievable imaging resolution possible with the current AFM-TD/APCI-MS system was evaluated using a stamped pattern of yellow ink on photo paper as test substrate. A bright field optical microscope image (Figure 4a) showed that the stamped ink pattern was composed of what appeared to be raised ink features on the paper surface that were about 10 μm × 10 μm in size spaced about 5 μm apart. These inked regions were about 2−5 times larger than the mass spectral image pixel size obtained at surface scan rates of 10 μm/s. Figure 4b shows the interpolated mass spectral chemical image for the pigment yellow 74 obtained from this surface overlaid on the three-dimensional AFM surface topography image of the same region that was acquired prior to the thermal desorption imaging. This 4D image corresponded well with the optical image. Moreover, the location of the ink as determined from the mass spectral data was shown only to be in the raised

Figure 2. Interface Optimization. (a) Normalized MS/MS peak area for pigment yellow 74 (m/z 387 → m/z 264, CE = 25%) in APCI positive ion mode versus gas flow rate into the ion source of the mass spectrometer, AFM probe temperature was 366 °C. (b) Averaged hole diameter in printed yellow ink on photo paper versus AFM probe temperature. (c) Integrated desorption peak signal areas for the product ion of pigment yellow 74, m/z 264 and the corresponding calculated crater volume for each individual probe sampling temperature. The AFM probe was held on the surface for 1.0 s for each spot sampled. Epson yellow ink printed on premium photo paper was used for all experiments. Error bars represent ±1 standard deviation calculated from 25 replicate measurements in (a) and (b) and from 10 replicate measurements in (c). Signal areas and crater volumes each normalized to the values obtained from the data at a probe temperature of 366 °C.

maximum signal levels were achieved at a gas flow rate of 16 mL/min. This optimum gas flow rate reflects a balance between greater effectiveness of sampling and analyte transport at higher flows and the higher ionization efficiency at lower flows owing to increased residence time in the ionization region. The 16 mL/min gas flow rate through the sampling capillary was used for all subsequent studies. The effect of AFM probe temperature at fixed sampling time on the diameter of desorption craters created and the corresponding mass spectral signal is illustrated by the data in Figures 2b,c. The actual hole size and signal levels achieved will vary with the material investigated, but the general trends observed with the yellow ink used here are expected to be similar for other materials. The data in Figure 2b shows that using probe temperatures from 333 to 350 °C resulted in approximately 1 μm holes, while at 366 °C the diameter of the holes was greater than 3 μm. Supplemental Figure S2 shows AFM topography images from a 25-point spot sampling array using AFM tip temperatures of 350 and 366 °C that illustrate the reproducibility of desorption craters. The depth of the craters formed was ≈0.6 μm. The peak areas obtained by integrating the mass spectral signal from the desorption craters along with the desorption crater volumes calculated from the AFM topographic image are plotted in Figure 2c. There was a 1086

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Figure 4. (a) Optical image of yellow ink stamped onto photo paper. (b) Corresponding AFM surface topography image overlaid with the interpolated mass spectral chemical image using the extracted ion current from major product ion of pigment yellow 74 in yellow ink m/ z 264. The imaging data was acquired using MS/MS (m/z 387 → m/z 264, CE = 25%) in positive ion mode APCI with a fixed trap fill time of 100 ms, surface scan rate of 10 μm/s, lane spacing of 2 μm, and AFM probe tip temperature of 350 ◦C.

