Published: September 01, 2011 r2011 American Chemical Society 7888 dx.doi.org/10.1021/ac2017629 | Anal. Chem. 2011, 83, 7888–7894 ARTICLE pubs.acs.org/ac High Dynamic Range Bio-Molecular Ion Microscopy with the Timepix Detector Julia H. Jungmann, † Luke MacAleese, †,|| Jan Visser, ‡ Marc J. J. Vrakking, §,† and Ron M. A. Heeren* ,† † FOM Institute for Atomic and Molecular Physics (AMOLF), Science Park 104, 1098 XG Amsterdam, The Netherlands ‡ National Institute for Subatomic Physics (Nikhef), Science Park 105, 1098 XG Amsterdam, The Netherlands § Max-Born-Institute, Max Born Straße 2A, D-12489, Berlin, Germany b S Supporting Information M ass spectrometry imaging (MSI) 1À4 measurements aim at the identification and localization of molecules from com- plex surfaces. 5À11 High-spatial resolution and high-throughput studies have been demonstrated with ion microscope mass spectrometers. 12 Microprobe mode MSI has enabled high-spatial resolution studies with highly focused primary ion or laser desorption ionization surface probes, which have returned pixel sizes better than 4 and 7 μm, respectively. In particular, ultraviolet/infrared (UV/IR) lasers in matrix-assisted laser desorption ionization (MALDI) 6À8 and pulsed primary ion beams have delivered spatial resolving powers on this order of magnitude. 9 Decreasing the surface probe area bears the disadvantage of long measure- ment times. In microscope mode MSI, surface molecules are desorbed and ionized over a large sample area, typically 200À300 μm in diameter. The large field of view enables fast, large area image acquisition. 12 An ion microscope employs ion optics to project the desorbed and ionized surface compounds onto a position- sensitive detector. The initial ion distribution is magnified, and the lateral spatial organization of the surface molecules is maintained on their way to the detector. Molecular images are time-of-flight separated, as all ions are accelerated by the same electrical potential and a narrow distribution of ion kinetic energies is achieved by the instrument’s ion optics. The ion optical magnification returns images with a high spatial resolving power, on the order of 4 μm. 5 The spatial resolution is not limited by the ionization beam. It is determined by the quality and capabilities of the ion optics and the position-sensitive detector. For a given set of ion optics, the ultimate spatial resolution is limited by ion optical aberrations. However, a spatial resolution on the order of the ion optical aberrations is often inaccessible due to insufficient detector spatial resolution. We show that with a pixelated complementary metal-oxide semiconductor detector, the obtainable spatial resolving power is only limited by the ion optics. Microscope mode MSI enables fast, high-resolution, large area imaging provided that a fast, two-dimensional, time- and posi- tion-sensitive detector is used to record high-quality molecular images. 5 In existing implementations, the ions are projected on a position-sensitive detector assembly which consists of micro- channel plates (MCP) followed by (i) a phosphor screen and a charge coupled device (CCD) camera or (ii) a resistive/delay- line anode. In the latter case, the ion arrival time is precisely measured as well. 13 Various techniques have been developed where the ion arrival time is measured by decoupling of the time- of-flight (TOF) signal from the MCP detector. 14 In TOF-MS based MSI, specific molecular masses are distinguished and recorded by simultaneously acquiring the impact position and the TOF of the ion. However, the MCP, phosphor screen, CCD camera assembly cannot link the ion TOF (i.e., the molecular weight) and the spatial distribution. Different molecular masses Received: July 12, 2011 Accepted: September 1, 2011 ABSTRACT: Highly parallel, active pixel detectors enable novel detection capabilities for large biomolecules in time-of-flight (TOF) based mass spectrometry imaging (MSI). In this work, a 512 Â 512 pixel, bare Timepix assembly combined with chevron microchannel plates (MCP) captures time-resolved images of several m/z species in a single measurement. Mass-resolved ion images from Timepix measurements of peptide and protein standards demonstrate the capability to return both mass- spectral and localization information of biologically relevant analytes from matrix-assisted laser desorption ionization (MALDI) on a commercial ion microscope. The use of a MCP-Timepix assembly delivers an increased dynamic range of several orders of magnitude. The Timepix returns defined mass spectra already at subsaturation MCP gains, which prolongs the MCP lifetime and allows the gain to be optimized for image quality. The Timepix peak resolution is only limited by the resolution of the in-pixel measurement clock. Oligomers of the protein ubiquitin were measured up to 78 kDa.