Multimodal, in Situ Imaging of Ex Vivo Human Skin Reveals Decrease of Cholesterol Sulfate in the Neoepithelium during Acute Wound Healing.

Skin repair is a significant aspect of human health. While the makeup of healthy stratum corneum and epidermis is generally understood, the mobilization of molecular components during skin repair remains largely unknown. In the present work, we utilize multimodal, in situ, mass spectrometry, and immunofluorescence imaging for the characterization of newly formed epidermis, following an initial acute wound for the first 96 h of epithelization. In particular, TOF-SIMS and confirmatory MALDI FT-ICR MS (/MS) analysis permitted the mapping of several lipid classes, including phospholipids, neutral lipids, cholesterol, ceramides, and free fatty acids. Endogenous lipid species were localized in discrete epidermal skin layers, including the stratum corneum (SC), stratum granulosum (SG), stratum basale (SB), and dermis. Experiments revealed that healthy re-epithelializing skin is characterized by diminished cholesterol sulfate signal along the stratum corneum toward the migrating epithelial tongue. The spatial distribution and relative abundances of cholesterol sulfate are reported and correlated with the healing time. The multimodal imaging approach enabled in situ high-confidence chemical mapping based on accurate mass and fragmentation pattern of molecular components. The use of postanalysis immunofluorescence imaging from the same tissue confirmed the localization of endogenous lipid species and Filaggrin and Cav-1 proteins at high spatial resolution (approximately a few microns).

Mapping chemotherapeutic drug distribution in cancer cell spheroids using 2D-TOF-SIMS and LESA-TIMS-MS.

Three-dimensional (3D) cancer cell cultures grown in the form of spheroids are effective models for the study of in vivo-like processes simulating cancer tumor pharmacological dynamics and morphology. In this study, we show the advantages of Time-of-Flight Secondary Ion Mass Spectrometry (TOF-SIMS) combined with in situ Liquid Extraction Surface Analysis coupled to trapped Ion Mobility Spectrometry Mass Spectrometry (LESA-TIMS-TOF MS) for high spatial resolution mapping and quantitation of ABT-737, a chemotherapeutic drug, at the level of single human colon carcinoma cell spheroids (HCT 116 MCS). 2D-TOF-SIMS studies of consecutive sections (∼16 µm thick slices) showed that ABT-737 is homogenously distributed in the outer layers of the HCT 116 MCS. Complementary in situ LESA-TIMS-TOF MS/MS measurements confirmed the presence of the ABT-737 drug in the MCS slides by the observation of the molecular ion [M + H]+m/z and mobility, and the charateristic fragmentation pattern. LESA-TIMS-TOF MS allowed a quantitative assessment of the ABT-737 drug of the control MCS slice spiked with ABT-737 standard over the 0.4-4.1 ng range and MCS treated starting at 10 µM for 24 h. These experiments showcase an effective protocol for unambigous characterization and 3D mapping of chemotherapeutic drug distribution at the single MCS level.

Three Dimensional Secondary Ion Mass Spectrometry Imaging (3D-SIMS) of Aedes aegypti ovarian follicles.

The mobilization of nutrient reserves into the ovaries of Aedes aegypti mosquitoes after sugar-feeding plays a vital role in the female's reproductive maturation. In the present work, three-dimensional secondary ion mass spectrometry imaging (3D-SIMS) was used to generate ultrahigh spatial resolution (~1 µm) chemical maps and study the composition and spatial distribution of lipids at the single ovarian follicle level (~100 µm in size). 3D-Mass Spectrometry Imaging (3D-MSI) allowed the identification of cellular types in the follicle (oocyte, nurse and follicular cells) using endogenous markers, and revealed that most of the triacyglycerides (TGs) were compartmentalized in the oocyte region. By comparing follicles from water-fed and sugar-fed females (n=2), 3D-MSI-Time of Flight-SIMS showed that TGs were more abundant in ovarian follicles of sugar-fed females; despite relative sample reproducibility per feeding condition, more biological replicates will better support the trends observed. While the current 3D-MSI-TOF-SIMS does not permit MS/MS analysis of the lipid species, complementary LC-MS/MS analysis of the ovarian follicles aided tentative lipid assignments of the SIMS data. The combination of these MS approaches is giving us a first glimpse of the distribution of functionally relevant ovarian lipid molecules at the cellular level. These new tools can be used to investigate the roles of different lipids on follicle fitness and overall mosquito reproductive output.

Submicron 3-D mass spectrometry imaging reveals an asymmetric molecular distribution on chemotaxing cells.

Background: Dictyostelium discoideum is a ~10 µm diameter unicellular eukaryote that lives on soil surfaces. When starved, D. discoideum cells aggregate into streams of cells in a process called chemotaxis. In this report, we studied D. discoideum cells during chemotaxis using 3D - mass spectrometry imaging (3D-MSI). Methods: The 3D-MSI consisted of the sequential generation of 2D molecular maps using burst alignment coupled to delayed extraction time-of flight secondary ion mass spectrometry (TOF-SIMS) combined with a soft sputtering beam to access the different layers. Results: Molecular maps with sub-cellular high spatial resolution (~300 nm) indicated the presence of ions at m/z = 221 and 236 at the front and sides, but reduced levels at the back, of cells moving toward of aggregation streams. The 3D-MSI also detected an ion at m/z = 240 at the edges and back, but reduced levels at the front, of aggregating cells. Other ions showed an even distribution across the cells. Conclusions: Together, these results demonstrate the utility of sub-micron MSI to study eukaryotic chemotaxis.

Analysis of Chemotherapeutic Drug Delivery at the Single Cell Level Using 3D-MSI-TOF-SIMS.

In this work, we show the advantages of label-free, tridimensional mass spectrometry imaging using dual beam analysis (25 keV Bi3+) and depth profiling (20 keV with a distribution centered at Ar1500+) coupled to time of flight secondary ion mass spectrometry (3D-MSI-TOF-SIMS) for the study of A-172 human glioblastoma cell line treated with B-cell lymphoma 2 (Bcl-2) inhibitor ABT-737. The high spatial (~250 nm) and high mass resolution (m/Δm ~10,000) of TOF-SIMS permitted the localization and identification of the intact, unlabeled drug molecular ion (m/z 811.26 C42H44ClN6O5S2- [M - H]-) as well as characteristic fragment ions. We propose a novel approach based on the inspection of the drug secondary ion yield, which showed a good correlation with the drug concentration during cell treatment at therapeutic dosages (0-200 µM with 4 h incubation). Chemical maps using endogenous molecular markers showed that the ABT-737 is mainly localized in subsurface regions and absent in the nucleus. A semiquantitative workflow is proposed to account for the biological cell diversity based on the spatial distribution of endogenous molecular markers (e.g., nuclei and cytoplasm) and secondary ion confirmation based on the ratio of drug-specific fragments to molecular ion as a function of the therapeutic dosage. Graphical Abstract ᅟ.