Laser ablation inductively coupled plasma mass spectrometry for imaging of copper, zinc, and platinum in thin sections of a kidney from a mouse treated with cis-platin

Platinum complexes are used for the treatment of several types of cancer. High platinum concentrations in the target tissue and low concentrations in dose-limiting tissue structures such as renal tubules are desirable to assure selective toxicity. Microlocal analysis of platinum distribution in tissue sections may thus contribute to the optimization of platinum therapy. Scanning laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) was used to produce images of element distribution in 14-μm thin sections of kidney tissue from a mouse treated with cis-platin 60 min prior to victimization. The sample surface was scanned (raster area 300 mm²) with a focused laser beam (wavelength 266 nm, diameter of laser crater 50 μm, inter line distance 50 μm and laser power density 3 × 10⁹ W cm⁻²) in a cooled laser ablation chamber (about −15 °C) developed for these measurements. The laser ablation system was coupled to a double-focusing sector field ICP-MS. Ion intensities of ⁶³Cu⁺, ⁶⁴Zn⁺, and ¹⁹⁶Pt⁺ were measured within the tissue by LA-ICP-MS. Matrix-matched laboratory standards served for calibration of analytical data. The mass spectrometric analysis yielded an inhomogeneous distribution for Cu, Zn, and Pt in thin kidney sections. Copper was enriched in the capsule and outer cortex, zinc in the inner cortex and the platinum concentration followed a centripetal gradient with clear medullar enrichment. Thus, scanning LA-ICP-MS may be a useful tool in the preclinical development of new and less nephrotoxic platinum complexes.     

Lipid imaging in the zebra finch brain with secondary ion mass spectrometry

Lipids have diverse functions in the nervous system, but the study of their anatomical distributions in the intact brain is rather difficult using conventional methodologies. Here we demonstrate the application of high resolution time-of-flight (ToF) secondary ion mass spectrometry (SIMS) to image various lipid components and cholesterol across an entire brain section prepared from an adult zebra finch (Taeniopygia guttata), with a spatial resolution of 2.3 μm, resulting in the formation of 11.5 megapixel chemical images. The zebra finch is a songbird in which specific neural and developmental functions have been ascribed to discrete “song control nuclei” of the forebrain. We have observed a relative increase of palmitic acid C16:0 and oleic acid C18:1 in song control nuclei versus the surrounding tissue, while phosphate (PO₃⁻), representative of phospholipids, was lower in these regions. Cholesterol was present at a high level only in the white matter of the optic tectum. More diffuse distributions were observed for stearic, arachidonic, linolenic, and palmitoleic acids. The presented results illustrate that SIMS imaging is a useful approach for assessing changes in lipid content during song circuit development and song learning.     

Trypsin and MALDI matrix pre‐coated targets simplify sample preparation for mapping proteomic distributions within biological tissues by imaging mass spectrometry

Prefabricated surfaces containing α‐cyano‐4‐hydroxycinnamic acid and trypsin have been developed to facilitate enzymatic digestion of endogenous tissue proteins prior to matrix‐assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS). Tissue sections are placed onto slides that were previously coated with α‐cyano‐4‐hydroxycinnamic acid and trypsin. After incubation to promote enzymatic digestion, the tissue is analyzed by MALDI IMS to determine the spatial distribution of the tryptic fragments. The peptides detected in the MALDI IMS dataset were identified by Liquid chromatography‐tandem mass spectrometry/mass spectrometry. Protein identification was further confirmed by correlating the localization of unique tryptic fragments originating from common parent proteins. Using this procedure, proteins with molecular weights as large as 300 kDa were identified and their distributions were imaged in sections of rat brain. In particular, large proteins such as myristoylated alanine‐rich C‐kinase substrate (29.8 kDa) and spectrin alpha chain, non‐erythrocytic 1 (284 kDa) were detected that are not observed without trypsin. The pre‐coated targets simplify workflow and increase sample throughput by decreasing the sample preparation time. Further, the approach allows imaging at higher spatial resolution compared with robotic spotters that apply one drop at a time. Copyright © 2016 John Wiley & Sons, Ltd.     

