Abstract— Mass Spectrometric Imaging (MSI) allows the generation of 2D ion density maps that help visualize molecules present in sections of tissues and cells. The combination of spatial resolution and mass resolution results in very large and complex data sets. New capabilities are necessary for efficient analysis and interpretation of this data. This work details the development and application of the capability to process, visualize, query, and analyze spatial mass spectrometry data. Applications include the generation of 2D maps for selected spectra, the manipulation of the heat maps, and the identification of spectral peaks. Heat maps are generated by projecting the sum of intensity vs. time spectra of each pixel for selected m/z value or range. These capabilities take the form of a new interactive software toolkit, MSI QuickView. This software approach is a significant advance over the previous state-of-the art methods that required the conversion of the RAW data using one software, manual assembly of the data, and visualization in another software. I. INTRODUCTION Nanospray desorption electrospray ionization (nano- DESI) is a technique that allows imaging of completely hydrated biological materials with high spatial resolution and sensitivity [1,2]. This significant improvement in detection efficiency and spatial resolution will facilitate new MSI applications in clinical diagnosis, drug discovery, biochemistry, and molecular biology [2]. Imaging nano- DESI experiments, described elsewhere [1,2], utilized a LTQ/Orbitrap instrument and the signal was collected using Xcalibur (Thermo Scientific, Waltham, MA). Xcalibur is a Windows-based user interface that integrates instrument setup, acquisition and data processing for single lines of data from Thermo Scientific mass spectrometers. The typical work flow during data acquisition and processing is illustrated in Fig. 1a. Acquiring MSI data allows the creation of ion density maps for each m/z value or signal collected by Xcalibur [3]. However, a limitation of this approach is that data conversion using FireflyTM software (Prosolia Inc.) is limited to 400 scans/ line. In addition, for individual m/z *Research supported by U.S. Department of Energy under LDRD DE- AC05-76RL01830. J.P. Carson is with the Pacific Northwest National Laboratory, Richland, WA 99354 USA (phone: 509-371-6894; fax: 509-371-6946; e- mail: james.carson@pnnl.gov) M. Thomas, J. Laskin, D. Li, B.S. Heath, K. Hui, E. Liu, A.P Kuprat and K. Kleese van Dam are with the Pacific Northwest National Laboratory, Richland, WA 99354 USA (e-mail: {mathew.thomas, julia.laskin, dongsheng.li, brandi.heath, ellen.liu, katrina.hui, andrew.kuprat, kerstin.kleesevandam}@pnnl.gov) values that cannot be processed using Firefly, the line scans have to be manually exported and visualized using Origin Pro 8.5 (OriginLab Corporation, Northampton, MA) [1,2]. Thousands of intensity values are obtained for each particular mass to charge ratio. Due to the large amount of data, it becomes increasingly difficult and time-consuming to manually extract meaningful results from MSI experimentation. Another challenge is that during the Fourier Transform Ion Cyclotron Resonance Mass Spectroscopy (FTMS) mode of acquisition, which is high resolution mass acquisition, Xcalibur does not save the intensity values for each m/z at that resolution but instead selects only certain m/z values to make the file size manageable and less computationally intensive. Hence there is no certainty on which m/z values and how many m/z values will be present in each line of data. This paper presents a new approach for handling the data obtained from MSI including FTMS data. The primary objective of this approach is to be able to quickly visualize the data locally over the whole image during image acquisition itself and provide immediate feedback to the user. The method is implemented as a new software named MSI QuickView. The typical work flow using this approach is displayed in Fig. 1b. Visualization of High Resolution Spatial Mass Spectrometric Data during Acquisition* Mathew Thomas, Brandi S. Heath, Julia Laskin, Dongsheng Li, Ellen Liu, Katrina Hui, Andrew P. Kuprat, Kerstin Kleese van Dam, James P. Carson, Member, IEEE Figure 1. Flowchart depicting (a) the previous workflow without MSI QuickView and (b) the new approach with MSI QuickView. 34th Annual International Conference of the IEEE EMBS San Diego, California USA, 28 August - 1 September, 2012 5545 978-1-4577-1787-1/12/$26.00 ©2012 IEEE