Acquiring thin histological sections of açaí (Euterpe oleracea Mart.) seeds for molecular mapping by mass spectrometry imaging

Abstract

RATIONALE: Matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) of tissues became popular in the last decade. Consequently, adapting sample preparation methods for different materials turned to be a pivotal step for a successful analysis, due to the requirement of samples slices of 12-20 µm thickness. However, preparing thin sections compatible with MALDI-IMS for unusual samples is challenging, as existing histological protocols may not be suitable, thus requiring new methods. Açaí (Euterpe oleracea Mart.) seed is an example of a challenging material due to its toughness and resistance to crack. Therefore, our goal was to develop a methodology to obtain thin (<20 µm) and entire longitudinal sections of açaí seeds for MALDI-IMS analysis. METHODS: Different strategies were evaluated for obtaining thin cuts of seeds, being the combination of the following steps the most suitable option: (i) softening of seeds by water immersion for 24 h; (ii) longitudinal cut of seeds to obtain half-seeds using a razor blade and a hammer; (iii) half-seeds imbibition in gelatin; (iv) sectioning using a cryostat at -20 °C to obtain samples with <20 µm thickness; and (v) collection of samples in an indium tin oxide coated glass slide covered by a double-face copper tape to avoid sample wrapping and ensure adhesion after unfreezing. (iv) storage of samples in a -80 °C freezer, if necessary. RESULTS: As a result, this adapted sample preparation method enabled the analysis of açaí seeds by MALDI-IMS providing spatial distribution of carbohydrates in the endosperm. CONCLUSIONS: The adaptations developed for sample preparation will help investigate the metabolic and physiological properties of açaí seeds in future studies.

Phenolic Profile and Antioxidant Properties in Extracts of Developing Açaí (Euterpe oleracea Mart.) Seeds

“…Samples were prepared according to a previously published methodology with minor modifications . Briefly, S1, S2, S3, and S4 development seed stages were kept in deionized water.…”

“…The sectioning process used a cryostat (Leica CM1860-UV, Leica Biosystems, Nussloch, Germany) kept at −25 °C, and the embedded seed was attached to the holder with a cryomatrix. The lack of solid endosperm at S1 and S2 stages prevented sectioning of the entire tissue to obtain a representative sample, and the analyses were continued only with S3 and S4, as previously described . The matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) analyses were performed in a Solarix XR FT-ICR 7T (Bruker Daltonics, Germany) spectrometer with a nitrogen laser (337 nm).…”

“…Samples were prepared according to a previously published methodology with minor modifications. 25 Briefly, S1, S2, S3, and S4 development seed stages were kept in deionized water. Then, the samples were drilled and immersed in warm gelatin at 10% before being placed in a vacuumpumped oven to remove the air while filling the seed cavities with gelatin to guarantee a section of the entire tissues.…”

“…The lack of solid endosperm at S1 and S2 stages prevented sectioning of the entire tissue to obtain a representative sample, and the analyses were continued only with S3 and S4, as previously described. 25 The matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) analyses were performed in a Solarix XR FT-ICR 7T (Bruker Daltonics, Germany) spectrometer with a nitrogen laser (337 nm). Before MALDI-IMS analysis, the dihydroxybenzoic acid (DHB) matrix was added using a standard sublimation method.…”

“…In addition to the PC identification in extract samples of each developmental stage, we performed MALDI imaging mass spectrometry (MALDI-IMS) to provide information about the localization of those substances. However, the gelatin used as media to embed the sample according to a previously developed protocol 25 dislocated the analytes when in direct contact with the tissue and the hollow cavity (Figure A8) of S1 and S2. Thus, the spatial distribution analysis in those stages was not achieved with adequate precision.…”