Biological Tissue Imaging with a Position and Time Sensitive Pixelated Detector

We demonstrate the capabilities of a highly parallel, active pixel detector for large-area, mass spectrometric imaging of biological tissue sections. A bare Timepix assembly (512?×?512 pixels) is combined with chevron microchannel plates on an ion microscope matrix-assisted laser desorption time-of-flight mass spectrometer (MALDI TOF-MS). The detector assembly registers position- and time-resolved images of multiple m/z species in every measurement frame. We prove the applicability of the detection system to biomolecular mass spectrometry imaging on biologically relevant samples by mass-resolved images from Timepix measurements of a peptide?Cgrid benchmark sample and mouse testis tissue slices. Mass-spectral and localization information of analytes at physiologic concentrations are measured in MALDI-TOF-MS imaging experiments. We show a high spatial resolution (pixel size down to 740?×?740?nm² on the sample surface) and a spatial resolving power of 6???m with a microscope mode laser field of view of 100?C335???m. Automated, large-area imaging is demonstrated and the Timepix?? potential for fast, large-area image acquisition is highlighted.     

Mass spectrometry imaging with high resolution in mass and space (HR<Superscript>2</Superscript> MSI) for reliable investigation of drug compound distributions on the cellular level

Mass spectrometry (MS) imaging is a versatile method to analyze the spatial distribution of analytes in tissue sections. It provides unique features for the analysis of drug compounds in pharmacokinetic studies such as label-free detection and differentiation of compounds and metabolites. We have recently introduced a MS imaging method that combines high mass resolution and high spatial resolution in a single experiment, hence termed HR² MS imaging. In the present study, we applied this method to analyze the spatial distribution of the anti-cancer drugs imatinib and ifosfamide in individual mouse organs. The whole kidney of an animal dosed with imatinib was measured at 35 μm spatial resolution. Imatinib showed a well-defined distribution in the outer stripe of the outer medulla. This area was analyzed in more detail at 10 μm step size, which constitutes a tenfold increase in effective spatial resolution compared to previous studies of drug compounds. In parallel, ion images of phospholipids and heme were used to characterize the histological features of the tissue section and showed excellent agreement with histological staining of the kidney after MS imaging. Ifosfamide was analyzed in mouse kidney at 20 μm step size and was found to be accumulated in the inner medulla region. The identity of imatinib and ifosfamide was confirmed by on-tissue MS/MS measurements. All measurements including mass spectra from 10 μm pixels featured accurate mass (≤2 ppm root mean square) and mass resolving power of R = 30,000. Selected ion images were generated with a bin size of ∆m/z = 0.01 ensuring highly specific information. The ability of the method to cover larger areas was demonstrated by imaging a compound in the intestinal tract of a rat whole-body tissue section at 200 μm step size. The described method represents a major improvement in terms of spatial resolution and specificity for the analysis of drug compounds in tissue sections.     

In situ isobaric lipid mapping by MALDI–ion mobility separation–mass spectrometry imaging

The highly diverse chemical structures of lipids make their analysis directly from biological tissue sections extremely challenging. Here, we report the in situ mapping and identification of lipids in a freshwater crustacean Gammarus fossarum using matrix‐assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) in combination with an additional separation dimension using ion mobility spectrometry (IMS). The high‐resolution trapped ion mobility spectrometry (TIMS) allowed efficient separation of isobaric/isomeric lipids showing distinct spatial distributions. The structures of the lipids were further characterized by MS/MS analysis. It is demonstrated that MALDI MSI with mobility separation is a powerful tool for distinguishing and localizing isobaric/isomeric lipids.     

Direct profiling of myelinated and demyelinated regions in mouse brain by imaging mass spectrometry

One of the newly developed imaging mass spectrometry (IMS) technologies utilizes matrix-assisted laser desorption/ionization (MALDI) mass spectrometry to map proteins in thin tissue sections. In this study, we evaluated the power of MALDI IMS as we developed it in our (Bruker) MALDI TOF (Reflex IV) and TOF-TOF (Ultraflex II) systems to study myelin patterns in the mouse central nervous system under normal and pathological conditions. MALDI IMS was applied to assess myelin basic protein (MBP) isoform-specific profiles in different regions throughout the mouse brain. The distribution of ions of m/z 14,144 and 18,447 displayed a striking resemblance with white matter histology and were identified as MBP isoform 8 and 5, respectively. In addition, we demonstrated a significant reduction of the MBP-8 peak intensity upon MALDI IMS analysis of focal ethidium bromide-induced demyelinated brain areas. Our MS images were validated by immunohistochemistry using MBP antibodies. This study underscores the potential of MALDI IMS to study the contribution of MBP to demyelinating diseases.     

