The Total Ion Current (TIC) graph (Figure ) illustrates that high TICs with excellent stabilities were achieved at 375°C. However, the trend of TIC does not represent the ion intensities of individual cellular species. To further evaluate the influence of temperature of the ion transfer capillary on MSI measurements of cellular species, experiments were conducted for m/z 786.2191, an intense ion commonly observed from cells and tissues. Our results indicate that 400°C resulted in significantly improved ion intensities (Figure .). Upon acquiring the optimized experimental conditions, we conducted DESI-MSI studies to achieve single-cell resolution (Figure ). OVCAR-8 cell line was used as the model system. Cells were cultured and attached onto glass coverslips with 50 x 50 µm grid (cat.no:10817, ibidi, Gräfelfing, Germany). To minimize the influence of matrix effect (e.g., induced by salts in the cell culture medium) on ionization efficiency, cells on the coverslips were washed twice with isotonic ammonium formate solution (0.144 M) prior to analysis. A pixel size of 10 µm x 10 µm was used to achieve high-quality MS images with spatially resolved single cells. Figure shows a photo of attached cells that were taken using a brightfield microscope (cat.no: 12575252, Fisher Scientific, Waltham, MA, USA) at 20x magnification. MS images obtained from DESI-MSI with 10 µm x 10 µm pixel size that show the abundances of select ions, which were tentatively annotated (mass error < 8 ppm) based on searching their measured m/z values on LipidMaps (). Darker areas indicate lower abundances, whereas brighter areas indicate higher abundance (Figure ). Excellent correlations of single cells' locations can be observed by comparing the microscopy photo and MS images. Although a finer pixel size of 5 µm x 5 µm has been tested, we were unable to obtain improved quality of MS images, likely due to inadequate signal intensities from smaller sampling areas using our current mass spectrometer. In summary, we have successfully utilized the commercial DESI-MSI technique to obtain MS images of cultured mammalian cells at the single-cell resolution. Experiments were performed using Waters DESI XS course coupled to a Waters Synapt G2Si mass spectrometer to analyze OVCAR-8 cells attached on glass coverslips. A heated ion transfer capillary was implemented into the DESI XS source. Comprehensive experiments were carried out to optimize experimental conditions, including the temperature of ion transfer capillary, distance between sprayer and surface, and solvent flowrate. This established method allowed us to observe the metabolites and their spatial distribution within individual cells with minimal sample preparation. Our method can be readily adopted by other researchers who use the similar instrument with little or no modification. Other analytical methods, such as ion mobility and tandem MS (MS/MS), can be implemented to provide more molecular information. The DESI-MSI technique with the singlecell resolution holds significant potential for advancing our understanding of cellular heterogeneity and the metabolic diversity within individual cells, providing valuable insights into dynamic biological processes, disease mechanisms, and potential therapeutic targets. It is worth noting that our current MSI results using smaller pixel sizes were unsatisfying, very likely due to the inadequate detection sensitivity of the mass spectrometer used in this work. Utilizing later generations of mass spectrometers compatible with the newer version of DESI XS, which is implemented with heated ion transfer capillary, can likely improve its performance for high resolution MSI studies. In addition, implementing new developments in DESI-based methods, such as using He or H2 to replace N2 as the nebulization gas and combining ammonia additive with post-photoionization , for improved ion intensities can potentially promote DESI-MSI studies of single cells.