Constant-Distance Mode Nanospray Desorption Electrospray

Dec 15, 2016 - 2005, 13 (1) 20– 26 DOI: 10.1016/j.tim.2004.11.006. [Crossref], [PubMed] ..... Noah B. Schorr , Zachary T. Gossage , Joaquín Rodríg...
1 downloads 0 Views 1MB Size
Subscriber access provided by UNIV OF REGINA

Article

Constant-Distance Mode Nanospray Desorption Electrospray Ionization Mass Spectrometry Imaging of Biological Samples with Complex Topography Son N. Nguyen, Andrey V. Liyu, Rosalie K. Chu, Christopher R. Anderton, and Julia Laskin Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b03293 • Publication Date (Web): 15 Dec 2016 Downloaded from http://pubs.acs.org on December 16, 2016

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Analytical Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Analytical Chemistry

Constant-Distance Mode Nanospray Desorption Electrospray Ionization Mass Spectrometry Imaging of Biological Samples with Complex Topography Son N. Nguyen1, Andrey V. Liyu2, Rosalie K. Chu2, Christopher R. Anderton2 and Julia Laskin1 1. Physical Sciences Division, Pacific Northwest National Laboratory, Richland, WA, USA 2. Environmental and Molecular Sciences Laboratory, Pacific Northwest National Laboratory, Richland, WA, USA



address correspondence to: Julia Laskin Physical Sciences Division Pacific Northwest National Laboratory PO Box 999, K8-88 Richland, WA 99352 Tel: (509)-3716136 Fax: (509)-3716139 Email: [email protected]

1 ACS Paragon Plus Environment

Analytical Chemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ABSTRACT A new approach for constant-distance mode mass spectrometry imaging (MSI) of biological samples using nanospray desorption electrospray ionization (nano-DESI) was developed by integrating a shearforce probe with the nano-DESI probe. The technical concept and basic instrumental setup, as well as the general operation of the system are described. Mechanical dampening of resonant oscillations due to the presence of shear forces between the probe and the sample surface enabled the constantdistance imaging mode via a computer-controlled closed-feedback loop. The capability of simultaneous chemical and topographic imaging of complex biological samples was demonstrated using living Bacillus subtilis ATCC 49760 colonies on agar plates. The constant-distance mode nano-DESI MSI enabled imaging of many metabolites, including non-ribosomal peptides (surfactin, plipastatin, and iturin) on the surface of living bacterial colonies, ranging in diameter from 10 mm to 13 mm with height variations up to 0.8 mm above the agar plate. Co-registration of ion images to topographic images provided highercontrast images. Based on this effort, constant-mode nano-DESI MSI proved to be ideally suited for imaging biological samples of complex topography in their native states.

KEYWORDS: nanospray desorption electrospray ionization (nano-DESI), mass spectrometry

imaging, shear force microscopy, constant distance, topography profiling, Bacillus subtilis ATCC 49760

2 ACS Paragon Plus Environment

Page 2 of 24

Page 3 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Analytical Chemistry

INTRODUCTION Since 1975, mass spectrometry (MS) has been widely applied to the characterization and identification of bacterial samples.1 Since then, matrix-assisted laser desorption/ionization (MALDI) MS has been regularly used not only for the identification of bacterial strains,2,3 but also for mass spectrometry imaging (MSI) of metabolites released by colonies.4–6 MALDI MSI requires matrix deposition and drying the sample prior to analysis,7 which precludes imaging of live microbial communities. Furthermore, matrix application may result in suppression of metabolite signals in the lower mass range by ions originating from the matrix and formation of matrix adducts of endogenous metabolites extracted from the sample.7 The development of ambient ionization techniques, such as desorption electrospray ionization (DESI)8 and nanospray desorption electrospray ionization (nano-DESI),9 has provided unique opportunities for the analysis of complex chemical and biological samples in their native states.10–12 Because of the high pressure of the nebulizing gas directed toward the sample, DESI analysis of microbial samples directly from the agar-medium plates where they are grown is challenging. Instead, imprinting the colony onto a hydrophilic membrane is often used for DESI imaging of metabolites produced by the colony.13,14 On the other hand, direct liquid extraction techniques15 have proven valuable for metabolic profiling of living bacterial colonies on agar plates.16–21 For example, Watrous et al. demonstrated the spatial profiling of microbial communities using nano-DESI and introduced metabolic networks as a powerful approach for identifying microbial metabolites using tandem mass spectrometry (MS/MS) experiments.16 Lanekoff et al. used spatially resolved nano-DESI to study molecules diffusing from living Synechococcus sp. PCC 7002 colonies to the surrounding agar in a time-course experiment.18 In that study, one-dimensional chemical gradients of metabolites secreted by the colony were mapped using line scans. In a later study, Watrous et al.17 used nano-DESI for imaging metabolites released from single microbial colonies as well as a mixed biofilm of S. oneidensis MR-1 and Bacillus subtilis 3610.17 In that study, samples were partially dehydrated prior to analysis and manual adjustment of the sample’s position relative to the nano-DESI probe was used to follow the topography of the colonies.17 As a result, the spatial resolution was rather low (~1 mm), limited by the complex surface morphology of live microbial colonies.17 In many direct liquid extraction techniques,10,15 the distance between the probe and the sample must be controlled with a high level of precision, especially when imaging samples with complex surface morphology or at high spatial resolution. MSI at a constant probe-to-surface distance enables 3 ACS Paragon Plus Environment

