Controlled-Resonant Surface Tapping-Mode Scanning Probe

Mar 7, 2014 - Fax: 865-576-8559. ... The system was used for lane scanning and chemical imaging of the cationic dye crystal violet in inked lines on g...
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Controlled-Resonant Surface Tapping-Mode Scanning Probe Electrospray Ionization Mass Spectrometry Imaging Matthias Lorenz, Olga S. Ovchinnikova, Vilmos Kertesz, and Gary J. Van Berkel* Organic and Biological Mass Spectrometry Group, Chemical Sciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831-6131, United States S Supporting Information *

ABSTRACT: This paper reports on the advancement of a controlled-resonant surface tapping-mode single capillary liquid junction extraction/ESI emitter for mass spectrometry imaging. The basic instrumental setup and the general operation of the system were discussed, and optimized performance metrics were presented. The ability to spot sample, lane scan, and chemically image in an automated and controlled fashion were demonstrated. Rapid, automated spot sampling was demonstrated for a variety of compound types, including the cationic dye basic blue 7, the oligosaccharide cellopentaose, and the protein equine heart cytochrome c. The system was used for lane scanning and chemical imaging of the cationic dye crystal violet in inked lines on glass and for lipid distributions in mouse brain thin tissue sections. Imaging of the lipids in mouse brain tissue under optimized conditions provided a spatial resolution of approximately 35 μm based on the ability to distinguish between features observed both in the optical and mass spectral chemical images. The sampling spatial resolution of this system was comparable to the best resolution that has been reported for other types of atmospheric pressure liquid extraction-based surface sampling/ ionization techniques used for mass spectrometry imaging.

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desorption region would be a major factor required to improve the achievable spatial resolution of DESI. The other established types of liquid-extraction-based techniques for MSI use a wall-less probe-to-surface liquid junction approach for surface sampling and separate the extraction and ionization processes in time or space. The liquid microjunction surface sampling probe (LMJ-SSP) was the first sampling system of this type.7 Through the use of a concentric, dual-capillary geometry, an extraction solvent is pumped toward the surface through the annulus of the two capillaries to form a probe-to-surface liquid junction 10−50 μm thick spanning the width of the outer capillary (typically 650 μm). Extracted material in this liquid junction is continually aspirated from the surface through the inner capillary, which also serves as the emitter for a conventional atmospheric pressure liquid introduction ionization source like electrospray ionization (ESI)7 or atmospheric pressure chemical ionization

everal liquid extraction-based approaches for atmospheric pressure surface sampling and ionization that can be used for mass spectrometry imaging (MSI) have been advanced over the past decade.1,2 Desorption electrospray ionization (DESI) is arguably the most well-known and widely used technique of this type.3 In DESI, charged droplets and gas from a pneumatically assisted electrospray source are directed at the surface of interest. Soluble components at the surface dissolve into a resulting thin film of liquid on the surface. Subsequent charged droplet/gas impact sputters secondary charged droplets from the liquid film which ultimately form gas phase ions via an electrospray-like process and are drawn into the mass spectrometer.4 The best spatial resolution for MSI using DESI has been reported to be 35−40 μm, as defined by the size of the smallest-known surface feature distinguishable in the resulting chemical images.5,6 Achieving this resolution required a careful optimization of a number of instrumental variables, including solvent composition and flow rate, to minimize the size of the effective DESI impact plume desorption/ionization area, and to minimize analyte redistribution on the surface during an analysis. Shrinking further the size of this effective © 2014 American Chemical Society

Received: December 31, 2013 Accepted: February 25, 2014 Published: March 7, 2014 3146

