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This work was supported by funds from the National Institutes of Health (1R01AI177469), National Science Foundation (2305182), Chan Zuckerberg Initiative, and Department of Defense (DoD OC220161).

OVCAR-8 cells were obtained from the American Type Culture Collection (ATCC). All cell culture procedures were performed in accordance with institutional biosafety guidelines. Cells were routinely tested for mycoplasma contamination using a mycoplasma detection kit according to the manufacturer’s instructions.
NOTE: Perform all procedures in accordance with institutional biosafety guidelines. Wear appropriate personal protective equipment. Handle organic solvents in a chemical fume hood.
1. Culture and prepare OVCAR-8 cells on gridded coverslips 
<ol>
	<li>Prepare complete medium by supplementing RPMI-1640 medium with 10% fetal bovine serum and 1% penicillin–streptomycin. Warm the medium to 37°C.</li>
	<li>Maintain OVCAR-8 cells in 10 cm cell culture dishes containing 10 mL of complete medium. Incubate the cells at 37°C in a humidified incubator with 5% CO2.</li>
	<li>Passage the cells when approximately 80% of the culture surface is visually occupied by adherent cells, typically every 2 days under the described culture conditions.
	<ol>
		<li>Aspirate the medium from the 10 cm culture dish and rinse the cells once with 5 mL of 1× phosphate-buffered saline.</li>
		<li>Add 2 mL of 0.25% trypsin–ethylenediaminetetraacetic acid to the culture dish. Incubate the cells at 37°C for approximately 3 min, or until the majority of cells visibly detach from the culture surface when examined under a microscope.</li>
		<li>Add 8 mL of pre-warmed complete medium to the dish to neutralize the trypsin. Using a 10 mL serological pipette, gently pipette the cell suspension up and down approximately 15–20 times to disperse the cells.</li>
		<li>Transfer 1 mL of the suspension into 9 mL of fresh complete medium.</li>
		<li>Count the cells using an automated cell counter. Load 10 µL of the resuspended cell suspension directly into the counting slide without additional dilution and perform cell counting according to the manufacturer’s instructions. 
		​NOTE: Cell viability dyes such as trypan blue were not used in this protocol. Assess cell condition visually by monitoring cell morphology and attachment before trypsinization.</li>
	</ol>
	</li>
	<li>Seed the cells on the coverslips.
	<ol>
		<li>Dilute the cells to a final concentration of ~1 × 106 cells/mL using complete medium. Calculate the required volume of cell suspension using C1V1 = C2V2, where C1 is the counted cell concentration, V1 is the volume of cell suspension required, C2 is the target concentration, and V2 is the desired final volume.</li>
		<li>Place individual 28 mm gridded glass coverslips with a 50 µm grid spacing into wells of a 6-well plate. 
		NOTE: Autoclave the glass coverslips before use for cell culture. Place the grid side facing downward to minimize interference with cell growth.</li>
		<li>Add 2 mL of complete medium and 200 µL of the cell suspension (~2 × 105 cells/well) to each well. Gently rock the 6-well plate in an “X” pattern to evenly distribute the cells across the coverslip surface.</li>
		<li>Incubate the cells for approximately 15–20 h to allow cell attachment. 
		NOTE: Monitor cell morphology routinely using a microscope to ensure normal cell growth. Examine potential mycoplasma contamination using a mycoplasma detection kit if the cells grow poorly.</li>
	</ol>
	</li>
</ol>
2. Cell washing with ammonium formate (AF) solution 
NOTE: Prepare fresh AF solution before each use.
<ol>
	<li>Prepare isotonic AF wash buffer immediately before use. Weigh 0.45 g of AF and dissolve it in 50 mL of LC/MS-grade water to prepare a final AF concentration of 0.14 M (0.9% w/w). Do not adjust the pH5,45. 
	NOTE: Use the freshly prepared AF solution on the same day of preparation. Keep the solution ice-cold (0°C–4°C) until use.</li>
	<li>Aspirate the culture medium from each well of the 6-well plate. Immediately add 2 mL of cold AF solution to each well.</li>
	<li>Using sterile forceps, gently lift each coverslip containing adherent cells from the well while maintaining the cell-coated surface facing upward. Briefly place the coverslip for 2–3 s into a separate well pre-filled with 2 mL of cold 0.9% AF solution.</li>
	<li>Repeat the rinsing process for a total of two rinses using fresh AF solution for each rinse. After the final rinse, remove the coverslip from the AF solution and allow it to air dry at room temperature in a chemical fume hood for approximately 20–30 min before DESI–MSI analysis. 
	NOTE: Rinse the cells gently to avoid disturbing the cells during the washing step.</li>
</ol>
3. Fabrication of modified FAIMS-based MS interface 
<ol>
	<li>Disassemble the FAIMS Pro electrode ion source housing adaptor to access the internal electrode region. Confirm that the adaptor is compatible with Thermo Orbitrap mass spectrometers equipped with the FAIMS Pro interface configuration.</li>
	<li>Remove the FAIMS electrode components, including the internal wires, electrode assemblies, O-rings, and supporting internal structures. Retain the external housing shell, mounting flanges, and the interface detection pin required for mass spectrometer recognition of the adaptor.</li>
