Imaging mass spectrometry combines the chemical specificity and parallel detection of mass spectrometry with microscopic imaging capabilities. The ability to simultaneously obtain images from all analytes detected, from atomic to macromolecular ions, allows the analyst to probe the chemical organization of a sample and to correlate this with physical features. The sensitivity of the ionization step, sample preparation, the spatial resolution, and the speed of the technique are all important parameters that affect the type of information obtained. Recently, significant progress has been made in each of these steps for both secondary ion mass spectrometry (SIMS) and matrix-assisted laser desorption/ionization (MALDI) imaging of biological samples. Examples demonstrating localization of proteins in tumors, a reduction of lamellar phospholipids in the region binding two single celled organisms, and sub-cellular distributions of several biomolecules have all contributed to an increasing upsurge in interest in imaging mass spectrometry. Here we review many of the instrumental developments and methodological approaches responsible for this increased interest, compare and contrast the information provided by SIMS and MALDI imaging, and discuss future possibilities.

Imaging mass spectrometry

The origin of ions in matrix-assisted laser desorption/ionization (MALDI) mass spectrometry is currently a matter of active research A number of chemical and physical pathways have been suggested for MALDI ion formation, including gas-phase photoionization, ion–molecule reactions, disproportionation, excited-state proton transfer, energy pooling, thermal ionization, and desorption of preformed ions These pathways and others are critically reviewed, and their varying roles in the wide variety of MALDI experiments are discussed An understanding of ionization pathways should help to maximize ion yields, control analyte charge states and fragmentation, and gain access to new classes of analytes © 1999 John Wiley & Sons, Inc Mass Spec Rev 17: 337–366, 1998

Ion formation in MALDI mass spectrometry

A matrix-assisted laser desorption ionization (MALDI) “event” constitutes a complex process, involving optical and mechanical phenomena as well as thermodynamic and physicochemical processes of phase transition and ionization. A successful MALDI analysis encompasses several crucial steps: sample preparation, excitation of sample and disintegration of the condensed phase, generation and separation of charges and ionization of analyte molecules, and, finally, extraction, separation according to the massto-charge ratio of the ions in the mass spectrometer, and detection. Despite the rapid acceptance of the method in chemistry and biomedicine after its introduction by Karas and Hillenkamp,1-4 the underlying mechanisms have been less well understood for a long time and a more comprehensive picture has only recently begun to emerge. Systematic variation of one or more of the relevant “input” parameters is one of the most straightforward experimental tools to obtain insight into the involved mechanisms, and has consequently been utilized in a large number of fundamental studies. The role of the different relevant irradiation (laser) parameters in the desorption/ionization process, as well as those of the matrix and the preparation protocol, have been addressed in these investigations. These included, for example, the role of the laser wavelength, pulse duration, and laser fluence (laser energy per pulse and unit area) for the laser parameters (addressed in section III), and the type of (co-)crystallization and the matter of the incorporation of analyte molecules into matrix crystals for the material side (section IV). In other work, the dynamical parameters of the expanding MALDI particle “plume” have been addressed (section V): the initial kinetic energies and energy distributions of molecules and ions, and the composition of the plume (ion-to-neutral ratio and ejection of particles and clusters versus the emission of molecular constituents). Several researchers have developed theoretical models for the desorption as well as the ionization * E-mail: dreisew@uni-muenster.de. Fax +49-251-8355121. Tel. +49-251-8356726. Klaus Dreisewerd was born in Beckum, Germany, in 1961. He received his diploma in Physics from the University of Münster in 1990 and his Ph.D. degree in 1995 under the supervision of Franz Hillenkamp. He moved to the Free University of Amsterdam as a post-doc in the molecular neurobiology department at the end of 1994 and returned to the Münster institute in 1997. His main current interests are in the field of MALDI fundamentals and that of MALDI-MS with pulsed infrared lasers (IRMALDI). 395 Chem. Rev. 2003, 103, 395−425

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The desorption process in MALDI.

1.1. Mass Spectrometry
A mass spectrometer is described as the smallest weighing scale in the world ever used.1 Mass spectrometry (MS) is a unique technique that has an interdisciplinary nature, which freely crosses the borders of physics, chemistry and biology. Mass spectrometry makes a great scientific tool due to its capabilities to determine the mass of large biomolecular complexes, individual biomolecules, small organic molecules as well as single atoms and their isotopes. Right from the time of its invention in the first decade of the 20th century, mass spectrometry has undergone tremendous improvements in terms of its sensitivity, resolution and mass range. It currently finds applications in all scientific disciplines such as chemistry, physics, biology, pharmacology, medicine, biochemistry and bio-agro-based industry.

