Mass Spectrometry Imaging of Arabidopsis thaliana Leaves at the Single-Cell Level by Infrared Laser Ablation Atmospheric Pressure Photoionization (LAAPPI).

In this study, we show that infrared laser ablation atmospheric pressure photoionization mass spectrometry (LAAPPI-MS) imaging with 70 µm lateral resolution allows for the analysis of Arabidopsis thaliana (A. thaliana) leaf substructures ranging from single-cell trichomes and the interveinal leaf lamina to primary, secondary, and tertiary veins. The method also showed its potential for depth profiling analysis for the first time by mapping analytes at the different depths of the leaf and spatially resolving the topmost trichomes and cuticular wax layer from the underlying tissues. Negative ion LAAPPI-MS detected many different flavonol glycosides, fatty acids, fatty acid esters, galactolipids, and glycosphingolipids, whose distributions varied significantly between the different substructures of A. thaliana leaves. The results show that LAAPPI-MS provides a highly promising new tool to study the role of metabolites in plants.

Soft X-ray Atmospheric Pressure Photoionization in Liquid Chromatography-Mass Spectrometry.

Soft X-ray atmospheric pressure photoionization (soft X-ray APPI) as an ionization method in liquid chromatography-mass spectrometry (LC-MS) is presented. The ionization mechanism was examined with selected test compounds in the negative ion mode, using soft X-ray APPI source emitting 4.9 keV photons. Test compounds with an acidic group were ionized by a proton transfer reaction, producing deprotonated molecules ([M - H]-), whereas compounds having positive electron affinity were ionized by a charge exchange reaction, producing negative molecular ions (M-•). Soft X-ray APPI does not require a dopant to achieve high ionization efficiency, which is an advantage compared with vacuum ultraviolet APPI with 10 eV photons, in which a dopant is needed to improve ionization efficiency. The energy of the soft X-ray photons is in the keV range, which is high enough to displace a valence electron and often also inner shell electrons from LC eluents and atmospheric gases, initiating an efficient ionization process in the negative ion mode.

Sub-100 µm Spatial Resolution Ambient Mass Spectrometry Imaging of Rodent Brain with Laser Ablation Atmospheric Pressure Photoionization (LAAPPI) and Laser Ablation Electrospray Ionization (LAESI).

In this study, we applied a new IR laser-beam-focusing technique to enable sub-100 µm spatial resolution in laser ablation atmospheric pressure photoionization (LAAPPI) and laser ablation electrospray ionization (LAESI) mass spectrometry imaging (MSI). After optimization of operational parameters, both LAAPPI- and LAESI-MSI with a spatial resolution of 70 µm produced high-quality MS images, which allowed accurate localization of metabolites and lipids in the mouse and rat brain. Negative and positive ion LAAPPI- and LAESI-MS detected many of the same metabolites and lipids in the brain. Many compounds were also detected either by LAAPPI- or LAESI-MS, indicating that LAAPPI and LAESI are more complementary than alternative methods.

Chemical profiles of birch and alder bark by ambient mass spectrometry.

Desorption atmospheric pressure photoionization (DAPPI) is an ambient mass spectrometry (MS) technique that allows the analysis of both polar and nonpolar compounds directly from the surfaces of various sample types. Here, DAPPI was used to study the chemical profiles in different parts of birch and alder tree barks. Four distinct fractions of Betula pendula (silver birch) bark were collected from three different developmental stages of the stem, after which the chemical profiles of the different tissue types were measured. Of special interest were triterpenoids, a class of important defensive substances, which are found in the bark of the silver birch. Additionally, the chemical profiles of lenticels and the surrounding surfaces in the phellem of B. pendula (silver birch), Alnus glutinosa (black alder), and Alnus incana (gray alder) were screened with DAPPI. Another ambient MS technique, laser ablation atmospheric pressure photoionization (LAAPPI), was further used for the mass spectrometry imaging of lenticels on the B. pendula phellem. All the studied birch bark fractions showed individual chemical profiles in DAPPI. The mass spectra from the young apical stem and the transition zone resembled each other more than the mature stem. Instead, the phellem was found to contain a high amount of triterpenoids in all the developmental stages of the stem. The most intense peaks in the DAPPI mass spectra of the birch bark fractions were those of betulin and lupeol. Betulinic and betulonic acid peaks were intense as well, and these compounds were detected especially in the lenticels of the tree samples. Graphical abstract.

A Simple Method for Improving the Spatial Resolution in Infrared Laser Ablation Mass Spectrometry Imaging.

In mass spectrometry imaging of tissues, the size of structures that can be distinguished is determined by the spatial resolution of the imaging technique. Here, the spatial resolution of IR laser ablation is markedly improved by increasing the distance between the laser and the focusing lens. As the distance between the laser and the lens is increased from 1 to 18 m, the ablation spot size decreases from 440 to 44 µm. This way, only the collimated center of the divergent laser beam is directed on the focusing lens, which results in better focusing of the beam. Part of the laser energy is lost at longer distance, but this is compensated by focusing of the radiation to a smaller area on the sample surface. The long distance can also be achieved by a set of mirrors, between which the radiation travels before it is directed to the focusing lens and the sample. This method for improving the spatial resolution can be utilized in mass spectrometry imaging of tissues by techniques that utilize IR laser ablation, such as laser ablation electrospray ionization, laser ablation atmospheric pressure photoionization, and matrix-assisted laser desorption electrospray ionization. Graphical Abstract ᅟ.