Development of capabilities for imaging mass spectrometry under ambient conditions with desorption electrospray ionization (DESI)

Aspects of the development of mass spectrometry over the past three decades are briefly reviewed and growth points in the subject are identified. Molecular imaging by mass spectrometry is one such growth area. The development of a capability for 2D chemical imaging of surfaces is described, based on the combination of a desorption electrospray ionization (DESI) ion source with an automated surface stage capable of x, y translational motion. The lateral resolution of this new system is found to be less than 200 microns, using a test ink pattern. Chemical imaging of surfaces is demonstrated using model examples of organic and biological systems: (i) imaging of a 2D pattern written in different colored inks on photographic paper and (ii) imaging of thin coronal sections of rat brain tissue fixed onto a glass microscope slide. In both cases, full mass spectra are recorded as a function of x,y-position on the surface. In the chemical imaging example, the distributions of the two different inks on the paper surface were mapped by tracking the abundance of the intact organic cation which characterizes each particular ink dye. In the tissue imaging example, distributions of specific lipids in coronal sections of rat brain tissue were followed from the abundance distributions in 2D space of the deprotonated lipid molecules recorded in the negative ion mass spectra. These latter distributions reveal distinct anatomical features of the rat brain. The results of these studies demonstrate the feasibility of performing surface imaging studies using DESI and show that at this stage of its development it has a lateral spatial resolution of a few hundred microns.     

High mass accuracy and high mass resolving power FT-ICR secondary ion mass spectrometry for biological tissue imaging

Biological tissue imaging by secondary ion mass spectrometry has seen rapid development with the commercial availability of polyatomic primary ion sources. Endogenous lipids and other small bio-molecules can now be routinely mapped on the sub-micrometer scale. Such experiments are typically performed on time-of-flight mass spectrometers for high sensitivity and high repetition rate imaging. However, such mass analyzers lack the mass resolving power to ensure separation of isobaric ions and the mass accuracy for elemental formula assignment based on exact mass measurement. We have recently reported a secondary ion mass spectrometer with the combination of a C₆₀ primary ion gun with a Fourier transform ion cyclotron resonance mass spectrometer (FT-ICR MS) for high mass resolving power, high mass measurement accuracy, and tandem mass spectrometry capabilities. In this work, high specificity and high sensitivity secondary ion FT-ICR MS was applied to chemical imaging of biological tissue. An entire rat brain tissue was measured with 150 μm spatial resolution (75 μm primary ion spot size) with mass resolving power (m/Δm _50%) of 67,500 (at m/z 750) and root-mean-square measurement accuracy less than two parts-per-million for intact phospholipids, small molecules and fragments. For the first time, ultra-high mass resolving power SIMS has been demonstrated, with m/Δm _50%?>?3,000,000. Higher spatial resolution capabilities of the platform were tested at a spatial resolution of 20 μm. The results represent order of magnitude improvements in mass resolving power and mass measurement accuracy for SIMS imaging and the promise of the platform for ultra-high mass resolving power and high spatial resolution imaging.

Desorption electrospray ionization imaging mass spectrometry of lipids in rat spinal cord