in which yellow-green crystals with visible dimensions of roughly 10 μm × 50 μm are observed using a bright field microscope. The AFM proximal probe was used in a spot sampling mode to thermally desorb material from an approximately 1 μm diameter area in a crystal-dense region of the colony. The base peak in the background subtracted full scan mass spectrum obtained was m/z 224 (Figure 5b). The background spectrum subtracted was that obtained while heating the AFM probe above the surface. This background subtraction eliminated from the spectrum signals due to chemical components in the laboratory air not derived from the sample. The MS/MS full scan product ion spectrum of m/z 224 showed m/z 207, owing to the loss of NH3, as the lone product ion formed (Figure 5c). This nominal precursor ion mass and product ion spectrum correlated well with published data for phenazine-1-carboxamide.37,38 Shown in Figure 5d is the background subtracted full scan mass spectrum obtained from an area on the colony several millimeters away from any visible crystals. The signal for m/z 224 was at least an order of magnitude lower at this location on the colony. These two spectra together demonstrate that there was a spatially distinct distribution of this phenazine in the colony. Confirmation for the identification of the species observed at m/z 224 as the protonated molecule of phenazine-1carboxamide was obtained by subjecting an extract of a portion of a GM17 colony (from the same agar gel plate) containing an abundance of yellow-green crystals to an HPLC separation with online electrospray ionization and high resolution, accurate mass, and tandem mass spectrometry analysis (Supplemental Figure 3). This established that the putatively identified phenazine-1-carboxamide was a major phenazine present in the growing colony and that the mass spectral data obtained by

Figure 3. Scan speed optimization. (a) Measured desorbed line width (vertical width) (▲) and pixel size (horizontal width) (●) as a function of scan speed in printed Epson yellow ink on premium photo paper and relative signal (□) of pigment yellow 74 as a function of AFM probe scan speed. These data were acquired using MS/MS of (m/z 387 → m/z 264, CE = 25) in positive ion mode APCI with a fixed trap fill time of 100 ms and an AFM probe tip temperature of 350 ◦ C. (b) AFM topography of the yellow ink surface after scanning three lines on the surface with the heated probe at 350 °C at a surface scan rate of 10 μm/s. (c) Corresponding line profile of the three desorption lines taken from (b) at the position indicated by the dashed line. Gas flow rate into the ion source was 16 mL/min.

regions on the surface. Both stamped features and blank spaces were clearly distinguishable. The quality of the chemical image suggested that even smaller features could be successfully resolved with the conditions used to acquire this data. Detection and Imaging of a Selected Metabolite in a Bacterial Colony. There has been significant recent interest in direct mass spectral analysis of bacteria using MALDI techniques33,34 as well as by atmospheric pressure liquid extraction techniques.35,36 The ability to identify and to image the distribution of a bacterial metabolite in a growing colony with the present methodology was demonstrated using the Populus rhizosphere isolate Pseudomonas sp. GM17 grown on agar medium. The photograph in Figure 5a shows a Pseudomonas sp. GM17 colony growing on agar for 2.5 weeks 1087

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presented suggest a locally high concentration of phenazine-1carboxamide in association with, or in close proximity to, the crystals we observed (Figure 5a, photograph inset). However, the exact composition of the crystals we observed in this case was not determined. Sequence comparisons43 indicate that a cluster of eight adjacent genes encoded in the Pseudomonas sp. GM17 genome (genes PMI20_01268 through PMI20_01261) is highly homologous at the protein level and in genomic structure (Supplemental Figure S4) to the well characterized phenazine-1-carboxamide biosynthetic loci of P.chlororaphis PCL1391.44 Thus, one can hypothesize that Pseudomonas sp. GM17 encodes genes with predicted functions for the enzymatic synthesis of phenazine-1-carboxamide.45 The identification of this gene cluster is consistent with our identification of phenazine-1-carboxamide in the GM17 colonies. The combined topographical AFM image and mass spectral chemical image targeting detection of phenazine-1-carboxamide in a 100 μm × 100 μm area of the Pseudomonas sp. GM17 colony in a crystal dense region is shown in Figure 6. The 3D AFM surface topography image of the colony surface was acquired prior to TD/ACPI-MS/MS imaging of the same region of the sample (MS/MS transition m/z 224 → m/z 207). This image overlay shows that there are spatially distinct variations in the abundance of the targeted phenazine (indicated by the red and yellow colors in the chemical image heat map), and the higher abundance regions appear to correlate most strongly with topographically elevated regions on the colony surface (indicated by the z dimension in the topography map). The pixel size for the AFM image was 0.2 μm × 0.8 μm and that for the mass spectral image was 2.5 μm × 2 μm. Overall, the combined image in Figure 6 demonstrates the capability to physically and chemically image a complicated biological sample such as a bacterial colony using this approach.