	<li>Modify the circular opening using standard machine-shop machining procedures. Enlarge the opening until it provides sufficient clearance around the protruding Orbitrap inlet/ion funnel assembly.</li>
	<li>Smooth and deburr the machined edge after cutting. Confirm that the modified opening allows unobstructed positioning of the housing adaptor on the mass spectrometer.</li>
	<li>Modify the length of two legs of the housing adaptor using a saw. Trim the legs to a final length of approximately 2 ¼ in to allow connection with the optical breadboard. 
	NOTE: Use appropriate machining tools and avoid damaging the structural integrity of the remaining housing.</li>
	<li>Prepare an aluminum optical breadboard (10 in × 12 in × 0.5 in, 1/4"-20 tapped holes). Position the breadboard horizontally beneath the modified FAIMS adaptor.</li>
	<li>Use two mounting brackets to connect the modified FAIMS adaptor to the optical breadboard. Trim the brackets as needed to reduce weight and allow proper positioning of the breadboard relative to the Orbitrap inlet.</li>
	<li>Secure the modified FAIMS interface onto the optical breadboard using mechanically compatible mounting hardware aligned with the 1/4"-20 tapped hole pattern of the breadboard. Ensure stable positioning during imaging experiments. 
	NOTE: Ensure that the interface is rigidly fixed to minimize vibration during stage movement and data acquisition.</li>
	<li>Machine or cut a shallow groove into the optical breadboard directly beneath the FAIMS housing contact region. Size and position the groove to provide sufficient clearance for stable seating and alignment of the modified FAIMS housing relative to the Orbitrap inlet. 
	NOTE: Size the groove to accommodate the base of the FAIMS housing adaptor and improve positional reproducibility.</li>
	<li>Align the mounted interface with the mass spectrometer inlet. Confirm that the retained mounting flanges seat properly on the instrument and that the optical breadboard remains level beneath the interface.</li>
	<li>Verify that the modified circular opening provides unobstructed clearance around the Orbitrap inlet/ion funnel assembly before installing the DESI sprayer.</li>
</ol>
4. Assemble the DESI sprayer and Orbitrap interface 
<ol>
	<li>Mount the DESI sprayer onto the integrated setup consisting of the customized FAIMS frame, motorized XYZ stage, gas regulator, and optical breadboard. Use a motorized stage controlled by a CONEX-MFACC controller.</li>
	<li>Define the x-axis as the raster scan direction from left to right and define the y-axis as the line-step direction from top to bottom. Set the z-axis position before acquisition and keep it fixed during imaging.</li>
	<li>Use a stage travel range of approximately 1 mm in the x-direction and 1 mm in the y-direction. Do not apply z-axis movement during acquisition.</li>
	<li>Position the DESI sprayer at an angle of approximately 70° relative to the sample surface. Place the sample directly below the sprayer and position the sprayer tip as close to the Orbitrap inlet as possible without physical contact.</li>
	<li>Connect the DESI grounding metal to a ground source beneath the mass spectrometer. Control raster movement using the stage-control software (Figure 1). 
	NOTE: The DESI sprayer used in this study is available only for users with DESI–MSI instruments.</li>
	<li>Use a motorized XYZ stage controller for stage movement and positioning. Connect the voltage line, nitrogen gas line, solvent line, and grounding line before operation.</li>
	<li>Install a custom-fabricated stainless-steel ion transfer tube (1/16 in OD × 0.033 in ID) designed to match the Orbitrap ion source inlet. Position the ion transfer tube such that it aligns directly with the Orbitrap inlet while extending outward sufficiently to allow unobstructed DESI plume access. 
	NOTE: Customize the ion transfer tube according to the mass spectrometer configuration. Fabricate the ion transfer tube using appropriate machining procedures.</li>
	<li>Use an ion transfer tube with a bent geometry to improve positioning of the DESI sprayer relative to the sample surface and Orbitrap inlet.</li>
	<li>Align the sprayer at an angle of approximately 70° relative to the sample surface so that the DESI spray plume intersects the Orbitrap inlet region without contacting the sample surface.</li>
	<li>Define a stable spray plume by the presence of stable mass spectra and consistent normalized level (NL) values during acquisition. Adjust the sprayer position until a stable ion signal is observed.</li>
	<li>Set the sprayer-to-surface distance to approximately 1.0–1.5 mm as a starting condition. Manually fine-adjust the sprayer position while monitoring NL values to achieve the highest stable ion signal.</li>
</ol>
<img alt="Thermo Orbitrap setup with FAIMS adaptor, nanoLC, DESI sprayer; for mass spec analysis." class="xfigimg" src="/files/ftp_upload/71691/71691fig1.jpg" data-altupdatedat="1788304762000" data-alt="Figure 1"> 
Figure 1. Integrated desorption electrospray ionization (DESI)–mass spectrometry imaging (MSI) setup coupled to an Orbitrap mass spectrometer. The DESI sprayer is mounted on an integrated platform consisting of a modified high-field asymmetric waveform ion mobility spectrometry (FAIMS) adaptor, a motorized XYZ stage, a gas regulator, and an optical breadboard. Solvent is delivered using a nano-liquid chromatography system, and nitrogen gas is supplied at approximately 12 psi for spray stabilization. The figure also shows the ionization voltage connection, solvent line, and positioning of the sprayer relative to the Orbitrap inlet. <a href="https://www.jove.com/files/ftp_upload/71691/71691fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