Analytical Chemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

simultaneous mapping of the sample topography and chemical composition and prevents the probe from either crashing or losing contact with the sample. Several approaches for constant-distance mode MSI have been reported in the literature. For example, constant-distance mode nano-DESI MSI of tissue sections was achieved by defining the plane in which this relatively flat sample resides and adjusting the sample height accordingly using an automated XYZ stage.22 In a separate study, an automated system was developed to control the probe-to-surface distance of a liquid micro junction surface sampling probe (LMJ-SSP) using an image-analysis-based concept.23 It tracked the distance between the sampling probe tip and its shadow on the surface by analyzing the image’s brightness. The average liquid microjunction thickness or 36-40 µm maintained using this approach enabled imaging of ink patterns on paper. Recent efforts by several groups have focused on combining ambient MSI and surface probe microscopy, which enables constant-distance mode imaging.24–28 For example, Van Berkel and coworkers24,25,28 successfully integrated a heated atomic force microscopy (AFM) probe with MSI and demonstrated the performance of this hyphenated technique for imaging inked patterns on paper and a Pseudomonas strain GM17 colony on agar. The heated AFM probe acted both as a force sensor for topographical imaging and for thermal desorption of analyte molecules from the substrate followed by either electrospray ionization (ESI)28 or atmospheric pressure chemical ionization (APCI) MS.24,25 Zenobi and co-workers also described an alternative way to integrate MSI and topographic methods.26,27 In these studies, scanning near-field optical microscopy (SNOM) was used for simultaneous mapping sample topography and laser desorption of material from surfaces which was ionized using either plasma ionization (PI)26 or electron ionization (EI) MS.27 The surface topography was obtained either before or after laser desorption with the same SNOM tip using shear force feedback mode. Herein, we report on the development of an alternative approach for simultaneous chemical and topographic imaging of living microbial communities and other samples of complex morphology using nano-DESI MSI. This approach relies on a shear-force-based distance control between the nano-DESI probe and the sample, and can be readily implemented on any mass spectrometer equipped with an ESI or other ambient ionization sources. In addition, shear force microscopy is compatible with any “proximity-probe”-based ionization technique. This development is inspired from earlier works by Brunner et al.29 and Katemann et al.,30 in which a piezoelectric shear force distance control for SNOM29 and scanning electrochemical microscopy (SECM)30 is described. The performance of the new hyphenated technique developed in this study is evaluated by examining chemical signatures of growing

4 ACS Paragon Plus Environment

Page 4 of 24

Page 5 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Analytical Chemistry

microbial colonies of Bacillus subtilis ATCC 49760 on agar plates. These proof-of-principle experiments demonstrate the utility of the shear force microscopy combined with nano-DESI MSI for simultaneous topographic and chemical imaging of complex biological samples of arbitrary morphology over a large area. MATERIALS AND METHODS Preparation of Bacterial Colonies. Colonies of Bacillus Subtilis ATCC 49760 (American Type Culture Collection, Manassas, VA) were prepared according to protocols described in the Supporting Information. Coupling Shear Force Microscopy with Nano-DESI MSI. The shear force scanning probe schematically shown in Figure 1 was fabricated by mechanically attaching two 3 x 3 x 0.55 mm piezoelectric ceramic plates (3.8 MHz, Steiner & Martins, Inc. Doral, FL) to a pulled fused silica capillary (Polymicro Technologies, L.L.C., Phoenix, AZ). The 20 µm OD probe tip was pulled from 0.8 mm x 0.2 mm (OD x ID) fused silica capillary using a laser-based micropipette puller system P-2000 (Sutter Instrument, Novato, CA). The top piezoelectric plate, driven by an Arbitrary Waveform Generator (Agilent 33220A, Agilent Technologies, Santa Clara, CA), induced a low-amplitude vibration of the probe tip. The second plate positioned closer to the sample detected the oscillation amplitude of the probe tip through a lock-in amplifier (Model SR865, Stanford Research Systems, Sunnyvale, CA) that operates in a frequency range of 1 mHz–2 MHz. The signal from the shear-force probe was converted via an analog-to-digital converter (NI USB 6009, National Instrument, Austin, TX) then processed and synchronized with the highresolution motorized XYZ sample stage (Zaber Technologies, Vancouver, BC) using custom-designed LabVIEW software.22 The shear-force probe was integrated into a customized nano-DESI source22 mounted onto an LTQ/Orbitrap XL mass spectrometer (Thermo Fisher Scientific, Waltham, MA) and positioned in close proximity (~5 µm) to the nano-DESI probe. The OD 150 µm x ID 50 µm fused silica capillaries were used to assemble the nano-DESI probe, composed of a primary capillary that delivers the extraction solvent to the sample and a secondary capillary that removes extracted analyte molecules and transfers them to a mass spectrometer inlet. Extraction solvent composed of 50% methanol (Fisher Scientific), 45% water, and 5% acetic acid (Fisher Scientific) was used in this study. Solvent was delivered through the primary capillary at a flow rate of 500 nl/min. A 3.5 kV potential was applied to the syringe needle to produce a Taylor cone at the tip of the secondary capillary positioned ~1 mm away from the instrument’s inlet. The heated capillary inlet was held at 30 V and 250°C. The capillaries (primary, secondary, and shear-force 5 ACS Paragon Plus Environment