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(APCI).8 Thus, sampling from a surface with the LMJ-SSP is analogous to an in situ microextraction and is applicable to all species that can be dissolved and aspirated into the probe and subsequently ionized by the respective ionization method being used. The technique named nanoDESI uses a similar liquid junction extraction approach but with smaller diameter solvent delivery and aspiration capillaries (e.g., 150 μm o.d. × 50 μm i.d.) positioned in a nonconcentric geometry.9 With respect to MSI, the size of the probe-to-surface liquid junction determines the best spatial resolution that can be achieved by either approach. With the LMJ-SSP to date, this liquid junction size has been in the 650 μm range.10,11 With nanoDESI significantly smaller liquid junctions have been reported capable of achieving a 10 μm wide extraction lane on a surface.12 Data acquired from model surfaces and real samples using appropriate surface scan speeds, mass spectral data acquisition rates, and lane step spacing indicated a pixel size of about 10 × 10 μm might be achieved.12−14 Further improvement in the spatial resolution using either LMJ-SSP or nanoDESI will require the use of smaller inner and outer diameter solvent delivery and aspiration capillaries. However, reducing the inner diameter of the aspiration capillary too much will negatively impact the robust operation of the probe (e.g., due simply to the frequency of plugging). One might predict that this size limit is not much smaller than the current 50 μm i.d. aspiration capillaries currently used for nanoDESI.13,14,20 Hiraoka and co-workers15,16 have developed a technique they call probe electrospray ionization (PESI), which uses a single sharp solid metal needle to sample the material’s surface which is then subsequently electrosprayed from the needle tip. This approach works best for surfaces that are wet,15 but with addition of solvent vapor near the needle tip16 or through the use of a liquid sheath flow,17 the approach has proven applicable to a wider variety of surfaces. The best imaging lateral resolution (pixel size) reported to date with this general approach was about 60 μm in freshly cut, hydrated mouse brain tissue. Given the appropriate detection sensitivity, the possibility exists with this approach for improving the imaging resolution by simply making the needle probe smaller. Recently, Otsuka et al.18 presented a single capillary liquid junction extraction/ESI emitter they named scanning probe electrospray ionization (SPESI). This geometry eliminates the aspiration/emitter capillary that is a primary factor in the ultimate resolution limit of any dual capillary, liquid junction surface sampling probe. The single capillary is used to supply solvent to form a liquid junction between the capillary and a sample surface. A bias voltage is applied to the solvent to generate an ESI from liquid that pools at the top of the capillary via capillary action, the surface tension of the liquid, and the force of the applied electric field. In the version most suitable for imaging, spontaneous vibration of the probe itself (termed tapping-mode) created an alternate liquid junction surface sampling/noncontact ESI situation at a rate of greater than 100 Hz. Data presented by Otsuka et al.,18 including a single manual lane scan across a thin section of mouse pancreas, indicated that sampling spot size and lane scan width (approximately 150 μm) was nearly equivalent to the outer diameter of the solvent delivery capillary. In their case, a blunt cut 150 μm o.d. × 50 μm i.d. fused silica capillary was used. Because the solvent delivery capillary in this design is not subject to the plugging or contamination issues of a sample collecting aspiration/emitter

capillary used in the LMJ-SSP or nanoDESI, one can envision making this delivery capillary much smaller. This would result in a smaller probe-to-surface liquid junction and thereby improve the spatial resolution. Furthermore, because the liquid junction to the surface is maintained for only a few milliseconds at a time, tapping mode SPESI might be a means to overcome some of the issues other liquid junction probes face when attempting to sample from an absorbant surface.19 Potentially most important, this tapping mode eliminates the need with the LMJ-SSP and with nanoDESI to precisely control the probe-tosurface liquid junction thickness. Both of these latter approaches require very flat surfaces and a precise preanalysis surface to probe alignment or a real time feedback mechanism to maintain the optimum liquid junction thickness during an analysis.10,20 In this work, we advance the basic concept of the original spontaneous tapping-mode SPESI system. Controlled-resonant tapping of the capillary was implemented using a mechanical relay and function generator; tapered tip fused silica capillaries of 20 μm i.d. were used to minimize the probe-to-surface liquid junction size improving spatial resolution, and computer controlled sample stage and data processing software were utilized to enable automated spot sampling and MSI. Inked and spotted features on glass and thin tissue sections mounted on glass slides were used as test substrates to study and optimize the performance of the system. The basic instrumental setup, the general operation of the system, and the optimization of performance metrics are discussed. On the basis of distinguishable features observed in the image of a mouse brain tissue, a MSI spatial resolution approaching 35 μm was achieved under optimized conditions.

