Prior to MSI single cell studies, we optimized experimental conditions using dried cell lysate on PTFE coated glass slides (ref: 041-MICRO-44, Scientific Device Laboratory, Des Plaines, IL, USA). First, we prepared the surface coated with dried cell lysates. To do this, we cultured OVCAR-8 human ovarian cancer cells, prepared cell lysate, and coated multiple spots on a glass slide with 10 µL of cell lysate on each spot and dried in air (Figure ). Second, we performed systematic experiments to optimize the temperature of the ion transfer capillary. Specifically, we wrapped the heating wire (22 gauge, Kanthal, Bethel, CT, USA), covered by a fiberglass mesh sleeve, around the ion transfer capillary (Figure ). This assembly was connected to a thermocouple (to monitor the temperature) and a 144W universal power supply (model: LGY-363000, Dong Guan Shi He Yu Tech, Dongguan, China), which allowed us to apply varying voltages to reach and maintain specific temperatures. We adjusted the voltage applied from the power supply to achieve a series of targeting temperatures: room temperature, 250, 275, 300, 325, 350, 375, 400, and 425°C. Third, for each temperature, the DESI XS stage was manually repositioned to manipulate the distance between the sprayer and spot coated with dried cell lysate on the slide. In general, a short distance can improve ion intensities, whereas an excessively short distance may cause the sprayer to scratch the sample. Last, we optimized the flowrate of solvent (methanol 95%/water 5% + formic acid 1%) delivered by the LC pump system (no column). Optimized experiment parameters include an ion transfer capillary temperature of 375°C, a distance of 1.2 mm between sprayer and surface, and a flowrate of 0.5 mL/min. Other parameters include scan rate of 20 µm/s, mass range of 100-1200 m/z, TOF mode, and positive ion mode.