5. Configure solvent delivery and spray conditions 
<ol>
	<li>Prepare fresh spray solvent consisting of methanol/water/formic acid (98:2:0.1, v/v/v) before DESI–MSI analysis. 
	NOTE: Other DESI-compatible solvent systems, including acetonitrile-based solvents and different methanol/water ratios, may be used after optimization. Methanol/water/formic acid (98:2:0.1, v/v/v) was used as the standard starting condition in this protocol because it provided stable plume formation, reduced solvent spreading, and reproducible positive-ion-mode lipid signals during single-cell DESI–MSI experiments. 
	CAUTION: Methanol is flammable and toxic. Formic acid is corrosive. Handle the solvents in a chemical fume hood.</li>
	<li>Deliver the solvent using an Ultimate 3000 nano-LC system operated as an isocratic solvent pump at 750 nL/min for the reported experiments. Connect the nano-LC outlet to the DESI sprayer using PEEK tubing (1/16 in OD × 0.005 in ID).</li>
	<li>Install a C18 column (e.g., nanoEase M/Z Peptide BEH; 130 Å, 1.7 µm, 300 µm × 150 mm) in-line to provide sufficient backpressure for stable spray formation at low flow rates.</li>
	<li>Set the mass spectrometer method to positive ion mode with Orbitrap detection, a spray voltage of 0.85 kV, Orbitrap resolution of 120,000 at m/z 200, and an m/z range of 150–1200.</li>
	<li>Set the normalized AGC target to 100%, maximum injection time to 100 ms, RF lens to 45%, and microscans to 1. Disable quadrupole isolation and source fragmentation.</li>
	<li>Supply nitrogen sheath gas from a nitrogen generator or liquid nitrogen source at approximately 12 psi to stabilize the DESI spray plume. Use 12 psi as the starting operating pressure and make minor manual adjustments only if needed to improve NL values and maintain a stable mass spectrum.</li>
</ol>
6. Perform raster imaging and pixel control 
<ol>
	<li>Identify a region of interest containing optically visible single cells using the gridded coverslips as a reference. Select isolated cells with clear visual separation from neighboring cells, intact adherent morphology, and no obvious overlap with debris or cell clusters.</li>
	<li>Define the raster region using Fance Stage Controller software46. Set the raster step size to 0.01 mm, the stage velocity to 0.01 mm/s, and the dwell time to 20 s.</li>
	<li>Use left-to-right raster scanning across each line followed by movement to the next line in the y-direction. Set the orthogonal step size between raster lines to 10 µm.</li>
	<li>Adjust the stage velocity according to the Orbitrap acquisition rate to achieve the desired scan-direction pixel dimension (e.g., approximately 2.5–6.5 µm). Calculate the scan-direction pixel dimension using the following relationship: scan-direction pixel dimension = stage velocity × acquisition time per scan.</li>
	<li>Using a stage velocity of 0.01 mm/s (10 µm/s), acquire 376 scans over 102 s to produce an acquisition time of 0.271 s per scan and a corresponding scan-direction pixel dimension of approximately 2.7 µm. Synchronize stage movement and mass spectral acquisition by setting the Orbitrap acquisition timing slightly shorter than the combined stage movement and dwell timing parameters. 
	NOTE: The orthogonal pixel dimension is determined by the programmed step size and the physical and mechanical limitations of the stage, whereas the scan-direction dimension is governed by stage velocity and spectral acquisition rate.</li>
	<li>Acquire imaging data over the selected mass range until completion of the predefined raster region. For a 1 mm × 1 mm imaging region acquired using a 0.01 mm line step and 0.01 mm/s stage velocity, the total acquisition time is approximately 4–5 h.</li>
	<li>Generate RAW data files during acquisition and stop acquisition after completion of the specified x–y raster coordinates and corresponding instrument method duration. Under the positive-ion-mode conditions described here, each raster line typically generates an approximately 50 MB RAW file, resulting in approximately 5 GB total data for 101 raster lines.</li>
</ol>
7. Optical-to-MS image registration 
<ol>
	<li>Capture brightfield images of the cells in the selected grid region after AF washing and drying and before DESI acquisition using a brightfield microscope equipped with a 10× objective.</li>
	<li>Convert RAW files to mzML files using msConvert from ProteoWizard48. Convert the mzML files to imzML format using imzML Converter49.</li>
	<li>Use centroid/profile conversion settings consistent with the acquired data format. Upload the imzML files directly to METASPACE (https://metaspace2020.eu)50 for image visualization.</li>
	<li>Do not apply normalization, hotspot removal, or additional image-processing corrections before visualization. Manually align the optical and MS images using the grid landmarks as positional references.</li>
	<li>Scale the optical image using the known image dimensions and grid spacing. Manually overlay the MS image to assess correspondence between optically visible cell positions and localized ion signals.</li>
	<li>Confirm single-cell localization by visual agreement between isolated cells in the brightfield image and corresponding MS ion distributions.</li>
</ol>