Imaging mass spectrometry allows for the direct investigation of tissue samples to identify specific biological compounds and determine their spatial distributions. Desorption electrospray ionization (DESI) mass spectrometry has been used for the imaging and analysis of rat spinal cord cross sections. Glycerophospholipids and sphingolipids, as well as fatty acids, were detected in both the negative and positive ion modes and identified through tandem mass spectrometry (MS/MS) product ion scans using collision-induced dissociation and accurate mass measurements. Differences in the relative abundances of lipids and free fatty acids were present between white and gray matter areas in both the negative and positive ion modes. DESI-MS images of the corresponding ions allow the determination of their spatial distributions within a cross section of the rat spinal cord, by scanning the DESI probe across the entire sample surface. Glycerophospholipids and sphingolipids were mostly detected in the white matter, while the free fatty acids were present in the gray matter. These results show parallels with reported distributions of lipids in studies of rat brain. This suggests that the spatial intensity distribution reflects relative concentration differences of the lipid and fatty acid compounds in the spinal cord tissue. The “butterfly” shape of the gray matter in the spinal cord cross section was resolved in the corresponding ion images, indicating that a lateral resolution of better than 200 μm was achieved. The selected ion images of lipids are directly correlated with anatomic features on the spinal cord corresponding to the white and the gray matter.     

Comparison of laser ablation-inductively coupled plasma-mass spectrometry and micro-X-ray fluorescence spectrometry for elemental imaging in Daphnia magna

Visualization of elemental distributions in thin sections of biological tissue is gaining importance in many disciplines of biological and medical research. Laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) and scanning micro-X-ray fluorescence spectrometry (micro-XRF) are two widely used microanalytical techniques for elemental mapping. This article compares the capabilities of the two techniques for imaging the distribution of selected elements in the model organism Daphnia magna in terms of detection power and spatial resolution. Sections with a thickness of 10 and 20 μm of the fresh water crustacean Daphnia magna were subjected to LA-ICP-MS and micro-XRF analysis. The elemental distributions obtained for Ca, P, S and Zn allow element-to-tissue correlation. LA-ICP-MS and micro-XRF offer similar limits of detection for the elements Ca and P and thus, allow a cross-validation of the imaging results. LA-ICP-MS was particularly sensitive for determining Zn (LOD 20 μg g⁻¹, 15 μm spot size) in Daphnia magna, while the detection power of micro-XRF was insufficient in this context. However, LA-ICP-MS was inadequate for the measurement of the S distributions, which could be better visualized with micro-XRF (LOD 160 μg g⁻¹, 5 s live time). Both techniques are thus complementary in providing an exhaustive chemical profiling of tissue samples.     

Single bubble growth during water electrolysis under microgravity

Single bubble evolution on a micro-electrode (Pt, φ = 0.2 mm) was observed in 0.36 M KOH solution under terrestrial (1-G) and microgravity (μ-G) environments. The bubble size during galvanostatic electrolysis (j = −2.6 × 10³ A m⁻²) was measured by CCD images, which allowed us to calculate the gas evolution efficiency, f_G, by comparison with the consumed charge. The efficiency under μ-G increased until 1 s after starting electrolysis and then reached constant value (f_G = 0.85), while, under 1-G, it showed a lower value and remarkably decreased 2 s after the beginning of the measurement. Such differences between μ-G and 1-G were explained by the mass transfer rate of the dissolved gas. Bubble-induced microconvection dominated the mass transfer under μ-G without any buoyancy force, on the other hand, the single-phase free convection (microscopic natural convection) influenced the bubble growth under 1-G.     

Three-dimensional visualization of protein expression in mouse brain structures using imaging mass spectrometry

We have developed a method to visualize matrix-assisted laser desorption ionization imaging mass spectrometry (MALDI IMS) data aligned with optically determinable tissue structures in three dimensions. Details of the methodology are exemplified using the 3-D reconstruction of myelin basic protein (MBP) in the corpus callosum of a mouse brain. In this procedure, optical images obtained from serial coronal sections are first aligned to each other to reconstruct a surface of the corpus callosum from segmented contours of the aligned images. The MALDI IMS data are then coregistered to the optical images and superimposed into the surface to create the final 3-D visualization. Correlating proteomic data with anatomical structures provides a more comprehensive understanding of healthy and pathological brain functions, and holds promise to be utilized in more complex anatomical arrangements.     

