Visualisation of microalgal lipid bodies through electron microscopy

Abstract

In this study, transmission electron microscopy (TEM) and cryo‐scanning electron microscopy (cryo‐SEM) were evaluated for their ability to detect lipid bodies in microalgae. To do so, Phaeodactylum tricornutum and Nannochloropsis oculata cells were harvested in both the mid‐exponential and early stationary growth phase. Two different cryo‐SEM cutting methods were compared: cryo‐planing and freeze‐fracturing. The results showed that, despite the longer preparation time, TEM visualisation preceded by cryo‐immobilisation allows a clear detection of lipid bodies, and is preferable to cryo‐SEM. Using freeze‐fracturing, lipid bodies were rarely detected. This was only feasible if crystalline layers in the internal structure, most likely related to sterol esters or di‐saturated triacylglycerols, were revealed. Furthermore, lipid bodies could not be detected using cryo‐planing. Cryo‐SEM is also not the preferred technique to recognise other organelles besides lipid bodies, yet it did reveal chloroplasts in both species and filament‐containing organelles in cryo‐planed Nannochloropsis oculata samples.This article is protected by copyright. All rights reserved Lipid bodies are cellular organelles, which serve as a storage for lipids inside the cell. Microalgae can accumulate up to more than 50% of their dry weight as lipids. These lipids can be of interest for application in the production of biofuel and for application in food products. Certain microalgae can grow fast and have a high yield, making them an interesting alternative lipid source. The amount of lipids in microalgae increases when the cells are grown under unfavourable conditions such as nutrient limitation. The goal of this study was to compare advanced microscopy techniques to visualise lipid bodies in two different microalgae species. The first technique is transmission electron microscopy (TEM), whereby samples are high‐pressure frozen, freeze‐substituted and cut into thin slices before visualisation. The second technique is cryo‐scanning electron microscopy (cryo‐SEM), preceded by two different cutting methods to reveal the sample's inner structure. After being quickly frozen, samples are either roughly broken (freeze‐fractured) or cut with a sharp knife to obtain a flat surface (cryo‐planing). For each microalgae species, cells in the mid‐exponential and early stationary growth phase were examined, lipid bodies are expected to be more numerous in the latter ones. The results showed that TEM visualisation preceded by cryo‐immobilisation allows the detection of lipid bodies, and is preferable to cryo‐SEM, despite the longer preparation time. Using freeze‐fracturing, only occasionally a few lipid bodies were detected as the fracturing plane needs to cross the lipid bodies revealing the internal structure (layers), which is necessary for lipid body identification. Cryo‐planing was not useful for lipid body detection in these samples as this technique resulted in flat cell surfaces. In addition, TEM reveals the highest amount of other cell organelles while cryo‐SEM could only reveal chloroplasts and filament‐containing organelles. Our findings could help other researchers in choosing an appropriate electron microscopy technique for visualising lipid bodies in biological samples or the microstructure of food matrices.