Analytical Chemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

probe) were positioned individually using high-resolution micromanipulators (XYZ 500MIM, Quater Research and Development, Bend, OR) and visualized using two Dino-Lite digital microscopes (AnMo Electronics Corporation, Sanchong, New Taipei, Taiwan). Data Collection and Image Processing. For details of imaging experiments/data processing methods, refer to Experimental section in Supporting Information. RESULTS AND DISCUSSION Precise control of the probe-to-surface distance in direct liquid extraction ambient ionization techniques enables reproducible extraction of analyte molecules from complex samples, which is critical for MSI and quantitative analysis.22,31 Furthermore, maintaining constant distance between the probe and the sample helps prevent the probe from crashing or losing contact with the surface, which is particularly important for robust MSI with high spatial resolution and imaging samples with complex morphology. In this study, we have developed an approach that combines scanning shear force microscopy with nanoDESI MSI for simultaneous imaging of the chemical composition and morphology of complex samples. Technical concept and optimization of constant-distance nano-DESI MSI. The combined shear force microscopy/nano-DESI MSI source is shown schematically in Figure 1. Shear force measurements are implemented using an approach described by Schuhmann and coworkers.30 Specifically, an oscillating tip comprising a shear-force probe is incorporated into the nano-DESI source22 to provide accurate measurement of the probe-to-sample distance during MSI experiments. Lateral tip oscillation, induced by a piezoelectric plate attached to the tip (agitation piezo), is monitored using a second piezoelectric plate (detection piezo) located closer to the sample surface. Voltage generated by the second piezo element is detected using a lock-in amplifier. Shear forces between the sample and the tip alter the amplitude of several oscillation modes, which affects the magnitude of the voltage measured at the corresponding frequencies. Constant-distance mode imaging is performed by selecting a frequency of an appropriate mode of oscillation and maintaining its amplitude at a constant value using a computercontrolled closed-feedback loop. Nano-DESI MSI is performed using a standard approach described in detail in previous studies.32 Initially, the shear force measurement was performed using the primary capillary of the nano-DESI probe (for more details of this setup, refer to the Supporting Information). Although generally feasible and well suited for imaging of solid-like samples such as thin tissue sections, this configuration does not give a stable feedback signal for mucous samples, e.g., bacterial colonies. Thus, this study employs a standalone shear-force probe positioned in close proximity to the nano-DESI probe using a high-resolution micromanipulator. The shear-force probe tip resides several micrometers 6 ACS Paragon Plus Environment

Page 6 of 24

Page 7 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Analytical Chemistry

below the lowest point of the nano-DESI probe. This approach produces a stable shear force signal from the probe over the extended period of time that is required for nano-DESI MSI experiments. Moreover, by using a standalone shear-force probe, the working distance can be adjusted to prevent the nano-DESI probe from getting too close to the sample surface, which is particularly important when imaging sticky surfaces of live bacterial colonies. In this study, several shear-force probe configurations are examined. We have determined that probes with smaller tip diameters are more sensitive to surface topography, indicating more efficient dampening of smaller diameter tips. However, highly sensitive shear-force probes are less stable because they sense even the smallest change in surface topography from dust particles or other types of defects. As a result, it may be difficult to maintain the stability of such probes during the imaging process. Similarly, shear-force probes with long, tapered tips tend to be more sensitive to surface topography, but they typically generate low signals because of the longer distance between the end of the tip and the detection piezo. We have found that the most stable shear-force probe has a tip diameter ranging from 5 to 30 µm and a tapered tip length of less than 10 mm. The position of the agitation piezo plate also plays an important role in determining shear-force probe performance. The best probe performance is obtained when the two piezo plates are positioned at the same angle relative to each other and separated by ~15 mm. Although higher signals can be obtained by positioning the agitation piezo closer to the detection piezo plate, it is difficult to observe significant shear force dampening on soft surfaces when the oscillation amplitude exceeds a certain value. In contrast, weak signals generated by moving the two piezo plates too far apart make it difficult to distinguish tip oscillations from noise. Because the entire nano-DESI source is attached to a mass spectrometer, vibrations from the vacuum pumps and other instrument components may interfere with shear force detection. In this study, noise signals associated with instrument vibration are minimized by tightly attaching the nano-DESI source to the mass spectrometer using two mechanical clamps. Furthermore, electrical noises from surrounding instruments, such as cables from microscope cameras or stage motors, also may affect the system. By shielding all wires and cables on the stage and rerouting them away from the shear-force probe, we have been able to substantially reduce the electrical noises. Lastly, the probe performance can also be affected by changes in the shear-force probe tip properties during the imaging experiment, resulting from picking up dust particles and/or sample debris. Although the probe can be easily cleaned using nitrogen gas, the settings must be checked before continuing imaging experiments.

7 ACS Paragon Plus Environment

Analytical Chemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

In order to understand the degree of control and X, Y, Z resolution, we compared topography of a mouse embryo tissue section obtained using our shear-force probe and a standard contact profilometer. The results of this comparison are shown in Figure S2. Good correspondence between topographic images acquired using two different approaches confirms the ability of the shear force probe to accurately map sample’s topography. Frequency scans and approach curve measurements. Shear force microscopy experiments are performed by monitoring the amplitude of a selected resonant probe oscillation that is strongly affected by shear forces between the sample and the probe. Before each imaging experiment, this resonant frequency is identified by acquiring probe excitation spectra away from (in air) and on the sample surface. Typical probe excitation spectra in a frequency range of 70 to 200 kHz are shown in Figure 2A. The top three traces in Figure 2A correspond to the excitation spectra acquired with the shear-force probe kept in air (dashed green trace), on the agar surface (blue trace), and on the colony (red trace). The bottom traces in Figure 2A correspond to difference spectra calculated by subtracting signals obtained in air from those on agar (black trace) and on the colony (gray trace). To assist resonant frequency selection for imaging experiments, difference spectra are automatically calculated by the LabVIEW program and plotted on the same graph. The spectra of two different sample surfaces of interest to this study (agar versus colony) are almost indistinguishable for frequencies below 170 kHz but different at higher frequencies. When comparing frequency spectra obtained in air and on both surfaces, it appears that signals from air and colony are almost identical, while significant dampening of the signal is observed on agar at higher frequencies (>170 kHz). Notably, some parts of a young colony used to generate these excitation spectra are wet and have a liquid-like surface, on which the dampening is expected to be much less pronounced than on a solid surface of agar gel. Difference spectra shown in Figure 2A indicate that some frequencies are sensitive to the agar surface but not to the colony and vice versa. Because nano-DESI MSI experiments probe both the agar and the colony, it is important to select a resonant frequency that shows the same response to both surfaces. Based on this criterion, two resonant frequencies that show similar response to both agar and colony, 158 kHz and 160 kHz, were identified as the best candidates for shear force microscopy measurements. Tip-down approach curves at both frequencies were acquired by constantly monitoring the signal amplitude of the detection piezo at a fixed frequency while moving the shear-force tip toward the surface. The signal amplitude was then plotted as a function of the probe-to-surface distance. Typical approach curves obtained at 158 kHz by moving the shear-force probe toward agar (blue trace) and 8 ACS Paragon Plus Environment

