The DESI‐MSI of the mouse brains was performed using a Waters DESI‐XS source coupled with a SELECT SERIES Cyclic Ion‐Mobility mass spectrometer. The data were collected in positive ion mode. The spatial resolution for the imaging was set at 125 μm, and the rate was set at 125 μm/s. A solution (90% methanol, 5% acetonitrile, 5% water, 0.01% formic acid, and 100 pg/μl of leucine enkephalin [Leu/Enk]) was delivered at 5 μl/min by a Hamilton syringe pump. The collected data were processed to produce the two‐dimensional images using Waters High‐Definition Imaging software. The mass accuracy of ondansetron (m/z 294.1601) was corrected by using Lue/Enk (556.2771) as the reference mass.
High definition imaging software
Manufactured by Waters Corporation
High‐Definition Imaging software is a software product designed to capture and process high-resolution images for various laboratory applications. It provides advanced imaging capabilities to support research and analysis tasks.
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3 protocols using high definition imaging software
1
DESI-MSI Imaging of Mouse Brains
2
MALDI-MSI N-Glycan Analysis of FFPE Lungs
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Formalin-fixed paraffin embedded (FFPE) lungs were processed as previously described.15 (link),16 (link) In brief, FFPE lungs were sectioned at 5 µm on glass slides. Tissues were dewaxed and rehydrated followed by an antigen retrieval process in citraconic anhydride buffer. Recombinant PNGase F (0.1 µg/mL) was applied by a M5 TM robotic Sprayer (HTX Technologies LLC, Chapel Hill, NC). Following incubation in a humidity chamber for 2 hours, slides were desiccated for 24 hours. The following day, 7 mg/mL of alpha-Cyano-4-hydroxycinnamic acid in 50% acetonitrile with 0.1% TFA were applied to each slide by the robotic sprayer. Slides were subsequently stored in a desiccator prior to MALDI-MSI analysis.
A Waters Synapt G2Si mass spectrometer (Waters Corporation, Millford, MA) equipped with a Nd:YAG UV laser with a spot size of 100 µm was used to detect N-glycans at X and Y coordinates of 100 µm. Following data acquisition, mass spectra were analyzed by High Definition Imaging Software (Waters Corporation) for mass range 500–3500 m/z. Three regions of interest were determined for each lung; pixel intensities were averaged and normalized by total ion current. Representative N-glycans were generated by Glycworkbench.
A Waters Synapt G2Si mass spectrometer (Waters Corporation, Millford, MA) equipped with a Nd:YAG UV laser with a spot size of 100 µm was used to detect N-glycans at X and Y coordinates of 100 µm. Following data acquisition, mass spectra were analyzed by High Definition Imaging Software (Waters Corporation) for mass range 500–3500 m/z. Three regions of interest were determined for each lung; pixel intensities were averaged and normalized by total ion current. Representative N-glycans were generated by Glycworkbench.
3
DESI-MSI Analysis of Liver Sections
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DESI-MSI analysis was performed using the Synapt G2-Si HDMS system (Waters Corporation, Manchester, UK) equipped with the 2D DESI ion source (Prosolia, Inc., Indianapolis, IN, USA), operated in negative ion sensitivity mode. S-3100-CA distribution on the liver sections was measured at the transition m/z 488.03/488.03 using methanol/water (95:5, v/v) containing 10 mM ammonium acetate as a spray solvent delivered at 2 µL/min. The spectra were acquired at trap collision energy 10 eV, sprayer voltage 4.5 kV, nitrogen nebulizing gas pressure 4.0 bar, surface scan rate 200 µm/sec, and spatial resolution 100 µm. The acquired spectra were analyzed with the use of High Definition Imaging software (Waters Corporation).
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