An automated platform has been developed for acquisition and visualization of mass spectrometry imaging (MSI) data using nanospray desorption electrospray ionization (nano-DESI). The new system enables robust operation of the nano-DESI imaging source over many hours by precisely controlling the distance between the sample and the nano-DESI probe. This is achieved by mounting the sample holder onto an automated XYZ stage, defining the tilt of the sample plane, and recalculating the vertical position of the stage at each point. This approach is useful for imaging of relatively flat samples such as thin tissue sections. Custom software called MSI QuickView was developed for visualization of large data sets generated in imaging experiments. MSI QuickView enables fast visualization of the imaging data during data acquisition and detailed processing after the entire image is acquired. The performance of the system is demonstrated by imaging rat brain tissue sections. Low background noise enables simultaneous detection of lipids and metabolites in the tissue section. High-resolution mass analysis combined with tandem mass spectometry (MS/MS) experiments enabled identification of the observed species. In addition, the high dynamic range (>2000) of the technique allowed us to generate ion images of low-abundance isobaric lipids. A high-spatial resolution image was acquired over a small region of the tissue section revealing the distribution of an abundant brain metabolite, creatine, on the boundary between the white and gray matter. The observed distribution is consistent with the literature data obtained using magnetic resonance spectroscopy.

Graphical abstract

Matrix effects in mass spectrometry imaging (MSI) may affect the observed molecular distribution in chemical and biological systems. In this study, we use mouse brain tissue of a middle cerebral artery occlusion (MCAO) stroke model to examine matrix effects in nanospray desorption electrospray ionization MSI (nano-DESI MSI). This is achieved by normalizing the intensity of the sodium and potassium adducts of endogenous phosphatidylcholine (PC) species to the intensity of the corresponding adduct of the PC standard supplied at a constant rate with the nano-DESI solvent. The use of MCAO model with an ischemic region localized to one hemisphere of the brain enables immediate comparison of matrix effects within one ion image. Furthermore, significant differences in sodium and potassium concentrations in the ischemic region in comparison with the healthy tissue allowed us to distinguish between two types of matrix effects. Specifically, we discuss matrix effects originating from variations in alkali metal concentrations and matrix effects originating from variations in the molecular composition of the tissue. Compensation for both types of matrix effects was achieved by normalizing the signals corresponding to endogenous PC to the signals of the standards. This approach, which does not introduce any complexity in sample preparation, efficiently compensates for signal variations resulting from differences in the local concentrations of sodium and potassium in tissue sections and from the complexity of the extracted analyte mixture derived from local variations in molecular composition.

Single cell metabolomics using mass spectrometry can contribute to understanding biological activities in health and disease.

Imaging mass spectrometry offers simultaneous spatially resolved detection of drugs, drug metabolites, and endogenous substances in a single experiment. This is important when evaluating effects of a drug on a complex organ system such as the brain, where there is a need to understand how regional drug distribution impacts function. Nanospray desorption electrospray ionization, nano-DESI, is a new ambient technique that enables spatially resolved analysis of a variety of samples without special sample pretreatment. This study introduces an experimental approach for accurate spatial mapping of drugs and metabolites in tissue sections by nano-DESI imaging. In this approach, an isotopically labeled standard is added to the nano-DESI solvent to compensate for matrix effects and ion suppression. The analyte image is obtained by normalizing the analyte signal to the signal of the standard in each pixel. We demonstrate that the presence of internal standard enables online quantification of analyte molecules extracted from tissue sections. Ion images are subsequently mapped to the anatomical brain regions in the analyzed section by use of an atlas mesh deformed to match the optical image of the section. Atlas-based registration accounts for the physical variability between animals, which is important for data interpretation. The new approach was used for mapping the distribution of nicotine in rat brain tissue sections following in vivo drug administration. We demonstrate the utility of nano-DESI imaging for sensitive detection of the drug in tissue sections with subfemtomole sensitivity in each pixel of a 27 μm × 150 μm area. Such sensitivity is necessary for spatially resolved detection of low-abundance molecules in complex matrices.

Mass spectrometry imaging (MSI) has been extensively used for determining spatial distributions of molecules in biological samples, and there is increasing interest in using MSI for quantification. Nanospray desorption electrospray ionization (nano-DESI) is an ambient MSI technique where a solvent is used for localized extraction of molecules followed by nanoelectrospray ionization. Doping the nano-DESI solvent with carefully selected standards enables online quantification during MSI experiments. In this proof-of-principle study, we demonstrate that this quantification approach can be extended to provide shotgun-like quantification of phospholipids in thin brain tissue sections. Specifically, two phosphatidylcholine (PC) standards were added to the nano-DESI solvent for simultaneous imaging and quantification of 22 endogenous PC species observed in nano-DESI MSI. Furthermore, by combining the quantitative data obtained in the individual pixels, we demonstrate quantification of these PC species in seven different regions of a rat brain tissue section.

Small molecule neurotransmitters are essential for the function of the nervous system, and neurotransmitter imbalances are often connected to neurological disorders. The ability to quantify such imbalances is important to provide insights into the biochemical mechanisms underlying the disorder. This proof-of-principle study presents online quantification of small molecule neurotransmitters, specifically acetylcholine, γ-aminobutyric acid (GABA) and glutamate, in rat brain tissue sections using nanospray desorption electrospray ionization (nano-DESI) mass spectrometry imaging. By incorporating deuterated internal standards in the nano-DESI solvent we show identification, accurate mapping, and quantification of these small neurotransmitters in rat brain tissue without introducing any additional sample preparation steps. We find that GABA is about twice as abundant in the medial septum-diagonal band complex (MSDB) as in the cortex, while glutamate is about twice as abundant in the cortex as compared to the MSDB. The study shows that nano-DESI is well suited for imaging of small molecule neurotransmitters in health and disease.

Dry eye syndrome is caused by a reduction in the volume or quality of tears. Here, we show that pituitary adenylate cyclase-activating polypeptide (PACAP)-null mice develop dry eye-like symptoms such as corneal keratinization and tear reduction. PACAP immunoreactivity is co-localized with a neuronal marker, and PACAP receptor (PAC1-R) immunoreactivity is observed in mouse infraorbital lacrimal gland acinar cells. PACAP eye drops stimulate tear secretion and increase cAMP and phosphorylated (p)-protein kinase A levels in the infraorbital lacrimal glands that could be inhibited by pre-treatment with a PAC1-R antagonist or an adenylate cyclase inhibitor. Moreover, these eye drops suppress corneal keratinization in PACAP-null mice. PACAP eye drops increase aquaporin 5 (AQP5) levels in the membrane and pAQP5 levels in the infraorbital lacrimal glands. AQP5 siRNA treatment of the infraorbital lacrimal gland attenuates PACAP-induced tear secretion. Based on these results, PACAP might be clinically useful to treat dry eye disorder.