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The authors have no conflicts of interest to disclose.

This protocol enables ambient single-cell DESI&#8211;MSI using high-mass-resolution Orbitrap mass spectrometers through the integration of precise stage control, nanoscale solvent delivery, optical targeting, and high-spatial-resolution mass analysis. The combination of these elements addresses multiple technical barriers that have historically limited the application of DESI&#8211;MSI at the single-cell level. In particular, coordinated control of spray stability, ion transmission, and acquisition timing enables reliable detection of molecular signals from extremely small sampling volumes corresponding to individual cells. A central factor governing performance in this workflow is the stability and confinement of the DESI spray plume. At micrometer-scale spatial resolutions, small perturbations in solvent flow rate, gas pressure, or sprayer positioning can significantly alter the effective sampling area. The use of nanoscale solvent delivery combined with appropriate backpressure improves spray stability and reduces solvent spreading, thereby enhancing spatial fidelity. In addition, maintaining a consistent sprayer-to-surface distance and angle is critical for reproducible ionization efficiency. Even minor deviations in geometry can lead to substantial variation in signal intensity, particularly when analyzing heterogeneous cellular samples. In practice, optimization of DESI spray conditions was performed empirically by monitoring total ion current, spectral stability, and NL values (typically around 105&#8211;106) during acquisition. Reproducible signal quality was achieved through iterative adjustment of sprayer position, solvent-flow stability, gas pressure, and alignment of the DESI plume relative to the Orbitrap inlet.
Ion transmission efficiency represents another key limitation at the single-cell scale because analyte abundance is inherently low within individual cells. At these spatial scales, efficient ion transmission may become a greater practical limitation than ionization efficiency in some implementations because only a very small fraction of desorbed ions ultimately reaches the mass spectrometer inlet. Efficient transfer of ions from the sampling region into the mass spectrometer inlet is therefore essential. The modified FAIMS housing adaptor used in this protocol provides an interface between the DESI spray source and a Thermo mass spectrometer, allowing compatibility with multiple instrument models, including Orbitrap Tribrid, Exploris, and Astral series platforms. Integration of the modified FAIMS housing adaptor with an optical breadboard also provides mechanical stability, thereby minimizing misalignment during extended imaging experiments. Synchronization between stage motion and mass spectral acquisition is equally critical for achieving high spatial resolution. In DESI&#8211;MSI, the pixel dimension in the scan direction is governed by the relationship between stage velocity and acquisition rate, whereas the orthogonal dimension is defined by the programmed step size. Accurate control of these parameters enables precise sampling. Because many mammalian cells typically range from approximately 5&#8211;20 &#181;m in diameter, pixel dimensions approaching only a few micrometers are required to minimize signal overlap between adjacent cells and improve localization of molecular features to individual cellular regions. However, the nominal pixel size does not necessarily correspond to the true sampling footprint because interaction of the solvent plume with the sample surface can introduce lateral spreading and partial signal overlap between adjacent pixels. Reported pixel dimensions should therefore be interpreted in the context of effective spatial resolution rather than strictly geometric definitions. The customized ion transfer tube used in this study was designed according to the geometry of the standard ion transfer tube to preserve ion transmission characteristics while enabling integration with the DESI source configuration.
High mass resolving power and mass accuracy provided by Orbitrap mass spectrometers are essential for resolving the complex mixture of metabolites and lipids present within individual cells. Many detected molecular features were tentatively annotated using accurate mass measurements and METASPACE analysis. Because multiple candidate assignments may correspond to a single measured m/z value, additional MS/MS validation and orthogonal molecular-confirmation strategies may further strengthen molecular identification in future studies. In positive ion mode, DESI spectra are often dominated by phospholipid species with closely spaced or isobaric m/z values. High-resolution Orbitrap detection enables differentiation of these species, thereby improving molecular specificity and reducing spectral ambiguity. This capability is particularly important for single-cell analyses, where reliance on a limited number of ions may lead to misinterpretation of cellular heterogeneity. This adaptability suggests that similar strategies may be applicable to other mass spectrometry platforms using corresponding interfaces with appropriate modifications. Despite these advantages, several limitations should be considered. First, the DESI source used in the current work is available only for users with DESI&#8211;MSI systems, and construction of DESI sprayer/Orbitrap systems may therefore be limited by DESI source availability. However, alternative DESI devices, including DEFFI, may also be implemented for high-spatial-resolution MSI studies42,47. Second, implementation of this platform requires significant customization, including machining of instrument interfaces and fabrication of mounting components. These requirements may limit accessibility for laboratories without experience in instrument development or mechanical fabrication. In the current implementation, the FAIMS interface housing served as a convenient mechanical mounting platform for positioning the DESI sprayer assembly relative to the Orbitrap inlet. Consequently, implementation of the described configuration is more practical for laboratories with access to FAIMS-compatible Orbitrap systems and basic machining support. However, the broader concept of mechanically coupling DESI sources to Orbitrap inlets can be achieved using alternative designs without modifying the FAIMS interface hardware. Third, although low solvent flow rates improve spatial confinement, they may also reduce overall ion yield, necessitating careful optimization to balance sensitivity and spatial resolution.
Another important consideration is the potential for analyte delocalization during sampling. Although DESI operates under ambient conditions and does not require matrix application, interaction of solvent droplets with the sample surface can lead to redistribution of analytes, particularly for highly soluble species. Minimizing solvent volume and optimizing spray conditions are therefore critical for preserving spatial integrity at the single-cell level. Similarly, the washing procedure using isotonic ammonium formate can reduce salt-associated ion suppression while maintaining cell integrity and preserving cellular species, as previously reported in single-cell MS workflows5.
Future developments may further improve the performance and applicability of this platform. For example, integration with faster data-acquisition systems could enable smaller effective pixel sizes and higher-throughput MSI experiments. Implementation of automated alignment systems or real-time feedback control may reduce variability and improve reproducibility. In addition, combining this approach with complementary techniques, such as fluorescence imaging or targeted labeling strategies, could enhance cell-type identification and enable multimodal analyses.
Beyond technical considerations, this platform has broad implications for biological and biomedical research. The ability to characterize molecular profiles at the level of individual cells under ambient conditions enables new opportunities for studying cellular heterogeneity in complex systems. DESI&#8211;MSI has already been successfully applied at the tissue level in studies of tumor microenvironments, neurological disease, host&#8211;pathogen interactions, and drug-resistant cellular populations12,15,16,35. Extending DESI&#8211;MSI to the single-cell level may further improve the ability to delineate localized chemical heterogeneity between neighboring cells, identify rare cellular subpopulations, and discover molecular markers that may otherwise be obscured in bulk tissue measurements. However, the present study was performed using a simplified monolayer cell-culture model, and additional studies are needed to demonstrate compatibility with other complex biological systems, including different cell models, tissues, organoids, and 3D cultures. The current workflow may provide a foundation for extending high-spatial-resolution DESI measurements toward increasingly heterogeneous biological environments following further validation and optimization. Because this method preserves spatial context while providing detailed molecular information, it can serve as a bridge between imaging and omics-based approaches. In summary, this protocol establishes a flexible and adaptable framework for achieving single-cell-resolution DESI&#8211;MSI on Orbitrap mass spectrometers. By addressing key challenges related to ionization efficiency, ion transmission, spatial control, and mass resolution, this approach enables detailed molecular characterization of individual cells. While further optimization and validation are needed, the methodology provides a foundation for expanding the use of ambient MSI in single-cell and spatial metabolomics studies.