Page 8 of 24

Page 9 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Analytical Chemistry

colony (red trace) are shown in Figure 2B. Similar results (data not shown) also have been obtained at 160 kHz. However, lower signals obtained at this frequency resulted in reduced sensitivity to surface topography. As a result, shear force microscopy experiments were performed at 158 kHz. The almost perfect overlap between the two approach curves shown in Figure 2B indicates that the selected resonant frequency provides the same response to both surfaces. For soft surfaces examined in this study, the approach curves are remarkably sharp, indicating that dampening of the shear forces starts when the shear-force probe is within 4 µm of the surface. Subsequent imaging experiments have been performed by fixing the voltage measured by the detection piezo at 80% of its maximum value. The precision of the system is determined by the repeatability of the stage motors, which is approximately 0.5 µm according to the manufacturer. Based on our current stage setup and the shape of the approach curves under these conditions, the shear-force probe is estimated to be positioned 2 ± 0.5 µm away from the surface. Similar value of repeatability was estimated by examining the approach curves acquired prior to each imaging experiment. Constant-distance mode nano-DESI imaging of Bacillus subtilis ATCC 49760. The performance and capabilities of shear force microscopy combined with nano-DESI MSI are demonstrated by simultaneous imaging the topography and chemical signatures of growing Bacillus subtilis ATCC 49760 colonies on agar plates. Bacillus subtilis is a Gram-positive bacterium naturally found in soil and vegetation, as well as on skin and in the digestive tract of livestock and humans.33 It has been extensively studied as a model organism for understanding microbial synthesis of antibiotics, high-value enzymes, and small metabolites,34–36 as well as sporulation.37–39 It also has been used as a model system for developing MSI approaches to study metabolic exchange between microbial communities.4,14,16,17,40–45 Bacillus subtilis is an ideal model system for evaluating shear-force-based nano-DESI MSI because it produces a wide range of well-characterized compounds and forms colonies of complex topography. In this work, nano-DESI MSI was used to examine molecular signatures of the domesticated strain of Bacillus subtilis ATCC 49760 at three time points: 1 day, 3 days, and 7 days (1D, 3D, and 7D) after inoculation on an LB agar plate. Images were acquired with a spatial resolution of better than 250 µm over 2-3 hours depending on the size of the colony. The relatively short acquisition time was selected to minimize the effect of drying and shrinking of agar during the experiments. In addition, the effect of drying agar on the imaging data was minimized by using relatively thick agar plates (~1 cm) and cutting out a piece of agar substantially larger than the colony. Since agar starts shrinking at the edges, this approach allowed us to acquire images before we observed any substantial effects of agar drying in the

9 ACS Paragon Plus Environment

Analytical Chemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 24

middle of the sample. Excitation spectra and approach curves were acquired for each sample. The best resonant frequency (selected as described earlier) was found to be reproducible between the samples and during probe life. To maintain the distance between the sample and shear-force probe, both the frequency and amplitude are fixed throughout the imaging experiment, and imaging is performed by moving the sample under the probe laterally while acquiring mass spectra. In the beginning of each line scan, the sample stage is slowly moved toward the shear-force probe while monitoring the signal generated by the probe at a fixed resonant frequency (for more details, refer to Movie 1 in Supporting Information). The approach is automatically stopped at a fixed amplitude set point (usually at 70–80% of the value obtained in air) that is maintained throughout the experiment using the closed-loop feedback system. The LabVIEW program developed in this study automatically calculates the slope (∆Vpiezo/∆z) at the set point, which correlates the shear force dampening of the tip oscillation amplitude to the displacement along the z-axis. This magnitude of the slope provides the feedback necessary for on-the-fly adjustments of the probe-to-surface distance according to the observed variations in the signal amplitude. As a result, nano-DESI MSI data are acquired at a constant probe-tosurface distance. For each line scan, topographic data are saved as a .csv text file. Figure 3 depicts the optical and topographic images, along with representative topographic line scans of the Bacillus subtilis ATCC 49760 colonies at different time points (1D, 3D, and 7D). The results show good correspondence between optical and topographic images. The youngest 1D colony (Figure 3A, 3B, and 3C) is ~10 mm in diameter and ~0.2 mm in height. No substantial increase in height and only a slight increase in diameter from ~10 mm to ~12 mm is observed for the 3D colony. Similarly, only a slight increase in diameter to ~13 mm is observed for the 7D colony. However, after 7 days, the surface topography of the colony becomes fairly rough. Specifically, line profile of the 7D colony (Figure 3H) shows substantial variations in colony height from ~0.2 mm to ~0.8 mm. Close examination of both optical and topographic images indicates that the 7D colony developed pronounced ridges. It has been reported that wrinkled structures observed in Bacillus subtilis biofilms are produced by lateral mechanical forces focused in space by localized cell death that results in vertical buckling of the rigid extracellular matrix46 composed of exopolysaccharide (EPS) and proteins.47,48 The mass spectral data of Bacillus ATCC 49760 acquired simultaneously with the topographic image show a number of signals unique to the colony (Figure S1). Figure 4 shows selected ion images obtained for the 1D, 3D, and 7D colonies, while a complete list of all the features observed in the nano-DESI MSI of Bacillus subtilis ATCC 49760 is presented in Table S1. Changes in the metabolic output as a function of