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EXPERIMENTAL SECTION Chemicals and Materials. Liquid chromatography−mass spectrometry (LC−MS) Optima grade methanol with 0.1% formic acid, and water with 0.1% formic acid were purchased from Fisher Scientific (Pittsburgh, PA). Glass slides were purchased from Fisher Scientific. A black ball point pen containing the dye crystal violet was purchased locally. RainX (ITW Global Brands, Houston, TX) was purchased locally and applied to a frosted glass microscope slide to make the surface hydrophobic. Fused silica tapered tip emitters with a 150 μm o.d. and 20 μm i.d. tapered down to 20 ± 3 μm over a distance of ∼210 μm were purchased from New Objective (Woburn, MA). Cellopentaose and equine horse heart cytochrome C > 95% were obtained from Sigma Aldrich (St. Louis, MO). Coronal mouse brain tissue sections (40 μm thick) thaw mounted on glass slides were obtained from Protea Biosciences Inc. (Morgantown, WV). Deconvolution of multiply charged full scan mass spectra was accomplished using the software tool Magtran (Amgen Inc. Thousand oaks, CA).21 Straight ink lines were drawn on the frosted part of a glass microscope slide with a black ball point pen. Optical images of the surfaces analyzed were obtained using a Dino-Lite Premier 2 digital microscope (Big C, Torrance, CA) and associated software, or a Nikon Biophot upright microscope (Tokyo, Japan) and associated software. Experimental Setup. The schematic of the experimental setup is shown in Figure 1a. An LTQ XL linear ion trap mass spectrometer was used for all experiments (Thermo Scientific, San Jose, CA). The normal atmospheric sampling capillary into the mass spectrometer was replaced with a slightly curved extended version (ca. 20 mm) typically used for DESI 3147

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(Figure 1b) as well as the proper position in front of the sampling capillary into the mass spectrometer (Figure 1c). High voltage (4−5 kV) from the LTQ XL ESI supply was applied to the solvent through a gold wire (0.64 mm diameter) that was terminated inside one arm of a PEEK tee contacting the liquid at a distance 11 cm from the fused silica capillary tapered tip. A 50 μL Hamilton Gastight 1705 syringe (Reno, NV) driven by a Harvard Apparatus syringe pump (Holliston, MA) was used to pump the solvent through the system. To accomplish the controlled tapping mode SPESI, a DG535 pulse delay generator (Stanford Research Systems, Sunnyvale, CA) was used to apply a pulse train (4 ms wide, 5 V pulses) to the relay to drive the oscillation of the capillary. In operation, the frequency of the pulse train was selected to be slightly off resonance for the capillary (100−110 Hz) to allow for adjusting the physical amplitude of the capillary motion. An LED light driven with 10 μs square pulses (DG535 pulse delay generator) was used to strobe illuminate the capillary from the back, enabling the turning points in the capillary oscillation to be visualized as the shadow image of the capillary (Figure 1d). The capillary made contact with the surface at one turning point and electrosprayed in the vicinity of the atmospheric sampling capillary of the mass spectrometer close to the other turning point. The emitter capillary was within ∼500 μm from the end of sampling capillary at the upper turning point. The position of the probe relative to the surface was monitored using a second Optem Zoom 70XL zoom lens connected to a Costar SIC400N CCD camera. Data Collection for Imaging. The data collection procedure for the imaging experiments was similar to that which we have utilized for other atmospheric pressure surface sampling/ionization techniques.5,22 Briefly, at the beginning of an imaging experiment, the surface was moved so that the capillary was positioned at one corner of the area of interest and tapping mode contact of the taper tip capillary/emitter with the surface was initiated. After that, stage movements were conducted under computer control. The first lane was scanned by moving the surface parallel to the x axis at the selected forward surface scan rate. At the end of the first lane, the surface was pulled back from the capillary 1 mm and the surface was moved to the beginning of the first lane. When the beginning of the first lane was reached, the surface was moved parallel to the y axis to achieve the required lane spacing distance and then moved forward to reestablish the tapping mode contact with the surface. The subsequent lanes were scanned in a similar fashion. Data for each lane scan was collected into individual data files. Movement of the stage was synchronized with the corresponding mass spectral data by triggering the start of the data collection at the beginning of a lane scan using the stage control software (HandsFree Surface Analysis).23 Imaging data was collected in full scan mode. Raw data files were converted into a BioMap compatible format using a software developed in-house (BioMap Converter for RAW Data). Chemical images of selected mass-to-charge ratios were visualized by importing the resulting file into the BioMap software package (Novartis Institutes for BioMedical Research, Basel, Switzerland).

