Overcoming the challenges of preserving lipid‐rich Cannabis sativa L. glandular trichomes for transmission electron microscopy.

Cannabis glandular trichomes produce and store an abundance of lipidic specialised metabolites (e.g. cannabinoids and terpenes) that are consumed by humans for medicinal and recreational purposes. Due to a lack of genetic resources and inherent autofluorescence of cannabis glandular trichomes, our knowledge of cannabinoid trafficking and secretion is limited to transmission electron microscopy (TEM). Advances in cryofixation methods has resulted in ultrastructural observations closer to the 'natural state' of the living cell, and recent reports of cryofixed cannabis trichome ultrastructure challenge the long‐standing model of cannabinoid trafficking proposed by ultrastructural reports using chemically fixed samples. Here, we compare the ultrastructural morphology of cannabis glandular trichomes preserved using conventional chemical fixation and ultrarapid cryofixation. We show that chemical fixation results in amorphous metabolite inclusions surrounding the organelles of glandular trichomes that were not present in cryofixed samples. Vacuolar morphology in cryofixed samples exhibited homogenous electron density, while chemically fixed samples contained a flocculent electron dense periphery and electron lucent lumen. In contrast to the apparent advantages of cryopreservation, fine details of cell wall fibre orientation could be observed in chemically fixed glandular trichomes that were not seen in cryofixed samples. Our data suggest that chemical fixation results in intracellular artefacts that impact the interpretation of lipid production and trafficking, while enabling greater detail of extracellular polysaccharide organisation. LAY DESCRIPTION: For millennia humans have grown and consumed cannabis flowers for use in traditional medicine and intoxication, due to the plant's active ingredients called cannabinoids. The most well‐known cannabinoid is tetrahydrocannabidiol (THC). Despite this long history, and recent relaxation of cannabis uses for medicinal and recreational consumption in many parts of the world, little is known about how the plant makes the cannabinoids in its cells. Small mushroom‐shaped structures on the flower surface, called glandular trichomes, produce and store the cannabinoids. To image inside the cells of THC‐rich cannabis flowers, it is necessary to use electron microscopy, and this has revealed complex internal membrane networks and specialised cell structures in these tiny biofactories. In this study, we explored different ways to preserve the cannabis glandular trichomes for electron microscopy, using either cryogenic preservation or chemical crosslinking, in order to determine the benefits and drawbacks of each method. The chemical crosslinking method produced dramatic re‐arrangement of cell contents that were not seen in the cryofixation, where the near‐instantaneous immobilisation of the cell structure led to exceptional preservation of the THC‐producing cells. Despite the benefits of using cryofixation for preserving the complex cell structures, it was not as useful for seeing details of the cell walls surrounding the glandular trichome. Together, our data show stark differences in cannabis glandular trichome cell structure that result from cryofixation and chemical fixation. This is important because the chemical fixation protocol was historically used and informed previous models of cannabinoid production and trafficking. Cryofixation produces exciting new models of THC production in cannabis, due to its high‐fidelity preservation of cell structures. In contrast, studies of cell walls may still benefit from the traditional chemical cross‐linking technique of sample preservation for electron microscopy. https://www.cell.com/current‐biology/fulltext/S0960‐9822(22)01115‐0 [ABSTRACT FROM AUTHOR]