Desorption electrospray ionization mass spectrometry imaging (DESI-MSI) is a powerful method for generating chemical maps of surfaces and tissues at ambient conditions without the need for sample preparation. In DESI-MSI, a continuous stream of microdroplets impacts a sample affixed to a XY translation stage (cDESI-MSI). Upon impact, analyte molecules are extracted from the surface into secondary microdroplets, from which gas-phase ions are eventually formed. We have developed a pulsed DESI-MSI source (pDESI-MSI) that demonstrates approx. 5-10 times improvement in sensitivity compared to cDESI-MSI. The pDESI-MSI source is designed so that desorption only occurs during the ion accumulation time (IT) of an ion trap mass spectrometer, which increases sampling efficiency (SE) close to 100%. In addition, pulsing the primary microdroplet spray reduces the total volume of deposited solvent per pixel, minimizing the 'washing effect' and improving spatial resolution. This capability is particularly advantageous for coupling DESI-MSI to high-resolution ion trap instruments that have lengthy transient acquisition times (e.g. LTQ-Orbitrap XL: IT = 0.5 s, m/z-resolution = 100,000, scan time = approx. 2.30 s, pDESI-MSI increases SE from approx. 22% to 100%). pDESI-MSI offers the potential to acquire multiple DESI-MSI scan types per pixel without degrading spatial resolution, thereby providing more chemical information and better spatial accuracy of co-registered images. Implementation of pDESI-MSI also suggests a potential avenue for improving the performance of other continuous spray-based ambient ionization MSI techniques.