Figure 1. Experimental setup and operation. (a) Schematic illustration showing the automated sample stage in front of the mass spectrometer equipped with an extended atmospheric sampling capillary, the modified relay on a printed circuit breadboard support with PEEK tubing sleeves guiding the fused silica capillary to the sample surface. Schematic illustrations of the phenomena at the extremes of the capillary oscillation showing (b) surface to capillary liquid junction formation and sample extraction and (c) ESI of the liquid extract from the tip of the capillary toward the mass spectrometer inlet. (d) A strobe light illumination photograph of the oscillating capillary emphasizing the turning points of the capillary motion at the sample surface and at the sampling capillary into the mass spectrometer. (e) Microscope image of the tapered tip of the fused silica capillary labeled with physical dimensions and the probe-to-surface contact angle α.

(Prosolia, Indianapolis, IN). Samples were positioned on a MS2000 x−y−z robotic platform (Applied Scientific Instrumentation Inc., Eugene, OR) that was used to manipulate, using manual joystick or automated control, the sample surface relative to the fixed x−y position tapping capillary and atmospheric pressure to vacuum sampling capillary of the mass spectrometer. An Optem Zoom 70XL zoom lens (Qioptiq Inc., Rochester, NY) connected to a Costar SIC400N CCD camera (Costar Inc., Anaheim, CA) was used to monitor the liquid microjunction from the underside of transparent samples utilizing a mirror to bend the optical path by 90°. A 5 V relay (QUAZ-SS-105D, Newark, Palatine, IL) was used to drive the oscillation of the fused silica taper tip solvent delivery/ESI capillary. The top plastic encasing on the relay was removed to expose the metal armature. The open circuit contact on the relay was removed to allow unrestricted oscillation of the spring-loaded armature under an AC bias. Two 10 mm pieces of 1/16″ o.d. × 0.007″ i.d. PEEK tubing were glued to the armature as well as to a printed circuit panel breadboard holding the relay securing the capillary to the relay. The fused silica taper tip emitter was fed through the PEEK tubing with 6 mm exposed past the armature providing the capillary enough range of motion to reach the sample surface

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RESULTS AND DISCUSSION Instrumental Setup and General Operation. The schematic illustration in Figure 1a shows the complete controlled-resonant tapping mode SPESI setup. A 150 μm o.d. × 20 μm i.d. taper tip capillary (Figure 1e) was used for

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gram shown in Figure 2a. The signal for the dye appeared very rapidly after the tip and liquid first contacted the surface; the