10 ACS Paragon Plus Environment

Page 11 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Analytical Chemistry

the colony’s age also are observed in the spatial distribution of lipopeptides, including surfactin, plipastatin (also known as fengycin), and iturin. These molecules are well-known signature metabolites produced by Bacillus subtilis. Surfactins, non-ribosomal lipopeptides named because of their exceptional surfactant activity, are cyclic lipopeptides known to have antibacterial, antiviral, antifungal, antimycoplasma, and hemolytic activities.45,49–51 Iturins and plipastatins are two other types of lipopeptide antibiotics produced by Bacillus subtilis. Ion images obtained for these species for Bacillus ATCC 49760 colonies at different time points indicate that both surfactins and iturins are produced in high abundance at the earliest time point and localized mainly to the colony’s interior, while plipastatin signals only appear in the 7D colony. Interestingly, all lipopeptides secreted by the 7D colony are strongly enhanced on the radial ridges. This localization is consistent with the previously reported role of surfactins in the growth of aerial structures, or the “wrinkles,” in Bacillus subtilis colonies.52 In addition, the tips of such raised structures are known to serve as preferential sites of sporulation—hence, the accumulation sites of newly produced surfactants.53 For example, it has been demonstrated that domesticated or mutated strains of Bacillus subtilis wild type without surfactant-producing genes failed to form wrinkle structures.52,54 Proof-of-principle experiments presented in this study clearly demonstrate the unique capability of the combined shear force microscopy and nano-DESI MSI for simultaneous imaging of the topography and chemical composition of complex biological samples. It is noteworthy that this approach affords the ability to spatially profile both live microbial colonies and the surrounding agar with height variations across the entire sample, reaching ~0.8 mm. Furthermore, this approach can be used for imaging complex systems over a broad range of lateral dimensions, from micrometers to centimeters. Such flexibility is difficult to achieve using state-of-the-art topographic instruments, including AFM and SNOM. Based on our current stage setup and typical experimental conditions (Scan rate:

40µm/s; electronic response rate: 25 ms; approach curve slope: 0.73 V/µm), we estimate the maximum shear-force probe response rate of 128 µm/s, which enables imaging of surfaces with a maximum angle of ±73o. Although the maximum vertical range (z axis) of our stage is 25mm, the available measurement range is roughly 10mm when we factor in the space occupied by the nano-DESI capillaries and sample holder. Finally we note that the quality of ion images (i.e., higher spatial resolution and stability) is vastly improved by using the constant-distance mode imaging approach, which ensures that the nano-DESI probe follows the surface of the sample without crashing or losing contact. Further improvement in the 11 ACS Paragon Plus Environment

Analytical Chemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 24

contrast of nano-DESI MSI data may be obtained by overlaying ion images with topographic images of the sample surface as shown in Figure 4B, 4D. The combination of chemical ion and topographical images illustrates the correlation between high abundant regions of surfactin-C15 and the elevated regions, or “ridges”, on the colony surface. The fully automated shear force microscopy/nano-DESI MSI approach provides a wealth of information about both the phenotype and chemotype of complex topography samples, which will be used in future studies for chemical imaging of microbial and fungal communities, tissue sections, plant roots, and other biological systems.

CONCLUSIONS In this study, we present a new approach for simultaneous chemical and topographic imaging of biological samples of complex topography using a combination of nano-DESI MSI with shear force microscopy. This approach relies on accurate control of the distance between the nano-DESI probe and the sample using feedback generated by a specially designed shear-force probe. The constant-distance mode nano-DESI MSI capabilities are demonstrated by simultaneously imaging the topography and chemical composition of Bacillus subtilis ATCC 49760 colonies growing on agar plates at different time points. The colonies feature diameters ranging from 10 mm to 13 mm and heights from 0.2 mm to 0.8 mm for the 1D and 7D samples, respectively. The formation of complex aerial structures is observed for the older 7D colonies. The mass spectral data contain many features unique to the colony, including signature lipopeptides (e.g., surfactin, plipastatin, and iturin). The distribution of lipopeptides on the sample surface depends on the colony’s age. Specifically, although a relatively uniform distribution of lipopeptides was observed on the 1D colony’s surface, these compounds are found to be localized to the ridges present in the 7D colony. Furthermore, we demonstrate substantial improvement in the ion image contrast by overlaying ion and topography images. The integrated system developed in this study offers new capabilities for chemical and topography imaging of complex samples in their native states over a broad range of heights and areas, from micrometer up to several centimeters. Future work will focus on nano-DESI imaging of tissue sections with high spatial resolution using shear force measurement as a feedback. We anticipate that, when combined with a sensitive mass analyzer, this approach will enable imaging of biological samples with submicron resolution.

12 ACS Paragon Plus Environment

Page 13 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Analytical Chemistry

ACKNOWLEDGEMENTS The research described in this paper is part of the Chemical Imaging Initiative at Pacific Northwest National Laboratory (PNNL). The research was conducted under the Laboratory Directed Research and Development Program at PNNL, a multi-program national laboratory operated by Battelle for the U.S. Department of Energy (DOE) under Contract DE-AC05-76RL01830. The authors thank Mark Engelhard for help with tissue profilometry. The work was performed using EMSL, a national scientific user facility sponsored by the DOE’s Office of Biological and Environmental Research and located at PNNL.