Comparative imaging of P,S, Fe,Cu, Zn and C in thin sections of rat brain tumor as well as control tissues by laser ablation inductively coupled plasma mass spectrometry

Laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) was used for quantitative imaging of selected elements (P, S, Fe, Cu, Zn and C) in thin sections of rat brain samples (thickness 20 μm). The sample surface was scanned (raster area ~ 2 cm²) with a focused laser beam (wavelength 266 nm, diameter of laser crater 50 μm, and irradiance 1 × 10⁹ W cm^− 2). The laser ablation system was coupled to a double-focusing sector field. The possibility was evaluated of using carbon (via measurement of ¹³C⁺) as an internal standard element for imaging element distribution as part of this method. The LA-ICP-MS images obtained for P, S, Fe Cu and Zn were quantified using synthetically prepared matrix-matched laboratory standards. Depending on the sample analyzed, concentrations of Cu and Zn in the control tissue were found to be in the range of 8–10 μg g^− 1 and 10–12 μg g^− 1, while in the tumor tissue these concentrations were in the range of 12–15 μg g^− 1 and 15–17 μg g^− 1, respectively. The measurements of P, S and Fe distribution revealed the depletion of these elements in tumor tissue. In all the samples, the shape of the tumor could be clearly distinguished from the surrounding healthy tissue by the depletion in carbon. Additional experiments were performed in order to study the influence of the water content of the analyzed tissue on the intensity signal of the analyte. The results of these measurements show the linear correlation (R² = 0.9604) between the intensity of analyte and amount of water in the sample. The growth of a brain tumor was thus studied for the first time by imaging mass spectrometry.     

MALDI imaging mass spectrometry of β‐ and γ‐crystallins in the ocular lens

Lens crystallin proteins make up 90% of expressed proteins in the ocular lens and are primarily responsible for maintaining lens transparency and establishing the gradient of refractive index necessary for proper focusing of images onto the retina. Age‐related modifications to lens crystallins have been linked to insolubilization and cataractogenesis in human lenses. Matrix‐assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) has been shown to provide spatial maps of such age‐related modifications. Previous work demonstrated that, under standard protein IMS conditions, α‐crystallin signals dominated the mass spectrum and age‐related modifications to α‐crystallins could be mapped. In the current study, a new sample preparation method was optimized to allow imaging of β‐ and γ‐crystallins in ocular lens tissue. Acquired images showed that γ‐crystallins were localized predominately in the lens nucleus whereas β‐crystallins were primarily localized to the lens cortex. Age‐related modifications such as truncation, acetylation, and carbamylation were identified and spatially mapped. Protein identifications were determined by top‐down proteomics analysis of lens proteins extracted from tissue sections and analyzed by LC‐MS/MS with electron transfer dissociation. This new sample preparation method combined with the standard method allows the major lens crystallins to be mapped by MALDI IMS.     

Simultaneous determination of gibberellic acid, indole-3-acetic acid and abscisic acid in wheat extracts by solid-phase extraction and liquid chromatography-electrospray tandem mass spectrometry

A liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed for simultaneous determination of three representative phytohormones in plant samples: gibberellic acid (GA₃), indole-3-acetic acid (IAA) and abscisic acid (ABA). A solid-phase extraction (SPE) pretreatment method was used to concentrate and purify the three phytohormones of different groups from plant samples. The separation was carried out on a C₁₈ reversed-phase column, using methanol/water containing 0.2% formic acid (50:50, v/v) as the isocratic mobile phase at the flow-rate of 1.0 mL min⁻¹, and the three phytohormones were eluted within 7 min. A linear ion trap mass spectrometer equipped with electrospray ionization source was operated in negative ion mode. Selective reaction monitoring (SRM) was employed for quantitative measurement. The SRM transitions monitored were as 345 → 239, 301 for GA₃, 174 → 130 for IAA and 263 → 153, 219 for ABA. Good linearities were found within the ranges of 5–200 μg mL⁻¹ for IAA and 0.005–10 μg mL⁻¹ for ABA and GA₃. Their detection limits based on a signal-to-noise ratio of three were 0.005 μg mL⁻¹, 2.2 μg mL⁻¹ and 0.003 μg mL⁻¹ for GA₃, IAA and ABA, respectively. Good recoveries from 95.5% to 102.4% for the three phytohormones were obtained. The results demonstrated that the SPE-LC–MS/MS method developed is highly effective for analyzing trace amounts of the three phytohormones in plant samples.     