both solvent delivery (typically 25−100 nL/min) to the surface (Figure 1b) and as the electrospray emitter (Figure 1c). The high voltage necessary for electrospray was made by an electrical contact to the liquid upstream of the taper tip capillary. This capillary was held in a PEEK tubing sleeve connected to a mechanical relay. A function generator supplying a square wave signal was used to adjust the frequency and amplitude of the motion of this capillary. For the experimental conditions used here, the resonance frequency of the spring-loaded relay armature with the attached PEEK sleeve and the mounted taper tip capillary was approximately 120−140 Hz. The exact frequency depended on the individual capillary used owing to slight variations in lengths and mounting. In actual operation, the frequency of the oscillation was set slightly off resonance (e.g., 100−110 Hz) to provide the desired amplitude of the capillary motion. Figure 1c shows a strobe light enabled photograph of the tip oscillation between tapping the surface at one turning point and electrospraying toward the atmospheric sampling capillary of the mass spectrometer close to the other turning point. The emitter capillary was within ∼500 μm from the end of sampling capillary at the upper turning point. In the photograph shown, the amplitude of motion spanned ∼5.5 mm. Both a close positioning of the taper tip capillary of the transfer capillary into the mass spectrometer at the upper movement range and relatively larger amplitude of motion was found to be important for generation of a consistent electrospray and effective sampling of the ions produced. Figure 1e shows a close up photograph of the taper tip capillary. The small inner diameter (20 μm) and taper tip shape of this capillary were two important factors for minimizing the liquid junction spot size on a surface and for forming a reproducible and small volume of electrospray liquid on the tip of the capillary (see below). Under these basic operation conditions, the full scan mass spectrum obtained showed a steady state total ion current profile whether or not the oscillating capillary touched a surface to be sampled (Figure S1 of the Supporting Information). The majority of ions in the spectrum were from solvent clusters or contaminants common in the solvents being used. When a clean glass slide was moved up to just touch the oscillating capillary at the lower turning point in the motion, an increase in total ion current, as large as 25%, was sometimes observed. This increase did not appear to relate to extraction of material from the surface. There was no change in the observable ions in the spectrum, and the ion current increase was steady state rather than transient as might be expected for extraction of material from the surface. This effect might be due to factors that positively impact the electrospray process or sampling of ions into the mass spectrometer. Further investigation of the effect with other surface types and solvents will be required to fully understand this observation. Spot Sampling from a Surface. A layer of blue Sharpie marker, containing the cationic dye basic blue 7 (m/z 478), was used to demonstrate the capability of this controlled-resonant tapping mode SPESI system to spot sample or chemically profile specific locations on a surface. The system was operated so that the sample stage automatically moved the sample surface up to where it just touched the oscillating capillary tip at the lower turning point in the capillary oscillation; it remained in that position for 1 s, and then retracted the sample away from the capillary and moved laterally to the next sampling location. This sampling procedure was repeated three consecutive times, providing the extracted ion current chrono-

Figure 2. Surface sampling in spot sampling mode. (a) Extracted ion current chronogram for m/z 478 from 3 consecutive, 1 s spot samples at different locations on a layer of blue Sharpie marker on glass. (b) Full scan mass spectrum obtained from the first of the 3 spot sampling experiments shown in panel (a). Solvent: 50/50/0.1 (v/v/v) methanol/water/formic acid at 75 nL/min. (c) Full scan mass spectrum from a single 10 s spot sampling of cellopentaose (828 Da, 0.3 nmol spotted on a glass slide). Solvent: 80/20/0.1 (v/v/v) methanol/water/formic acid at 50 nL/min. (d) Full scan mass spectrum from a single 10 s spot sample of horse heart cytochrome c (12360.2 Da, 0.8 pmol spotted on a glass slide). Solvent: 50/50/0.1 (v/v/v) methanol/water/formic acid at 100 nL/min. Ion trap injection time is 10 ms in all cases.

signal reached a relatively steady state and then rapidly decreased to background levels when the surface was pulled away. The sampling time on the surface was nominally 1 s and the total ion signal profile for the dye was about 1.4 s, indicating fast sample washout and little carryover. The full scan mass spectrum obtained from the first spot sampling experiment in this sequence is shown in Figure 2b. The intact molecular cation of the dye (m/z 478) was observed as the base peak in the spectrum. Sampling in a similar manner from spotted samples of cellopentaose and horse heart cyctochrome c yielded the spectra shown in Figure 2 (panels c and d, respectively). Cellopenatose is a 5-glucose residue oligosaccharide that is a product of the hydrolysis or degradation of cellulose. Under ESI conditions, the molecule tends to form the sodiated molecule, (M + Na)+, as we observed here, even under acidic solvent conditions.24 The sodium in this case comes from traces in the sample itself, the glass surface, or from the solvent systems. The cytochrome c spectrum shows a bimodal charge 3149