13 ACS Paragon Plus Environment

Analytical Chemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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

14 ACS Paragon Plus Environment

Page 14 of 24

Page 15 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Analytical Chemistry

FIGURE LEGENDS Figure 1. Experimental setup. Schematic illustration of the constant-distance mode nano-DESI MSI. The custom nano-DESI stage was modified by adding a shear-force probe positioned in close proximity to the nano-DESI probe composed of the primary and secondary capillaries. The shear-force probe was fabricated by attaching two piezoelectric stages to a pulled silica capillary with OD 0.8 mm and ID 0.2 mm. The upper stage (agitation piezo), near the tip holder and connected to a waveform generator, was used to induce tip oscillation. The detection piezo, positioned closer to the pulled tip of the shear-force probe and connected to a lock-in amplifier, was used to measure the amplitude of the shear-force probe vibration. The whole system was operated using a computer-controlled closed-feedback loop. Figure 2. Excitation spectra representing the amplitude of the shear-force probe vibration as a function of the agitation frequency (70–200 kHz). (A) Excitation spectra acquired with the probe kept in air (dashed green trace), positioned on the agar surface (blue trace), and positioned on top of a 1 day-old (1D) Bacillus ATCC 49760 colony (red trace). Black trace represents the difference spectrum between air and agar, while gray trace is a difference spectrum between air and colony. The arrow indicates the resonant frequency selected for imaging experiments. At this frequency, signals obtained from both the agar and colony have the same amplitude that is substantially different from the signal amplitude obtained in air. Panel (B) shows the amplitude of the shear-force probe vibration at a frequency of 158 kHz as a function of the distance between the sample and the probe. Almost identical sharp approach curves were obtained on agar (blue trace) and colony (red trace). Figure 3. Optical (A), (D), and (G) and topography (C), (F), and (I) images obtained for the Bacillus ATCC 49760 colonies at different time points. (B), (E), and (H) feature representative line profiles across topography images. The results are shown for 1 day-old 1D (A, B, C), 3 day-old 3D (D, E, F), and 7 day-old 7D (G, H, I) colonies. Good correspondence is observed between topography and optical images. The growing colonies have diameters ranging from 10 mm to 13 mm and heights ranging from 0.2 mm to 0.8 mm. Figure 4. Results of nano-DESI MSI analysis of signature molecules secreted by Bacillus ATCC 49760 at different time points: 1 day-old (1D) sample (left column), 3 day-old (3D) sample (middle column), and day-old (7D) sample (right column). Near-field microscopy three-dimensional surface topography image of 3 day-old (3D) (A) and 7 day-old (7D) (C) and overlaid (B) and (D) with the interpolated chemical ion image of surfactin-C15 (m/z 1058.6). Scale bars are 2 mm. Intensity scale bar ranges from 0 (dark blue) to 100% (dark red) signal intensity of an individual peak.

15 ACS Paragon Plus Environment

Analytical Chemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

REFERENCES (1)

Anhalt, J.; Fenselau, C. Anal. Chem. 1975, 500 (2), 219–225.

(2)

Seng, P.; Drancourt, M.; Gouriet, F.; La Scola, B.; Fournier, P.-E.; Rolain, J. M.; Raoult, D. Clin Infect Dis 2009, 49 (4), 543–551.

(3)

Bizzini, A.; Durussel, C.; Bille, J.; Greub, G.; Prod’hom, G. J. Clin. Microbiol. 2010, 48 (5), 1549– 1554.

(4)

Liu, W.-T.; Yang, Y.-L.; Xu, Y.; Lamsa, A.; Haste, N. M.; Yang, J. Y.; Ng, J.; Gonzalez, D.; Ellermeier, C. D.; Straight, P. D.; Pevzner, P. A.; Pogliano, J.; Nizet, V.; Pogliano, K.; Dorrestein, P. C. Proc. Natl. Acad. Sci. U. S. A. 2010, 107 (37), 16286–16290.

(5)

Watrous, J. D.; Dorrestein, P. C. Nat. Rev. Microbiol. 2011, 9 (9), 683–694.

(6)

Moree, W. J.; Phelan, V. V; Wu, C.-H.; Bandeira, N.; Cornett, D. S.; Duggan, B. M.; Dorrestein, P. C. Proc. Natl. Acad. Sci. U. S. A. 2012, 109 (34), 13811–13816.

(7)

Yang, J. Y.; Phelan, V. V.; Simkovsky, R.; Watrous, J. D.; Trial, R. M.; Fleming, T. C.; Wenter, R.; Moore, B. S.; Golden, S. S.; Pogliano, K.; Dorrestein, P. C. J. Bacteriol. 2012, 194 (22), 6023–6028.

(8)

Takáts, Z.; Wiseman, J. M.; Gologan, B.; Cooks, R. G. Science (80-. ). 2004, 306 (5695), 471–473.

(9)

Roach, P. J.; Laskin, J.; Laskin, A. Analyst 2010, 135 (9), 2233–2236.

(10)

Venter, A. R.; Douglass, K. A.; Shelley, J. T.; Hasman, G.; Honarvar, E. Anal. Chem. 2014, 86 (1), 233–249.

(11)

Badu-Tawiah, A. K.; Eberlin, L. S.; Ouyang, Z.; Cooks, R. G. Annu. Rev. Phys. Chem. 2013, 64 (1), 481–505.

(12)

Espy, R. D.; Teunissen, S. F.; Manicke, N. E.; Ren, Y.; Ouyang, Z.; van Asten, A.; Cooks, R. G. Anal. Chem. 2014, 86 (15), 7712–7718.

(13)

Song, Y.; Talaty, N.; Datsenko, K.; Wanner, B. L.; Cooks, R. G. Analyst 2009, 134 (5), 838–841.

(14)

Watrous, J.; Hendricks, N.; Meehan, M.; Dorrestein, P. C. Anal. Chem. 2010, 82 (5), 1598–1600.

(15)

Laskin, J.; Lanekoff, I. Anal. Chem. 2016, 88 (1), 52–73.

(16)

Watrous, J.; Roach, P.; Alexandrov, T.; Heath, B. S.; Yang, J. Y.; Kersten, R. D.; van der Voort, M.; Pogliano, K.; Gross, H.; Raaijmakers, J. M.; Moore, B. S.; Laskin, J.; Bandeira, N.; Dorrestein, P. C. 16 ACS Paragon Plus Environment

Page 16 of 24

Page 17 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Analytical Chemistry

Proc. Natl. Acad. Sci. U. S. A. 2012, 109 (26), E1743–E1752. (17)

Watrous, J.; Roach, P. J.; Heath, B. S.; Alexandrov, T.; Laskin, J.; Dorrestein, P. C. Anal. Chem. 2013, 85 (21), 10385–10391.