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CONCLUSIONS In this paper, we reported on the development of a hybrid atmospheric pressure AFM/MSI system that utilized nano-TA probes for thermal desorption surface sampling with subsequent atmospheric pressure chemical ionization and mass analysis. This combination of AFM and MS provided the capability to obtain topographical and chemical images of the same sample using a single instrument platform. A coregistered mass spectral chemical image and atomic force microscopy topographical image were obtained from inked patterns on paper with ink features approximately ∼5−10 μm in size, spaced ∼5−10 μm apart. The same type of coregistered images were also obtained from a living bacterial colony of Pseudomonas species GM17 on an agar gel, targeting mass spectral detection of the specific bacterial metabolite phenazine1-carboxamide. Spatial resolution of the topography images based on pixel size (0.2 μm × 0.8 μm) was better than the resolution of the mass spectral images (pixel size 2.5 μm × 2.0 μm), which were limited by current mass spectral data acquisition rate and system detection levels. Even so, this small pixel size is superior to the very best reported to date for MSI using any type of AP surface sampling and ionization method.46,47 The use of a mass analyzer with faster full scan or tandem mass spectral data acquisition rates combined with even modest improvements in detection levels will most likely allow submicrometer scale pixel sizes to be realized for MSI using this approach. In any case, the coregistration of topography, physical and detailed chemical images of a surface obtained

Figure 5. (a) Optical image of Pseudomonas sp. GM17 bacteria colony grown on an agar gel surface. Arrow indicates yellow-green crystals growing inside the colony with inset photograph showing a close-up view of the crystals. (b) Background subtracted, averaged full scan mass spectrum of a 1.0 s heating event in a crystal dense region of the colony. Inset shows the structure of phenazine-1-carboxyamide (MW = 223). (c) MS/MS full scan product ion spectrum of m/z 224 (CE = 35%) from this general location. (d) Background subtracted, averaged full scan mass spectrum of a 1.0 s heating event in an area on the colony several millimeters away from any visible crystals. Signal levels in panel (b) are normalized to those in panel (d).

the TD/APCI process were representative of the sample composition at the different points of analysis. Phenazines are produced by multiple Pseudomonas species and other bacterial species, and they are recognized as performing multiple roles in mediating the interaction of the bacteria with its environment.39 In pure form, various phenazines form yellow or green crystals,40,41 and crystals of some phenazines have been observed to form directly in aged agar cultures.42 The data 1088