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state distribution spanning the range from (M + 17H)17+ to (M + 7H)7+. One might expect that the spotting conditions, the sampling procedure, the solvent system, and the mass spectrometer settings might all influence the nature of the charge states observed for this or other proteins by this methodology. In any case, the calculated average molecular mass of this protein following N-terminal methionine cleavage and acetylation is 12360.2 Da and the mass determined from the spectrum in Figure 2d was 12360.7 Da. In general, the data in Figure 2 demonstrates that rapid, automated spot sampling is possible with this approach and a wide variety of compound types can be analyzed. As long as the material of interest can be sampled from the surface by a liquid−solid extraction mechanism, and ionized by ESI, this approach can be used successfully for rapid spot sampling from surfaces. Lane Scans along a Surface. Initial tests of lane scanning using this controlled-resonant tapping mode SPESI system were carried out using inked lines (ca. 100 μm wide) from a black ballpoint pen (containing the cationic crystal violet, m/z 372) drawn on a frosted glass surface (Figure S2 of the Supporting Information). The optical bright field image of the lines is shown in Figure S2a of the Supporting Information. The chemical image was rendered from the tandem mass spectrometry signal for crystal violet (m/z 372 → 356) in a series of 51 lane scans acquired at a surface scan rate of 100 μm/s, with a lane spacing of 40 μm, using 80/20/0.1 (v/v/v) methanol/water/formic acid as the solvent at a flow of 50 nL/ min (Figure S2b of the Supporting Information). Under these conditions, there was a good correlation between the optical image and the chemical image. The chemical image retained the same approximate line width of 100 μm measured in the optical image. Moreover, the chemical image showed no evidence of significant sample drag or carryover as the tapping probe passed through the inked lines under the conditions used. Overall, these data demonstrate the ability to perform unattended chemical imaging with this technique. Optimization of Liquid Junction Spot Size for Lane Scanning and Imaging. Studies were undertaken to better understand and optimize the achievable spatial resolution of lane scanning and thus MSI with this technique. The size of the liquid junction or the area sampled by this junction should be a main factor in determining the spatial resolution of this MSI technique. Thus, minimizing the size of the spot wetted on the surface during the sampling process would be expected to enhance the sampling resolution. With a fixed tapping capillary size (20 μm i.d.) and solvent composition [50/50/0.1 (v/v/v) methanol/water/formic acid], the shape and area wetted on a hydrophobic, frosted glass slide surface were measured as a function of solvent flow rate and surface to tapping capillary contact angle, α. This measurement was achieved by viewing the wetted spot optically from beneath the sample. The sizes of the spots wetted certainly will change depending on the solvents and surface type, but the trends reported here are expected to be general. The data presented in Figure 3a show the measurement results for a fixed capillary contact angle of 29° with a single solvent [50/50/0.1 (v/v/v) methanol/water/formic acid] at flow rates from 25−100 nL/min. With this relatively steep contact angle, the wetted area observed on the surface was always circular in this flow rate range [see Figure 3c(I)]. Generally, as the flow rate increased, the size of the wetted area was observed to increase. This increase was most dramatic from

Figure 3. Optimization of liquid junction spot size. (a) Surface area wetted versus solvent flow rate at a fixed capillary to surface contact angle (∼29°). (b) Surface area wetted versus capillary to surface contact angle at a solvent flow rate of 50 nL/min. Overlaid is the pictorial representation of the evolution of the wetted area as a function of the contact angle. (c) Pictorial representation of the position of capillary relative to the surface and the shape of the wetted area at 29° (I) and 23° (II), as indicated in panel (b). The solvent was 50/50/0.1 (v/v/v) methanol/water/formic acid.