Introduction of “soft” ionization sources such as electrospray ionization (ESI) by J.B. Fenn et al.2 and matrix-assisted laser desorption/ionization (MALDI) by M. Karas et al.3 in 1980s revolutionized mass spectrometry as it offered the capability to analyze large intact biomolecules. As such MS became an irreplaceable tool for the biological sciences. The development of both ESI and MALDI made possible the ionization of smaller biomolecules such as drugs and metabolites as well larger biomolecules such as lipids, peptides and even proteins.2,4 The molecular weight (Mw) ranges we use in this review are defined as follows. The low Mw range includes elements and molecules from 1 to 500 Da. Molecules with Mw between 500 and 2000 Da fall in the medium Mw range. All molecules with Mw > 2000 Da are considered to be part of the high molecular weight class. It goes beyond the scope of this review to cover all developments in MS. Rather this review focuses on one of the latest, rapidly developing innovations in MS namely mass spectrometric imaging (MSI). This young technique takes benefit from all methodological and technological developments in general MS over the last decades. Over the last twenty years MSI has transformed from an esoteric, specialist technology studied by few researchers only to a technique that now finds itself at the center stage of mainstream MS. Over the last years the technology has matured to find applications in many different areas, with instrument developments taken up by all MS instrument manufacturers resulting in a rapid rise of the number of research groups active in this area. A thorough review of the area is therefore timely and needed to offer a starting point for all newcomers to the field. In this paper we describe and review approximately 20 years of MSI developement from the perspective of its application to biomedical imaging. We will emphasize the key research steps and pitfalls that determine the outcome of biological applications of this relatively new label free biomolecular imaging technique in life sciences.

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Mass spectrometric imaging for biomedical tissue analysis.

Mass spectrometry of intact synthetic polymers has been limited to field desorption (FD) MS for many years. More recently, soft ionization techniques such as electrospray (ESI) and matrix-assisted laser desorption/ionization (MALDI) and the revival of time-of-flight analyzers created new opportunities for the characterization of polymers. In this review emphasis is put on MALDI time-of-flight mass spectrometry of polymers. The selection of an appropriate MALDI matrix, cationization salt and sample preparation techniques are critical success factors for obtaining a reliable mass spectrum and to infer structural information such as monomer mass(es) and end-groups. However even under optimized conditions mass discrimination in the analysis of polydisperse polymers and specific oligomer discrimination might occur. Hence hyphenated techniques such as size exclusion chromatography (SEC)/MALDI have been developed. Many different polymer applications appeared in recent literature, but most studies deal with homopolymers. Many challenges remain, particularly in the fields of halogenated polymers, polyolefines, copolymers, blends, and in the sequencing of block-copolymers. © 1999 John Wiley & Sons, Inc., Mass Spec Rev 18: 309–344, 1999

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Maldi time-of-flight mass spectrometry of synthetic polymers

A new methodology, matrix-enhanced secondary ion mass spectrometry (ME-SIMS), is reported for the molecular analysis of biomaterials. The technique applies static secondary ion mass spectrometry (SSIMS) techniques to samples prepared in a solid organic matrix similar to sample preparations used in matrix-assisted laser desorption/ionization (MALDI). Molecular ions are observed in this ion beam sputtering of organic mixtures for peptides and oligonucleotides up to masses on the order of 10 000 Da. This matrix-enhanced SIMS exhibits substantial increases in the ionization efficiency of selected analyte molecules compared to conventional SSIMS processes. Thus, higher mass peptides, proteins, and nucleic acids become accessible to near-surface analysis by ion beam techniques, and subpicomole sensitivity has been demonstrated. A number of matrices were examined for their efficiency in ME-SIMS applications, and these initial matrix studies focused on common MALDI matrices and their isomers. The results of this ...

Matrix-enhanced secondary ion mass spectrometry : a method for molecular analysis of solid surfaces

The pulsed laser thin‐film deposition process can enable preparation of thin films of complex composition with good control over the film stoichiometry The film compositions are similar to that of the target pellet and as a consequence this technique appears to be an ideal method for preparing high Tc thin films on a variety of substratesThe factors which contribute to this beneficial phenomenon have been explored by a laser ionization mass spectrometry (LIMS) and a post ablation ionization (PAI) neutral velocity analysis technique in order to determine the mass and velocities of the laser ejected material In addition, x‐ray absorption measurements on films deposited onto substrates at room temperature were performed in order to identify the presence of short‐range crystalline order in the films Both of these studies rule out the ejection of stoichiometric clusters of material from the pellet during the laser ablation/deposition process Instead, binary and ternary suboxides are emitted from the targe

Nature of the pulsed laser process for the deposition of high Tc superconducting thin films

MALDI MS is a powerful tool for determining molecular-weight distributions and structures of synthetic organic polymers.

Robert W. Odom

Papers

Matrix-enhanced secondary ion mass spectrometry : a method for molecular analysis of solid surfaces

Nature of the pulsed laser process for the deposition of high Tc superconducting thin films

Peer Reviewed: Characterizing Synthetic Polymers by MALDI MS

Quantitative image acquisition system for ion microscopy based on the resistive anode encoder

A rule-building expert system for classification of mass spectra