Metabolomics seeks to comprehensively characterize small-molecule metabolites that reflect the functional state of biological systems1,2,3,4,5. Because metabolites are direct products of enzymatic activity, they provide a dynamic readout of cellular phenotype and biochemical regulation. However, conventional metabolomic workflows typically rely on bulk extraction methods that average signals across thousands to millions of cells, thereby obscuring cellular heterogeneity6,7,8.
Single-cell metabolomics addresses this limitation by enabling molecular profiling at the level of individual cells, revealing metabolic diversity within complex populations and improving mechanistic understanding of disease progression, drug response, and cellular differentiation. Mass spectrometry (MS) is an indispensable tool for molecular analysis of biological samples. Recent advancements in MS techniques, including sample preparation, instrumentation, and data analysis, enable MS metabolomics studies to be performed at the single-cell level9,10,11,12.
MS imaging (MSI), which enables spatially resolved molecular analysis of biological samples, integrates precise sample motion, minimized sampling, efficient ionization, and data visualization by acquiring a full mass spectrum at each defined spatial coordinate across a sample surface12,13,14. MS images are reconstructed by plotting the intensity of selected mass-to-charge ratio (m/z) values across x&#8211;y coordinates, thereby preserving chemical spatial information within complex biological systems12,13,15,16,17,18. Achieving spatially resolved single-cell metabolomics requires analytical platforms capable of preserving molecular localization while maintaining sufficient sensitivity and mass resolution to detect low-abundance species9,12,19,20.
Single-cell MSI presents additional challenges because individual cells typically range from 5 to 20 &#181;m in diameter8,21. At this spatial scale, analyte abundance per sampling event is extremely limited. Therefore, multiple factors, including accurate control of sample motion, minimized sampling, high ionization efficiency, and efficient ion transmission, are equally essential to obtain interpretable molecular information from individual cells14,22. Matrix-assisted laser desorption/ionization (MALDI)&#8211;MSI, a vacuum-based technique routinely used for high-spatial-resolution MSI studies, requires matrix deposition and vacuum operation; however, matrix crystallization in MALDI can introduce analyte delocalization and variability at the single-cell scale23,24,25,26,27. In contrast, ambient MSI approaches operate under ambient conditions, and samples maintain near-native chemical states because little or no sample preparation is required. Among ambient MSI methods, DESI28, nano-DESI21, pneumatically assisted nano-DESI29, and Single-probe8,30,31,32,33,34,35, have previously been used for single-cell MSI studies. Although techniques based on micro-liquid extraction, including nano-DESI, pneumatically assisted nano-DESI, and Single-probe, provide high sampling and ionization efficiency, they rely on maintaining a stable liquid bridge at the sampling probe tip, whereas perturbation of the liquid bridge, such as changes in surface morphology or probe clogging, can affect the quality of MSI experiments5,8,21,30,35.
DESI is an ambient ionization technique in which charged solvent droplets impact a surface, extract analytes, and generate secondary droplets containing ionized species36. DESI provides non-contact, clog-free sampling with compatibility for raster-based imaging workflows, making it well suited for high-resolution, multi-ion molecular mapping of optically identified single cells when coupled to high-resolving-power Orbitrap platforms20,37,38,39,40. DESI is widely used for tissue imaging but has rarely been applied to single-cell MSI, largely due to technical challenges20,28. Recent studies performed using a commercial setup consisting of a Waters DESI XS coupled to a SELECT SERIES Cyclic IMS mass spectrometer demonstrated that single-cell resolution can be achieved for multiple cell lines. Achieving high spatial resolution in DESI&#8211;MSI requires coordinated control of ionization efficiency, ion transmission, acquisition rate, and sprayer alignment12,20,28. In particular, advancements in the design of new generations of DESI sprayers, including DESI XS and desorption electro-flow focusing ionization (DEFFI), have significantly reduced the sampling area41,42. In addition, reducing solvent flow rates, such as nanoliter-per-minute flow rates delivered by nano-liquid chromatography (LC) pumps, can substantially improve spatial confinement to achieve single-cell molecular delineation28. Although this approach achieved single-cell resolution MSI, it relies on instrumentation from a specific vendor (Waters). Broader implementation of single-cell DESI&#8211;MSI therefore requires strategies that can be integrated with widely available high-resolution mass spectrometers, such as Orbitrap systems, to provide high sensitivity, mass accuracy, and resolving power. Combining DESI with Orbitrap mass spectrometers provides new opportunities in single-cell MSI studies, including differentiation of closely spaced and isobaric species in complex cellular extracts20,43,44.
This protocol describes a modular strategy for integrating a Waters DESI XS sprayer with Orbitrap mass spectrometers for single-cell MSI studies. The workflow integrates gridded coverslips for cell attachment and optical correlation, a motorized XYZ stage for controlled raster scanning, nanoscale solvent delivery for stable spray formation, and high-resolution Orbitrap detection. Although a Thermo Orbitrap Tribrid Lumos mass spectrometer was selected as the platform, many other Orbitrap mass spectrometer models equipped with compatible source-interface geometries may potentially be coupled with similar customized DESI XS configurations following appropriate mechanical adaptation and optimization.