25 to 50 nL/min, where the diameter of the wetted spot doubled from about 30 to 62 μm, increasing the wetted area by approximately a factor of 4 (from ∼0.7 × 103 to 3.0 × 103 μm2). Further increasing the flow rate from 50 to 100 nL/min increased only modestly the wetted spot size (∼62 to ∼70 μm) and area (∼3.0 × 103 to ∼3.8 × 103 μm2). At flow rates above 100 nL/min, the size and shape of the wetted spot started to increase significantly (data not shown). Smallest wetted spot size was achieved at the 25 nL/min, but this flow rate proved impractical for robust operation of the system. At this low flow rate, wetting of the surface was observed often to be a discontinuous process due most likely to intermittent solvent flow because of pulsing of the syringe pump used or more likely due to the relatively large inner diameter tapping capillary. A smaller internal diameter capillary (≪20 μm i.d.) might alleviate this problem while producing an even smaller wetted spot area. Robust operation of the system was found at flow rates from 50−100 nL/min. The optimum flow rate can be expected to change depending on the exact nature of the surface (e.g., hydrophobic versus absorbent) and the solvent(s) used, though those effects were not systematically tested in this work. The data presented in Figure 3b show a separate set of measurement results for capillary contact angles from ∼23° to ∼29° for the same solvent system at a flow rate of 50 nL/min. This range of contact angles was determined in other experiments (data not shown) to encompass the optimized operational range. These data show that as the contact angle was decreased from about ∼29° to ∼26°, the size of the wetted spot decreased by less than a factor of 2, but the shape changed from circular to elliptical (minor axis of ellipse at 26° was ∼30 μm). A further decrease in the contact angle to ∼23° resulted in 3150

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an increase in the elliptical contact area by just over a factor of 2. This evolution in the size and the shape of the wetted spot with contact angle is illustrated in Figure 3c. On the basis of these observations, all our subsequent lane scanning and imaging experiments employed a capillary-to-surface contact angle of ∼26° and solvent flow rates between 50 and 100 nL/ min (see below). Chemical Imaging of Endogenous Compounds in Tissue. Figure 4a shows the averaged mass spectrum from a

Figure 5. Imaging endogenous compounds in mouse brain tissue. (a) Optical bright field image of mouse brain tissue on glass. Major regions of the brain are labeled. (b) Zoomed in optical image of the boxed area shown in panel (a). (c) Chemical image of the mouse brain tissue showing the RGB combined distribution of three ions, m/z 734.7 (red) m/z 788.7 (green), and m/z 808.5 (blue), extracted from the full scan mass spectral data. (d) Zoomed in chemical image of the boxed region shown in panel (c). Ion trap fill time of 10 ms, surface scan rate of 100 μm/s, 151 lane scans, and lane spacing of 20 μm. Solvent: 50/ 50/0.1 (v/v/v) methanol/water/formic acid at 85 nL/min flow, capillary-to-surface contact angle of ∼26°. Total imaging time = 2.5 h.

Figure 4. (a) Averaged full scan mass spectrum (m/z 100−1000) from a single lane scan across a mouse brain tissue. (b) Zoomed in view of the same spectrum from m/z 700−880. Ion trap injection time of 10 ms and surface scan rate of 100 μm/s. Solvent: 50/50/0.1 (v/v/v) methanol/water/formic acid at 85 nL/min flow rate.

over m/z 734.6 (red) in the granular layer giving this area a purple color. Figure 5 (panels b and d) show the zoomed-in regions from both the optical and chemical images, respectively, around a physical feature of ∼35 μm in diameter that is clearly resolved in both images. On the basis of the ability to distinguish this feature in the chemical image and the corresponding optical image, the spatial resolution for this chemical image was estimated to be ∼35 μm.

