In mass spectrometry imaging, spatial resolution is pushed to its limits with the use of ion microscope mass spectrometric imaging systems. An ion microscope magnifies and then projects the original spatial distribution of ions from a sample surface onto a position-sensitive detector, while retaining time-of-flight mass separation capabilities. Here, a new type of position-sensitive detector based on a chevron microchannel plate stack in combination with a 512 ϫ 512 complementary metal-oxide-semiconductor based pixel detector is coupled to an ion microscope. Spatial resolving power better than 6 m is demonstrated by secondary ion mass spectrometry and 8 -10 m spatial resolving power is achieved with laser desorption ionization. A detailed evaluation of key performance criteria such as spatial resolution, acquisition speed, and data handling is presented. (J Am Soc Mass Spectrom 2010, 21, 2023-2030 © 2010 Published by Elsevier Inc. on behalf of American Society for Mass Spectrometry M ass spectrometry imaging (MSI) [1-3] measurements allow the visualization of the spatial structure and identification of the molecular masses from complex surfaces. High spatial resolution is accomplished with ion-microscope mass spectrometers, where ions are extracted from the sample surface and projected onto a position-sensitive detector. A spatial resolution better than 4 m has been reported using UV/IR laser surface probes in matrix assisted laser desorption ionization (MALDI) [4 -8]. A pulsed primary ion beam as a surface probe can achieve higher spatial resolving powers (1 m) [9,10]. The spatial resolution can be further improved by using a more focused primary ion/laser desorption ionization surface probe. However, fragmentation of the surface molecules and long measurement times are undesired side effects of decreasing the surface probe area. For instance, at a 2 ϫ 2 m pixel size (4 m lateral resolution) and a sample size of 1 ϫ 1 mm, a typical measurement comprises 250,000 measurement points and can last several hours. An alternate approach to increase the spatial resolution is the use of microscope mode MSI. In the microscope mode, surface molecules are desorbed and ionized over a large sample area, typically 200 -300 m in diameter. An ion microscope employs ion optics to project the ionized surface compounds onto a position-sensitive detector while magnifying the image and retaining the spatial information defined by the sample surface. With a field of view of 200 ϫ 200 m and a sample size of 1 ϫ 1 mm, a microscope mode imaging experiment involves 25 measurement points and retains the 4 m lateral resolution given the corresponding ion optical magnification factor. The ion optical magnification allows high-resolution images to be obtained independent of the ionization source. Microscope mode MSI enables fast, highresolution large area imaging provided that an adequate, i.e., fast and position-sensitive, detector is used to record high quality molecular images [4].Position-sensitive detectors most commonly used for microsc...