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Figure 6. AFM surface topography image of a 100 μm × 100 μm region of the Pseudomonas sp GM17 bacteria colony growing on agar gel overlaid with the interpolated mass spectral chemical image constructed for phenazine-1-carboxamide using the major MS/MS product ion at m/z 207. Imaging data was acquired using positive ion mode APCI with a fixed trap fill time of 100 ms, surface scan rate of 10 μm/s, lane spacing of 2 μm, and AFM probe tip temperature of 350 ◦C. (2) Scanning Probe Microscopy of Functional Materials; Kalinin, S. V., Gruverman, A., Eds.; Springer: New York, NY, 2010. (3) Flores, S. M.; Toca-Herrera, J. L. Nanoscale 2009, 1, 40−49. (4) Lucas, M.; Reido, E. Rev. Sci. Instrum. 2012, 83, 061101. (5) McDonnell, L. A.; Heeren, R. M. A. Mass Spectrom. Rev. 2007, 26, 606−643. (6) Flectcher, J. S.; Lockyer, N. P.; Vickerman, J. C. Mass Spectrom. Rev. 2011, 30, 142−174. (7) Pól, J.; Strohalm, M.; Havlíček, V.; Volný, M. Histochem. Cell. Bio. 2010, 134, 423−443. (8) Watrous, J. D.; Alexandrov, T.; Dorrestein, P. C. J. Mass Spectrom. 2011, 46, 209−222. (9) Norris, J. L.; Caprioli, R. M. Chem. Rev. 2013, 113, 2309−2342. (10) Nemes, P.; Vertes, A. Trends Anal. Chem. 2012, 34, 22−34. (11) Wu, C.; Dill, A. L.; Eberlin, L. S.; Cooks, R. G.; Ifa, D. R. Mass Spectrom. Rev. 2013, 32, 218−243. (12) Adams, F.; Barbante, C. Talanta 2012, 102, 16−25. (13) Masyuko, R.; Lanni, E. J.; Sweedler, J. V.; Bohn, P. W. Analyst 2013, 138, 1924−1939. (14) Whitby, J. A.; Ö stlund, F.; Horvath, P.; Gabureac, M.; Riesterer, J. L.; Utke, I.; Hohl, M.; Sedlácě k, L.; Jiruše, J.; Friedli, V.; Bechelany, M.; Michler, J. Adv. Mat. Sci. Engineer. 2012, article no. 180437, 13 pages. (15) Wirtz, T.; Fleming, Y.; Gerard, M.; Gysin, U.; Glatzel, T.; Meyer, E.; Wegmann, U.; Maier, U.; Odriozola, A. H.; Uehli, D. Rev. Sci. Instrum. 2012, 83, 063702. (16) Wirtz, T.; Fleming, Y.; Gysin, U.; Glatzel, T.; Wegmann, U.; Meyer, E.; Maier, U.; Rychen, J. Surf. Interface Anal. 2013, 45, 513− 516. (17) Schmitz, T. A.; Gamez, G.; Setz, P. D.; Zhu, L.; Zenobi, R. Anal. Chem. 2008, 80, 6537−6544. (18) Price, D. M.; Reading, M.; Hammiche, A.; Pollock, H. M. Int. J. Pharm. 1999, 192, 85−96. (19) Price, D. M; Reading, M.; Hammiche, A.; Pollock, H. M. J. Thermal Anal. Calorimetry 2000, 60, 723−733. (20) Price, D. M.; Reading, M.; Lever, R. J.; Hammiche, A.; Polluck, H. M. Thermochim. Acta 2001, 367−368, 195−202. (21) Price, D. M.; Reading, M.; Smith, R. M.; Pollock, H. M.; Hammiche, A. J. Thermal Anal. Calorimetry 2001, 64, 309−314. (22) Reading, M.; Price, D. M.; Grandy, D. B.; Smith, R. M.; Bozec, L.; Conroy, M.; Hammiche, A.; Pollock, H. M. Macromol. Symp. 2001, 167, 45−62. (23) Ovchinnikova, O. S.; Nikiforov, M. P.; Bradshaw, J. A.; Jesse, S.; Van Berkel, G. J. ACS Nano 2011, 5, 5526−5531.

by this hybrid AFM and MSI instrument platform offers new possibilities for higher-resolution characterization and detailed understanding of the compositions of surfaces ranging from new materials to biological specimens.

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ASSOCIATED CONTENT

S Supporting Information *

Additional information as noted in the text. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Fax: 865-576-8559. Tel.: 865574-1922. Funding Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This manuscript has been authored by a contractor of the U.S. Government under contract No. DE-AC05−00OR22725. Accordingly, the U.S. Government retains a paid-up, nonexclusive, irrevocable, worldwide license to publish or reproduce the published form of this contribution, prepare derivative works, distribute copies to the public, and perform publicly and display publicly, or allow others to do so, for U.S. Government purposes. Instrumental implementation, fundamental and metric studies were supported by the Division of Chemical Sciences, Geosciences, and Biosciences, Office of Basic Energy Sciences, United States Department of Energy under Contract DEAC05-00OR22725 with Oak Ridge National Laboratory (ORNL), managed and operated by UT-Battelle, LLC. The work with and analysis of Pseudomonas species GM17 was funded by the Laboratory Directed Research and Development (LDRD) Program of ORNL.

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