Background

Plastoglobules are lipid–protein droplets in chloroplasts that play important roles in membrane remodeling, stress adaptation, and redox balance [1,2]. They are closely associated with thylakoid membranes and change dynamically in number and size in response to stress [3]. Proteomic studies in Arabidopsis thaliana have revealed a specialized set of enzymes involved in lipid metabolism, redox regulation, and hormone biosynthesis, supporting the view that plastoglobules function as metabolic platforms rather than passive storage sites [4–7]. Despite these insights, knowledge of plastoglobule lipid and protein composition remains limited outside of model systems.

In cyanobacteria, various inclusion bodies such as carboxysomes, glycogen granules, and polyhydroxy alkanoate (PHA) bodies are well studied, while lipid droplets have only recently attracted attention. These structures, now termed cyanoglobules, are widely observed across species and increase in abundance under nutrient stress conditions [8–10,16]. Early studies suggested they contained triacylglycerol, though recent work has shown that they also harbor unique plastoquinone derivatives [11,12,16]. Notably, the lipid composition of cyanoglobules can vary depending on environmental stress and species, reflecting their dynamic metabolic roles. Proteomic and lipidomic analyses indicate that cyanoglobules share core features with plastoglobules, including enrichment in prenyl lipids and conserved proteins, while also exhibiting unique attributes such as stress-induced carotenoid accumulation [2,7,16]. These findings suggest that cyanoglobules are metabolically active compartments with important roles in stress physiology and share an evolutionary relationship with plastoglobules [13,14,16].

Traditionally, plastoglobules and cyanoglobules have been studied using in situ transmission electron microscopy (TEM), which provides high-resolution imaging within the cellular context but does not readily allow evaluation of isolated lipid droplets or assessment of sample purity [15]. Biochemical methods such as thin-layer chromatography (TLC) or gas chromatography–mass spectrometry (GC–MS) have advanced our understanding of lipid composition, but they do not provide rapid or direct visualization of droplet morphology. Negative staining TEM of isolated droplets offers a valuable complementary approach, enabling direct imaging of droplet morphology, size, and purity with minimal preparation.

Here, we present a rapid and broadly applicable protocol for visualizing cyanoglobule lipid droplets by negative staining TEM. Unlike conventional resin embedding and thin-section TEM, which require time-consuming fixation, dehydration, and sectioning steps that may alter droplet morphology, negative staining enables direct observation of isolated structures in near-native form. This approach provides a straightforward workflow to directly assess droplet morphology and integrity immediately after isolation, thereby expanding the toolkit available for studying cyanobacterial lipid droplets. Beyond cyanobacteria, this method can also be applied to plastoglobules and potentially to cytosolic lipid droplets in diverse organisms. Its simplicity, speed, and reproducibility make it a powerful method for advancing the study of lipid droplet biology, stress adaptation, and evolutionary connections across the photosynthetic lineage.