<table><tbody><tr><td>&amp;minus;80 &amp;deg;C freezer</td><td>Eppendorf New Brunswick</td><td>U700</td><td>Storage of biological samples before preparation.</td></tr><tr><td>6&quot; vertical bracket for breadboards, 1/4&quot;-20 holes (2 pieces)</td><td>Millipore Sigma</td><td>VB01A</td><td>Mechanical support and stabilization of the custom sprayer-mount assembly</td></tr><tr><td>Acetonitrile</td><td>Fisher Chemical</td><td>240744</td><td>Organic solvent for DESI-compatible solvent preparation and optimization</td></tr><tr><td>Ammonium formate</td><td>Thermo Fisher Scientific</td><td>401152500</td><td>Preparation of isotonic ammonium formate wash buffer for cell washing prior to DESI&amp;ndash;MSI</td></tr><tr><td>Brightfield microscope</td><td>Fisher Scientific</td><td>12575252</td><td>Optical imaging and visualization of adherent cells before DESI&amp;ndash;MSI acquisition</td></tr><tr><td>C18 column</td><td>Waters</td><td>186009259</td><td>In-line backpressure generation for stable nano-flow DESI spray formation</td></tr><tr><td>Copper wire</td><td>N/A</td><td>N/A</td><td>Electrical connection and grounding during sprayer-mount fabrication</td></tr><tr><td>DESI XS sprayer</td><td>Waters</td><td>700014033</td><td>Ambient DESI ionization source for single-cell DESI&amp;ndash;MSI experiments</td></tr><tr><td>Epoxy resin</td><td>Devcon</td><td>20945</td><td>Mechanical stabilization and fixation of custom sprayer-mount components</td></tr><tr><td>FAIMS Pro electrode ion source housing adaptor</td><td>Thermo Fisher Scientific</td><td>98100-20046</td><td>Modification and fabrication of the custom Orbitrap-compatible DESI interface</td></tr><tr><td>Formic acid</td><td>Ward&amp;rsquo;s Science</td><td>470301-120</td><td>Acid modifier for DESI spray solvent preparation</td></tr><tr><td>Methanol</td><td>Fisher Chemical</td><td>A456-4 Optima</td><td>Organic solvent for DESI spray solvent preparation</td></tr><tr><td>Gas regulator</td><td>Millipore Sigma</td><td>23831-U</td><td>Regulation of nitrogen sheath-gas pressure during DESI operation</td></tr><tr><td>Gridded glass coverslip</td><td>Ibidi</td><td>10817</td><td>Cell culture substrate and positional reference for optical-to-MS image registration</td></tr><tr><td>Grounding wire</td><td>N/A</td><td>N/A</td><td>Electrical grounding connection for DESI sprayer and interface assembly</td></tr><tr><td>Incubator</td><td>HeraCell</td><td>51026282</td><td>Maintenance of mammalian cell cultures under controlled temperature and CO&lt;sub&gt;2&lt;/sub&gt; conditions</td></tr><tr><td>LC/MS-grade water</td><td>Thermo Fisher Scientific</td><td>W6500</td><td>Preparation of ammonium formate wash buffer and DESI spray solvent</td></tr><tr><td>METASPACE platform</td><td>METASPACE</td><td>https://metaspace2020.eu</td><td>Cloud-based MS image visualization and spatial metabolomics data analysis</td></tr><tr><td>Modified FAIMS interface</td><td>Thermo Fisher Scientific</td><td>98100-20046</td><td>Customized interface for coupling the DESI sprayer with Orbitrap mass spectrometers</td></tr><tr><td>Motorized XYZ stage</td><td>Newport Corporation</td><td>CONEX-MFACC</td><td>Controlled raster-stage movement during DESI&amp;ndash;MSI acquisition</td></tr><tr><td>MycoAlert PLUS mycoplasma detection kit</td><td>Lonza</td><td>LT07-703</td><td>Routine monitoring of mycoplasma contamination in cultured cells</td></tr><tr><td>Nano-liquid chromatography system</td><td>Thermo Fisher Scientific</td><td>5200.0355</td><td>Nano-flow solvent delivery system for DESI spray generation</td></tr><tr><td>Optical breadboard</td><td>Thorlabs</td><td>MB1012</td><td>Mechanical support platform for DESI sprayer and modified interface assembly</td></tr><tr><td>Orbitrap Fusion Lumos mass spectrometer</td><td>Thermo Fisher Scientific</td><td>UVPD</td><td>High-resolution Orbitrap mass spectrometer for DESI&amp;ndash;MSI data acquisition</td></tr><tr><td>PEEK tubing</td><td>IDEX</td><td>1535</td><td>Solvent-line connection between nano-LC system and DESI sprayer</td></tr><tr><td>Penicillin&amp;ndash;streptomycin</td><td>Gibco</td><td>15140-122</td><td>Antibiotic supplement for mammalian cell culture medium</td></tr><tr><td>Phosphate-buffered saline (PBS)</td><td>VWR</td><td>0780-50L</td><td>Washing solution for cultured cells during passaging</td></tr><tr><td>RPMI-1640 medium</td><td>Gibco</td><td>11875093</td><td>Mammalian cell culture medium for maintenance of OVCAR-8 cells</td></tr><tr><td>Stage-control software</td><td>Lasken Group</td><td>Version 6</td><td>Software control of raster scanning, stage movement, and imaging acquisition parameters</td></tr><tr><td>Stainless steel tubing</td><td>MicroGroup</td><td>316H16H</td><td>Custom-fabricated ion transfer tubing (1/16&quot; OD, 0.033&quot; ID) for Orbitrap interface integration</td></tr><tr><td>Syringe, 250 &amp;micro;L</td><td>Hamilton</td><td>1725LTN250UL</td><td>Solvent loading and nano-flow solvent delivery</td></tr><tr><td>Synthetic fetal bovine serum (FBS)</td><td>HyClone</td><td>SH3006603</td><td>Serum supplement for preparation of complete mammalian cell culture medium</td></tr><tr><td>TC20 Automated Cell Counter</td><td>Bio-Rad Laboratories</td><td>1450102EDU</td><td>Automated counting of cultured cells before seeding on coverslips</td></tr><tr><td>Tissue culture dish, 100 mm</td><td>Fisher Scientific</td><td>FB012924</td><td>Expansion and maintenance of adherent OVCAR-8 cell cultures</td></tr><tr><td>Trypsin-EDTA</td><td>Thermo Fisher Scientific</td><td>25200-072</td><td>Enzymatic detachment of adherent cells during passaging</td></tr><tr><td>UV-curing resin</td><td>Prime Dental</td><td>6.03</td><td>Fabrication and stabilization of custom DESI sprayer-mount components</td></tr><tr><td>Xcalibur software</td><td>Thermo Fisher Scientific</td><td>4.7</td><td>Instrument control, Orbitrap method setup, and mass spectral data acquisition</td></tr></tbody></table>

single-cell mass spectrometry imaging using desorption electrospray ionization coupled to orbitrap mass spectrometers