(18)

Lanekoff, I.; Geydebrekht, O.; Pinchuk, G. E.; Konopka, A. E.; Laskin, J. Analyst 2013, 138 (7), 1971–1978.

(19)

Traxler, M. F.; Watrous, J. D.; Alexandrov, T.; Dorrestein, P. C.; Kolter, R. MBio 2013, 4 (4), 1–12.

(20)

Nguyen, D. D.; Wu, C.-H.; Moree, W. J.; Lamsa, A.; Medema, M. H.; Zhao, X.; Gavilan, R. G.; Aparicio, M.; Atencio, L.; Jackson, C.; Ballesteros, J.; Sanchez, J.; Watrous, J. D.; Phelan, V. V.; van de Wiel, C.; Kersten, R. D.; Mehnaz, S.; De Mot, R.; Shank, E. A.; Charusanti, P.; Nagarajan, H.; Duggan, B. M.; Moore, B. S.; Bandeira, N.; Palsson, B. Ø.; Pogliano, K.; Gutiérrez, M.; Dorrestein, P. C. Proc. Natl. Acad. Sci. U. S. A. 2013, 110 (28), E2611–E2620.

(21)

Shih, C.-J.; Chen, P.-Y.; Liaw, C.-C.; Lai, Y.-M.; Yang, Y.-L. Nat. Prod. Rep. 2014, 31 (6), 739–755.

(22)

Lanekoff, I.; Heath, B. S.; Liyu, A.; Thomas, M.; Carson, J. P.; Laskin, J. Anal. Chem. 2012, 84 (19), 8351–8356.

(23)

Kertesz, V.; Ford, M. J.; Van Berkel, G. J. Anal. Chem. 2005, 77 (22), 7183–7189.

(24)

Ovchinnikova, O. S.; Kjoller, K.; Hurst, G. B.; Pelletier, D. A.; Van Berkel, G. J. Anal. Chem. 2014, 86 (2), 1083–1090.

(25)

Ovchinnikova, O. S.; Tai, T.; Bocharova, V.; Okatan, M. B.; Belianinov, A.; Kertesz, V.; Jesse, S.; Van Berkel, G. J. ACS Nano 2015, 9 (4), 4260–4269.

(26)

Nudnova, M. M.; Sigg, J.; Wallimann, P.; Zenobi, R. Anal. Chem. 2015, 87 (2), 1323–1329.

(27)

Schmitz, T. A.; Gamez, G.; Setz, P. D.; Zhu, L.; Zenobi, R. Anal. Chem. 2008, 80 (17), 6537–6544.

(28)

Ovchinnikova, O. S.; Nikiforov, M. P.; Bradshaw, J. A.; Jesse, S.; Van Berkel, G. J. ACS Nano 2011, 5 (7), 5526–5531.

(29)

Brunner, R.; Bietsch, a.; Hollricher, O.; Marti, O. Rev. Sci. Instrum. 1997, 68 (4), 1769–1772.

(30)

Ballesteros Katemann, B.; Schulte, A.; Schuhmann, W. Chem. - A Eur. J. 2003, 9 (9), 2025–2033.

(31)

Lanekoff, I.; Thomas, M.; Laskin, J. Anal. Chem. 2014, 86 (3), 1872–1880.

(32)

Laskin, J.; Heath, B. S.; Roach, P. J.; Cazares, L.; Semmes, O. J. Anal. Chem. 2012, 84 (1), 141–148. 17 ACS Paragon Plus Environment

Analytical Chemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(33)

Sonenshein, A. L.; Hoch, J. A.; Losick, R. Bacillus Subtilis and Other Gram-positive Bacteria: Biochemistry, Physiology, and Molecular Genetics; American Society Mic Series; American Society for Microbiology, 1993.

(34)

Rob Meima, Jan Maarten van Dijl, Siger Holsappel, and S. B. In Protein expression technologies: current status and future trends; Baneyx, F., Ed.; Horizon Bioscience, 2004; pp 199–252.

(35)

Perkins, John B., Markus Wyss, H.-P. H. and U. S. In The metabolic pathway engineering handbook: Fundamentals; Smolke, C., Ed.; CRC press, 2009; Vol. 1, pp 23–1.

(36)

Schallmey, M.; Singh, A.; Ward, O. P. Can. J. Microbiol. 2004, 50 (1), 1–17.

(37)

Errington, J. Nat Rev Microbiol 2003, 1 (2), 117–126.

(38)

Piggot, P. J.; Hilbert, D. W. Curr. Opin. Microbiol. 2004, 7 (6), 579–586.

(39)

Stragier, P.; Losick, R. Annu. Rev. Genet. 1996, 30, 297–41.

(40)

Gonzalez, D. J.; Haste, N. M.; Hollands, A.; Fleming, T. C.; Hamby, M.; Pogliano, K.; Nizet, V.; Dorrestein, P. C. Microbiology 2011, 157 (9), 2485–2492.

(41)

Watrous, J. D.; Phelan, V. V; Hsu, C.-C.; Moree, W. J.; Duggan, B. M.; Alexandrov, T.; Dorrestein, P. C. ISME J. 2013, 7 (4), 770–780.

(42)

Hsu, C. C.; Elnaggar, M. S.; Peng, Y.; Fang, J.; Sanchez, L. M.; Mascuch, S. J.; M??ller, K. A.; Alazzeh, E. K.; Pikula, J.; Quinn, R. A.; Zeng, Y.; Wolfe, B. E.; Dutton, R. J.; Gerwick, L.; Zhang, L.; Liu, X.; Mansson, M.; Dorrestein, P. C. Anal. Chem. 2013, 85 (15), 7014–7018.

(43)

Yang, Y.-L.; Xu, Y.; Straight, P.; Dorrestein, P. C. Nat. Chem. Biol. 2009, 5 (12), 885–887.

