For the first time macromolecular ion microscope images have been recorded using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Single-shot, mass-resolved images of the spatial distributions of intact peptide and protein ions over an area of 200 microm in diameter were obtained in less than 1 ms at a repetition rate of 12 Hz. The magnifying ion optics of the ion microscope allowed ion images to be obtained with a lateral resolution of 4 microm. These results prove the concept of high-resolution MALDI-MS imaging in microscope mode without the need for a tight laser focus and the accompanying sensitivity losses. The ion microscopy approach offers an improvement of several orders of magnitude in speed of acquisition compared to the conventional (microprobe) approach to MALDI-MS imaging.

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Imaging mass spectrometry provides both chemical information and the spatial distribution of each analyte detected. Here it is demonstrated how imaging mass spectrometry of tissue at subcellular resolution can be achieved by combining the high spatial resolution of secondary ion mass spectrometry (SIMS) with the sample preparation protocols of matrix-assisted laser desorption/ionization (MALDI). Despite mechanistic differences and sampling 10(5) times less material, matrix-enhanced (ME)-SIMS of tissue samples yields similar results to MALDI (up to m/z 2500), in agreement with previous studies on standard compounds. In this regard ME-SIMS represents an attractive alternative to polyatomic primary ions for increasing the molecular ion yield. ME-SIMS of whole organs and thin sections of the cerebral ganglia of Lymnaea stagnalis demonstrate the advantages of ME-SIMS for chemical imaging mass spectrometry. Subcellular distributions of cellular analytes are clearly obtained, and the matrix provides an in situ height map of the tissue, allowing the user to identify rapidly regions prone to topographical artifacts and to deconvolute topographical losses in mass resolution and signal-to-noise ratio.

Intense intact molecular ion signals have been obtained from phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, and phosphatidyiinositol using matrix-enhanced secondary ion mass spectrometry (ME-SIMS). It was found that the high-mass (m/z >500) regions of the ME-SIMS spectra closely resembled those obtained using matrix-assisted laser desorption/ionization (MALDI). Using high spatial resolution SIMS, a detailed investigation of dried-droplet samples was performed. Based on the detected Na+ and 2,5-DHB matrix signal intensities, different crystal types were distinguished, in addition to different sizes of crystals. Spatially mapping the pseudomolecular and fragment ions of the phospholipids revealed that the nature of the pseudomolecular ions formed, as well as the ratio of intact molecular to fragment ion, was dependent on the type and surface composition of the crystal. The observed chemical bias effects due to crystal heterogeneity and the resulting variation in desorption/ionization efficiency will complicate the interpretation of data obtained from matrix-assisted mass spectrometric (imaging) techniques and is an important factor in the "hot spot" phenomenon frequently encountered in MALDI experiments. In this respect, imaging SIMS was found to be a versatile tool to investigate the effects of the local physicochemical conditions on the detected molecular species.

Disjunct eddy covariance measurements of oxygenated volatile organic compounds fluxes from an alfalfa field before and after cutting [1] There is interest in and significant uncertainty about the emissions of oxygenated volatile organic compounds (oxVOCs) from vegetation to the atmosphere. Here, we measured the fluxes of selected oxVOCs from an alfalfa field, before, during, and after cutting, using a combination of disjunct eddy covariance and proton-transfer-reaction mass spectrometry. Over the course of 1 day a significant methanol flux of 4 mg m À2 h À1 was observed from undisturbed alfalfa with a maximum at 0800 LT, possibly caused by the evaporation of dew. A smaller release of hexenals during this day (0.04 mg m À2 h À1 ) demonstrated the sensitivity of the method. Other results suggested that acetaldehyde and acetone were released in the afternoon but were lost by dry deposition in the evening and morning; deposition velocities were estimated to be 0.2 cm s À1 (acetaldehyde) and 0.09 cm s À1 (acetone). After the alfalfa was cut the emissions of methanol, acetaldehyde, acetone, and hexenals were significantly enhanced and remained high for three days during which the alfalfa was drying. After a rainstorm the oxVOC emissions from the cut, wet alfalfa increased even more. Nighttime measurements yielded low oxVOC fluxes in general, but the high variability of the concentrations during the night and the high degree of correlation between different oxVOCs suggest that the nighttime releases of oxVOCs from alfalfa were nonzero. This work suggests that the global source of oxVOCs due to the production of hay is of minor importance. The emission flux of methanol from vegetation during the growing season may be very large on a global basis.

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Imaging mass spectrometry (IMS) allows the direct investigation of both the identity and the spatial distribution of the molecular content directly in tissue sections, single cells and many other biological surfaces. In this protocol, we present the steps required to retrieve the molecular information from tissue sections using matrix-enhanced (ME) and metal-assisted (MetA) secondary ion mass spectrometry (SIMS) as well as matrix-assisted laser desorption/ionization (MALDI) IMS. These techniques require specific sample preparation steps directed at optimal signal intensity with minimal redistribution or modification of the sample analytes. After careful sample preparation, different IMS methods offer a unique discovery tool in, for example, the investigation of (i) drug transport and uptake, (ii) biological processing steps and (iii) biomarker distributions. To extract the relevant information from the huge datasets produced by IMS, new bioinformatics approaches have been developed. The duration of the protocol is highly dependent on sample size and technique used, but on average takes approximately 5 h.