Faster and simpler determination of chlorophenols in water by fiber introduction mass spectrometry

Solid phase microextraction (SPME) of chlorophenols [2-chlorophenol (2CP), 2,4-dichlorophenol (24CP), 4-chloro-3-methylphenol (43CP), 2,4,6-tri-chlorophenol (246CP) and pentachlorophenol (PCP)] followed by direct mass spectrometric analysis has been performed by fiber introduction mass spectrometry (FIMS). Two SPME fibers (65 μm PDMS/DVB and 85 μm PA fibers) were tested, and FIMS was performed via selective ion monitoring (SIM). The extractions were evaluated at 10% ionic strength and pH 1. Best extraction times were determined for both fibers. Limits of detection (LOD) and limits of quantification (LOQ) for both fibers were in the low μg L⁻¹ range. Coefficients of correlation for the analytical curves showed linear responses and mineral water and river water samples spiked with 50 μg L⁻¹ presented high recoveries. FIMS, as compared to current EPA methods, is demonstrated to allow faster and simpler (elimination of pre-separation or derivatization steps) analysis of chlorophenols in water with the required sensitivity.     

A positron annihilation lifetime measurement system with an intense positron microbeam

An intense positron microbeam was formed using an electron linear accelerator. The beam is pulsed to apply positron lifetime spectroscopy to very small samples and to obtain positron lifetime images by scanning it. Positron lifetimes are measured with time resolution of <300 ps and with lateral spatial resolution of 30–100 μm. A counting rate of the γ-ray to measure positron lifetime is about 10³ s⁻¹ which is 10 times higher than that achieved by the radioisotope based microbeam.     

Absolute Quantification of Rifampicin by MALDI Imaging Mass Spectrometry Using Multiple TOF/TOF Events in a Single Laser Shot

Matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) allows for the visualization of molecular distributions within tissue sections. While providing excellent molecular specificity and spatial information, absolute quantification by MALDI IMS remains challenging. Especially in the low molecular weight region of the spectrum, analysis is complicated by matrix interferences and ionization suppression. Though tandem mass spectrometry (MS/MS) can be used to ensure chemical specificity and improve sensitivity by eliminating chemical noise, typical MALDI MS/MS modalities only scan for a single MS/MS event per laser shot. Herein, we describe TOF/TOF instrumentation that enables multiple fragmentation events to be performed in a single laser shot, allowing the intensity of the analyte to be referenced to the intensity of the internal standard in each laser shot while maintaining the benefits of MS/MS. This approach is illustrated by the quantitative analyses of rifampicin (RIF), an antibiotic used to treat tuberculosis, in pooled human plasma using rifapentine (RPT) as an internal standard. The results show greater than 4-fold improvements in relative standard deviation as well as improved coefficients of determination (R²) and accuracy (>93% quality controls, <9% relative errors). This technology is used as an imaging modality to measure absolute RIF concentrations in liver tissue from an animal dosed in vivo. Each microspot in the quantitative image measures the local RIF concentration in the tissue section, providing absolute pixel-to-pixel quantification from different tissue microenvironments. The average concentration determined by IMS is in agreement with the concentration determined by HPLC-MS/MS, showing a percent difference of 10.6%.

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Graphical Abstract ?

     

Tandem Mass Spectrometry Imaging Enables High Definition for Mapping Lipids in Tissues

Mass spectrometry imaging (MSI) of lipids in biological tissues is useful for correlating molecular distribution with pathological results, which could provide useful information for both biological research and disease diagnosis. It is well understood that the lipidome could not be clearly deciphered without tandem mass spectrometry analysis, but this is challenging to achieve in MSI due to the limitation in sample amount at each image spot. Here we develop a multiplexed MS² imaging (MS²I) method that can provide MS² images for 10 lipid species or more for each sampling spot, providing spatial structural lipidomic information. Coupling with on-tissue photochemical derivatization, imaging of 20 phospholipid C=C location isomers is also realized, showing enhanced molecular images with high definition in structure for mouse brain and human liver cancer tissue sections. Spatially mapped t-distributed stochastic neighbor embedding has also been adopted to visualize the tumor margin with enhancement by structural lipidomic information.