Confirmation of sub-cellular resolution using oversampling imaging mass spectrometry
The use of oversampling in MALDI (matrix-assisted laser desorption/ionization) imaging mass spectrometry (IMS) to improve lateral resolution is a common practice. However, its application is still controversial and recent studies reported a spot size–dependent change in the relative intensity of the spectra. Previously, using oversampling, we described the lipidome of the human colon epithelium, a 20–30 μm wide cell monolayer; even assessing the changes occurring within this monolayer associated with complex biological processes. Interestingly, the K-means analysis of those experiments unveiled the presence of a third epithelial cluster that anatomically matched the nuclei position. Taking into account the nucleus size (9–12 μm of diameter) and its distinctive lipidome, we decided to test whether this cluster was really of nuclear origin. Hence, the spectra obtained directly from tissue sections were compared with those recorded from the nuclei isolated from colon biopsies. The highest correlation coefficient was obtained when comparing the spectrum of the isolated nuclei with that of the tissue nuclear cluster, demonstrating the successful identification of the nuclear lipidome in the MALDI-IMS experiments run using oversampling and a lateral resolution of 10 μm/pixel. Importantly, it was established that phosphatidylinositol 38:4 nuclear levels remained stable along the colon crypt. That is, it mimicked neither the regular decrease observed in the epithelium nor the regular increase observed in the stroma, eliminating the chance of inter-pixel contamination. Altogether, besides confirming the usefulness of the oversampling technique, these results strongly reinforce the pivotal role IMS may have in promising fields such as single-cell analysis.
KeywordsMALDI imaging Lateral resolution Oversampling Colonocytes Nucleus
We are greatly thankful to the nurses and the medical doctors of the Gastroenterology Unit of Hospital Universitari de Son Espases (HUSE, Palma, Spain) and Hospital Comarcal d'Inca (Inca, Spain). Technical and human support provided by the Plataforma de Microscopía of IdISBa and by the Servicio de Lipidómica of the SGIKER (UPV/EHU, MICINN, GV/EJ, ESF) is gratefully acknowledged.
This study was funded by The Institute of Health Carlos III (CP12/03338), and the MINECO (Ministry of Economy and Competitiveness, RTC-2015-3693-1), and the Basque Government (IT1162-19) partially supported this study. The following institutions and projects supported authors’ contracts: AM-B, Institute of Health Carlos III (ISCIII, PI16/02200); JG, University of the Basque Country (UPV/EHU); JB-E, Govern Balear-Direcció General d'Innovació i Recerca (predoctoral fellowship, FPI/1787/2015); DHL and RF, Ministry of Economy and Competitiveness (RTC-2015-3693-1); and GB-C, ISCIII (Miguel Servet II program, CPII17/0005). Further, contracts and projects were co-funded either by the European Regional Development Fund (ERDF) or the European Social Fund (ESF).
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
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