(44)

Yang, Y. L.; Xu, Y.; Kersten, R. D.; Liu, W. T.; Meehan, M. J.; Moore, B. S.; Bandeira, N.; Dorrestein, P. C. Angew. Chemie - Int. Ed. 2011, 50 (26), 5839–5842.

(45)

Debois, D.; Hamze, K.; Guérineau, V.; Le Caër, J. P.; Holland, I. B.; Lopes, P.; Ouazzani, J.; Séror, S. J.; Brunelle, A.; Laprévote, O. Proteomics 2008, 8 (18), 3682–3691.

(46)

Asally, M.; Kittisopikul, M.; Rué, P.; Du, Y.; Hu, Z.; Çağatay, T.; Robinson, A. B.; Lu, H.; GarciaOjalvo, J.; Süel, G. M. Proc. Natl. Acad. Sci. U. S. A. 2012, 109 (46), 18891–18896.

(47)

Branda, S. S.; Vik, ??shild; Friedman, L.; Kolter, R. Trends Microbiol. 2005, 13 (1), 20–26.

(48)

Marvasi, M.; Visscher, P. T.; Casillas Martinez, L. FEMS Microbiol. Lett. 2010, 313 (1), 1–9.

18 ACS Paragon Plus Environment

Page 18 of 24

Page 19 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Analytical Chemistry

(49)

Pathak, K. V.; Keharia, H. 3 Biotech 2013, 4 (3), 283–295.

(50)

Peypoux, F.; Bonmatin, J. M.; Wallach, J. Appl. Microbiol. Biotechnol. 1999, 51 (5), 553–563.

(51)

Angelini, T. E.; Roper, M.; Kolter, R.; Weitz, D. a; Brenner, M. P. Proc. Natl. Acad. Sci. U. S. A. 2009, 106 (43), 18109–18113.

(52)

Straight, P. D.; Willey, J. M.; Kolter, R. J. Bacteriol. 2006, 188 (13), 4918–4925.

(53)

Branda, S. S.; González-Pastor, J. E.; Ben-Yehuda, S.; Losick, R.; Kolter, R. Proc. Natl. Acad. Sci. U. S. A. 2001, 98 (20), 11621–11626.

(54)

Branda, S. S.; González-Pastor, J. E.; Ben-Yehuda, S.; Losick, R.; Kolter, R. Proc. Natl. Acad. Sci. U. S. A. 2001, 98 (20), 11621–11626.

19 ACS Paragon Plus Environment

Analytical Chemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 1. Experimental setup. Schematic illustration of the constant-distance mode nano-DESI MSI. The custom nano-DESI stage was modified by adding a shear-force probe positioned in close proximity to the nano-DESI probe composed of the primary and secondary capillaries. The shear-force probe was fabricated by attaching two piezoelectric stages to a pulled silica capillary with OD 0.8 mm and ID 0.2 mm. The upper stage (agitation piezo), near the tip holder and connected to a waveform generator, was used to induce tip oscillation. The detection piezo, positioned closer to the pulled tip of the shear-force probe and connected to a lock-in amplifier, was used to measure the amplitude of the shear-force probe vibration. The whole system was operated using a computer-controlled closed-feedback loop. Figure 1, 112x71mm (300 x 300 DPI)

ACS Paragon Plus Environment

Page 20 of 24

Page 21 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Analytical Chemistry

Figure 2. Excitation spectra representing the amplitude of the shear-force probe vibration as a function of the agitation frequency (70–200 kHz). (A) Excitation spectra acquired with the probe kept in air (dashed green trace), positioned on the agar surface (blue trace), and positioned on top of a 1 day-old (1D) Bacillus ATCC 49760 colony (red trace). Black trace represents the difference spectrum between air and agar, while gray trace is a difference spectrum between air and colony. The arrow indicates the resonant frequency selected for imaging experiments. At this frequency, signals obtained from both the agar and colony have the same amplitude that is substantially different from the signal amplitude obtained in air. Panel (B) shows the amplitude of the shear-force probe vibration at a frequency of 158 kHz as a function of the distance between the sample and the probe. Almost identical sharp approach curves were obtained on agar (blue trace) and colony (red trace). Figure 2, Figure 2A, Figure 2B 111x147mm (300 x 300 DPI)

ACS Paragon Plus Environment

Analytical Chemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Paragon Plus Environment

Page 22 of 24

Page 23 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Analytical Chemistry

Figure 3. Optical (A), (D), and (G) and topography (C), (F), and (I) images obtained for the Bacillus ATCC 49760 colonies at different time points. (B), (E), and (H) feature representative line profiles across topography images. The results are shown for 1 day-old 1D (A, B, C), 3 day-old 3D (D, E, F), and 7 day-old 7D (G, H, I) colonies. Good correspondence is observed between topography and optical images. The growing colonies have diameters ranging from 10 mm to 13 mm and heights ranging from 0.2 mm to 0.8 mm. Figure 3, Figure 3A, Figure 3B 109x137mm (300 x 300 DPI)

ACS Paragon Plus Environment

Analytical Chemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 4. Results of nano-DESI MSI analysis of signature molecules secreted by Bacillus ATCC 49760 at different time points: 1 day-old (1D) sample (left column), 3 day-old (3D) sample (middle column), and day-old (7D) sample (right column). Near-field microscopy three-dimensional surface topography image of 3 day-old (3D) (A) and 7 day-old (7D) (C) and overlaid (B) and (D) with the interpolated chemical ion image of surfactin-C15 (m/z 1058.6). Scale bars are 2 mm. Intensity scale bar ranges from 0 (dark blue) to 100% (dark red) signal intensity of an individual peak. Figure 4; Figure 4B, 4D 88x44mm (300 x 300 DPI)

ACS Paragon Plus Environment

Page 24 of 24

Page 25 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Analytical Chemistry

For TOC only 44x23mm (300 x 300 DPI)

ACS Paragon Plus Environment