Single-Cell Mass Spectrometry Imaging Using Desorption Electrospray Ionization Coupled To Orbitrap Mass Spectrometers

Desorption electrospray ionization&#8211;mass spectrometry imaging (DESI&#8211;MSI) enables ambient, matrix-free molecular imaging with minimal sample preparation. This protocol describes a workflow for single-cell MSI of OVCAR-8 cells using DESI coupled to Orbitrap mass spectrometers. The system integrates a DESI sprayer, a modified mass spectrometer interface, a motorized XYZ stage, and an optical breadboard for high-spatial-resolution imaging. Cells are cultured on gridded glass coverslips, washed with ammonium formate solution to reduce salt interference, dried, and analyzed under ambient conditions. Solvent is delivered continuously using a nano-liquid chromatography system, and nebulizing nitrogen gas is regulated to maintain spray stability. The protocol also describes raster imaging, pixel control, and optical-to-mass spectrometry image registration for single-cell localization. High mass resolution and mass accuracy provided by Orbitrap detection enable differentiation of closely related molecular species and support single-cell metabolomic analysis. This protocol provides an adaptable strategy for implementing high-spatial-resolution single-cell MSI using multiple Orbitrap mass spectrometer platforms.

Successful execution of this protocol produces DESI–MSI datasets in which optically identified single cells spatially correspond to localized molecular ion signals (Figure 2). Brightfield microscopy images acquired before DESI–MSI analysis enable visual identification of individual OVCAR-8 cells and facilitate optical-to-MS image registration using the gridded coverslip as a positional reference. Representative DESI–MSI ion images demonstrate localized ion distributions that spatially correlate with individual cells identified in the corresponding brightfield image (Figure 2A–2D).
<img alt="Cell cluster fluorescence; image analysis; microscopy grid; emission intensity; spectral mapping." class="xfigimg" src="/files/ftp_upload/71691/71691fig2.jpg" data-altupdatedat="1788304762000" data-alt="Figure 2"> 
Figure 2. Comparison of brightfield microscopy and MSI of OVCAR-8 cells. (A) Brightfield microscopy image of OVCAR-8 cells acquired at 10× magnification. (B–D) MS images acquired using DESI–MSI with a pixel size of 5.5 µm × 10 µm. Representative ion distributions are shown for (B) m/z 317.111, (C) m/z 808.562, and (D) m/z 788.616. Scale bar = 50 µm. <a href="https://www.jove.com/files/ftp_upload/71691/71691fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Individual cells generate complete mass spectra containing multiple lipid-related ions and small molecules. Rather than relying on a single molecular marker, each cell exhibits a multi-ion molecular profile that functions as a chemical fingerprint. Representative ion images at m/z 317.111, m/z 808.562, and m/z 788.616 demonstrate heterogeneous molecular distributions across neighboring cells, indicating measurable metabolic heterogeneity at the single-cell level (Figure 2B–2D). METASPACE-based annotations suggested that ions detected at m/z 808.562 and m/z 788.616 were primarily associated with phosphatidylcholine and phosphatidylethanolamine lipid species, whereas m/z 317.111 corresponded to several candidate small molecules and dipeptide-related species. These results demonstrate that the platform enables detection of spatially localized molecular features from optically identified single cells under ambient conditions. Additional results, including tentatively annotated species, MS/MS-identified species, representative MS images, and mass spectra, are provided in previous studies20.
The integrated DESI–MSI platform consists of a DESI sprayer, a modified FAIMS adaptor, a motorized XYZ stage, and an optical breadboard mounted adjacent to the Orbitrap inlet (Figure 1). The setup enables stable solvent delivery, controlled stage movement, and alignment of the DESI spray plume with the mass spectrometer inlet for ambient single-cell imaging experiments. The customized ion transfer tube shown in Figure 3 enables ion transmission between the DESI source region and the Orbitrap inlet. The bent capillary geometry facilitates positioning of the DESI sprayer relative to the sample surface and inlet region while maintaining unobstructed ion transfer.
<img alt="Chromatography injection needle with ruler scale, used for sample introduction in analytical chemistry." class="xfigimg" src="/files/ftp_upload/71691/71691fig3.jpg" data-altupdatedat="1788304762000" data-alt="Figure 3"> 
Figure 3. Customized ion transfer tube for Orbitrap MSI. Photograph of the custom-fabricated ion transfer tube used to couple the DESI sprayer to the Orbitrap Fusion Lumos mass spectrometer. The figure shows the bent transfer capillary geometry and mounting assembly used for ion transmission during ambient single-cell MSI experiments. <a href="https://www.jove.com/files/ftp_upload/71691/71691fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Figure 4 shows the modified FAIMS adaptor integrated with the optical breadboard and motorized XYZ stage. The top view (Figure 4A) illustrates the positioning of the sample stage and modified adaptor relative to the Orbitrap inlet, whereas the bottom view (Figure 4B) shows the mounting brackets and stage assembly used to support the imaging platform. Mechanical stability of the integrated assembly is important for maintaining consistent raster movement and minimizing positional drift during imaging acquisition.
<img alt="Thermo Orbitrap Fusion Lumos diagram, modified FAIMS adaptor, optical setup, XYZ stage elements." class="xfigimg" src="/files/ftp_upload/71691/71691fig4.jpg" data-altupdatedat="1788304762000" data-alt="Figure 4"> 
Figure 4. Modified high-field asymmetric waveform ion mobility spectrometry (FAIMS) adaptor integrated with the optical breadboard and motorized XYZ stage. (A) Top view of the modified adaptor mounted adjacent to the Orbitrap inlet and sample stage. (B) Bottom view showing the modified brackets, optical breadboard, and motorized XYZ stage assembly used to support and align the imaging platform. <a href="https://www.jove.com/files/ftp_upload/71691/71691fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
High mass resolution provided by Orbitrap detection enables differentiation of closely spaced and isobaric lipid species, thereby improving molecular specificity and image contrast compared to lower-mass-resolution systems. Small pixel dimensions can be achieved through synchronization of stage velocity and spectral acquisition rate. For example, pixel dimensions as small as 2.7 µm × 10 µm have been achieved using rapid-acquisition Orbitrap platforms and optimized raster parameters42. High-spatial-resolution imaging enables molecular delineation of closely adjacent cells while minimizing signal overlap between neighboring cellular regions.
Successful DESI–MSI experiments are characterized by stable spray conditions, consistent ion intensity across raster lines, and spatial localization of molecular signals that correspond to individual cells in the optical image. Suboptimal experiments may produce diffuse ion distributions, low signal intensity, inconsistent raster patterns, or loss of cellular localization because of unstable spray formation, improper sprayer alignment, excessive solvent spreading, or sample movement during acquisition. Careful optimization of spray alignment, solvent flow rate, stage velocity, and sample positioning improves spatial fidelity and ion signal stability during single-cell imaging experiments.

