Desorption electrospray ionization (DESI) has emerged as a powerful technique for mass spectral analysis and imaging under ambient conditions. Synchronization of DESI (sDESI) with the ion injection period (IT)of low-duty cycle mass spectrometers has been previously shown to improve sensitivity and reduce the amount of sample depleted during the acquisition of each spectrum (viz. MS scan time). In this report, we describe the development and characterization of an sDESI mass spectrometry imaging source (sDESI-MSI). Our results show that synchronization of DESI with the IT of an LTQ Orbitrap-XL mass spectrometer improves spatial resolution by factors of ∼4−6. In addition, under certain experimental conditions, synchronization was essential to acquire distinct MS images of low-intensity endogenous FAs (< 5% relative intensity) in fingermarks at high sampling frequencies (step sizes ≤ 75 μm). The magnitudes of these improvements in performance depend on the properties of the microdroplet spray, sample, and surface. Simulations that model analyte movement during desorption and the “washing effect” replicate the experimental results with the washing parameter having the greatest impact on performance. Thus, synchronization improves spatial resolution and sensitivity by decreasing the percentage of the total MS scan time that analytes are influenced by the “washing effect”. Generally, synchronization of DESI with IT improves performance and expands the range of analytes, surfaces, and experimental conditions amenable to DESI-MSI, especially for analytes that are weakly attached to a surface.