single lane scan across a mouse brain tissue mounted on glass. The mass spectrum is particularly rich with tissue-related ions in the region from m/z 700 to 900 (Figure 4b). Given nominal mass resolution, focusing on only the most abundant peaks, and with reference to other MSI studies of mouse brain,25−27 the peaks observed in this region are presumed to be predominately phosphatidylcholine (PC) type lipids. Some tentative identifications, for example, might be the protonated molecules of PC (32:0) at m/z 734.7 and PC (34:1) at m/z 760.7. Further definitive identification of the species detected is beyond the scope of the proof-of-principle imaging capability presented in this work. Any one or more of these ion signals might be used to generate ion images of this brain tissue surface as illustrated by the data in Figure 5. Figure 5a shows the optical bright field image of a portion of the same brain tissue used to obtain the data in Figure 4. Different regions of the brain observed are labeled. A series of 151 lane scans across this mouse brain tissue section were obtained while recording full scan mass spectra. The wetted elliptical sampling spot from the tapping capillary was estimated to be approximately 33 × 68 μm on the basis of the data in Figure 3. The lane scans were used to generate the RGB (red green blue) combined chemical image of three ions, m/z 734.7 (red), m/z 788.7 (green), and m/z 808.5 (blue), each with distinctive distributions within the tissue (Figure 5c). The ion observed at m/z 788.7 [green, potentially (M + H)+ of PC (36:1)] was observed most abundantly in the arbo vitae section of the brain, while m/z 734.7 [red, potentially (M + H)+ of PC (32:0)] had higher abundance in the molecular layer giving a pink hue to this region. A third at m/z 808.5 (blue) dominates

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CONCLUSIONS In this paper, we reported on an advanced tapping-mode SPESI system. Spot sampling, lane scanning, and chemical imaging were carried out in an automated fashion on a variety of compounds, including basic blue 7, crystal violet, cellopentaose, equine heart cytochrome c, and endogenous lipids in mouse brain thin tissue sections. Spatial resolution for the mass spectral chemical images of the brain tissue determined on the basis of the ability to distinguish between features observed both in the optical and mass spectral chemical images was estimated to be approximately 35 μm. This spatial resolution is on par with the very best resolution reported by DESI5,6 and is within a factor of 3.5 for the best sampling resolution reported for nanoDESI.12 Some modifications to the system and further optimization should enable even better spatial resolution to be achieved. These modifications might include the use of smaller internal diameter tapping capillary to limit the wetted sampling area on the surface and chemical or physical modification of this capillary tip to better control the position and volume of the solution being electrosprayed. For example, hydrophobic/ hydrophilic coatings and/or fabrication of a solid point protrusion at the capillary tip might confine the electrospray, 3151

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limiting the solvent volume and flow path on top of the capillary. This could minimize dilution of the extract, increasing analyte signal, and further stabilize the electrospray process as well as minimize contamination possibilities on the sampling capillary. Additionally, incorporating a force-feedback loop into the controlled resonant system would enable control over the capillary/surface interactions during tapping and possibly be used to define physical characteristics of the surface while sampling, adding an additional degree of imaging information. Work will need to be done, however, to determine detection limits for this sampling system for trace compounds, especially in complicated mixtures (e.g., pharmaceuticals in dosed tissues). This should include investigations into the extraction efficiency of the approach and how it might be improved. In any case, the automated spot sampling, lane scanning, and chemical imaging of surfaces using a controlled-resonant surface tapping-mode single capillary liquid junction extraction/ESI emitter offers new possibilities for higher-resolution characterization of the composition of surfaces from materials to biological specimens.

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ASSOCIATED CONTENT

S Supporting Information *

Additional information as noted in 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]. Tel: 865-574-1922. Fax: 865576-8559. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Instrumental implementation and 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 contractor UT-Battelle, LLC. The tissue imaging work was supported by AB Sciex through a Cooperative Research and Development Agreement (CRADA NFE-10-02966).

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dx.doi.org/10.1021/ac404249j | Anal. Chem. 2014, 86, 3146−3152