This protocol describes single-cell mass spectrometry imaging of OVCAR-8 cells using desorption electrospray ionization coupled to Orbitrap mass spectrometers for high-spatial-resolution metabolomic analysis.

Desorption electrospray ionization&#8211;mass spectrometry imaging (DESI&#8211;MSI) enables ambient, matrix-free molecular imaging with minimal sample ...

single-cell-mass-spectrometry-imaging-using-desorption-electrospray

Research

JoVE Journal

Chemistry

Desorption electrospray ionization mass spectrometry (DESI-MS) is an ambient method by which samples, including biological tissues, can be imaged with minimal sample preparation. By rastering the sample below the ionization probe, this spray-based technique provides sufficient spatial resolution to discern molecular features of interest within tissue sections.

Imaging of Biological Tissues by Desorption Electrospray Ionization Mass Spectrometry

Immortalized cancer cell lines can be grown as 3D cell cultures, a valuable model for biological research. This protocol describes mass spectrometry imaging of 3D cell cultures, including improvements in the sample preparation platform. The goal of this protocol is to instruct users to prepare 3D cell cultures for mass spectrometry imaging analysis.

Sample Preparation Strategies for Mass Spectrometry Imaging of 3D Cell Culture Models

Here, we present protocols to perform both ambient mass spectrometry imaging (MSI) of tissues and in-situ live single cell MS (SCMS) analysis using the single-probe, which is a miniaturized multifunctional device for MS analysis.

Applications of the Single-probe: Mass Spectrometry Imaging and Single Cell Analysis under Ambient Conditions

A mass spectrometry imaging (MSI) source operated at atmospheric pressure was developed by coupling mid-infrared laser desorption and electrospray post-ionization. Exogenous ice matrix was used as the energy-absorbing matrix to facilitate resonant desorption of tissue-related material. This manuscript provides a step-by-step protocol for performing IR-MALDESI MSI of whole-body neonatal mouse.

Whole-body Mass Spectrometry Imaging by Infrared Matrix-assisted Laser Desorption Electrospray Ionization (IR-MALDESI)

Proteome characterization of ocular microvascular beds is pivotal for in-depth understanding of many ocular pathologies in humans. This study demonstrates an effective, rapid, and robust method for protein extraction and sample preparation from small blood vessels employing the porcine short posterior ciliary arteries as model vessels for mass-spectrometry-based proteomics analyses.

Sample Preparation for Mass-spectrometry-based Proteomics Analysis of Ocular Microvessels

An integrated cell manipulation platform is developed for use in conjunction with a single-probe mass spectrometry setup for the on-line analysis of individual suspension cells under ambient conditions.

Integrated Cell Manipulation Platform Coupled with the Single-probe for Mass Spectrometry Analysis of Drugs and Metabolites in Single Suspension Cells

This article introduces sample preparation methods for a unique real-time analytical method based on the ambient mass spectrometry. This method lets us perform real-time analysis of the biological molecules in vivo without any special pretreatments.

Sample Preparation for Probe Electrospray Ionization Mass Spectrometry

Ion mobility spectrometry (IMS) is an interesting complement to mass spectrometry for the characterization of biomolecules, notably because it is sensitive to isomerism. This protocol describes a tandem IMS (IMS/IMS) experiment, which allows the isolation of a molecule and the generation of the mobility profiles of its fragments.

Using a Cyclic Ion Mobility Spectrometer for Tandem Ion Mobility Experiments

Here we describe a mass spectrometry-based proteomic characterization of cell lineages with known tissue fates in the vertebrate Xenopus laevis embryo.

Cell-Lineage Guided Mass Spectrometry Proteomics in the Developing (Frog) Embryo

This protocol outlines the use of microMS for fluorescence-guided, single-cell MALDI-2 mass spectrometry, enabling enhanced molecular profiling of primary rat neuronal cells.

Fluorescence-Guided Matrix-assisted Laser Desorption/Ionization with Laser-Induced Postionization Mass Spectrometry of Individual Rat Neural Cells

This protocol aims to preserve cell metabolites for single-cell mass spectrometry (SCMS) by combining volatile salt solution washing, rapid liquid nitrogen quenching, freeze-drying, and −8 °C storage. It demonstrates that liquid nitrogen quenching is essential for maintaining metabolite profiles, while prolonged cold storage should be minimized to prevent metabolic alterations.

Sample Preparation for Single Cell Mass Spectrometry Metabolomics Studies: Combined Cell Washing, Quenching, Drying, and Storage

University of Oklahoma.  This protocol describes single-cell mass spectrometry imaging of OVCAR-8 cells using desorption electrospray ionization coupled to Orbitrap mass spectrometers for high-spatial-resolution metabolomic analysis.

Single-Cell Mass Spectrometry Imaging Using Desorption Electrospray Ionization Coupled To Orbitrap Mass Spectrometers

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