Many macromolecules, including transmembrane proteins and apolipoproteins, interact with lipids and membranes and play crucial roles in diverse biological processes. Understanding macromolecule–lipid interactions at the structural level is essential for elucidating their functions and mechanisms. However, determining the structure of macromolecules, particularly proteins in their lipid-bound state, has traditionally been challenging for X-ray crystallography due to the conformational and compositional heterogeneity of macromolecule–lipid complexes. Transmission electron microscopy (TEM) offers a unique capability to directly visualizing individual macromolecular particles in the presence of lipid interactions. Among TEM techniques, negative staining (NS) is a fast and widely used approach for imaging macromolecules. However, conventional NS protocols often introduce artifacts in lipid-associated protein samples, such as rouleaux formation in lipoproteins, which can compromise structural interpretation. To address these limitations, Ren and colleagues developed an optimized negative staining (OpNS) protocol by refining earlier methods and validating the results against cryo-electron microscopy (cryo-EM) images of lipoproteins embedded in vitrified ice. This optimized protocol minimizes artifacts and produces “near-native” particle images with high quality. It enables more accurate structural analysis, particularly for three-dimensional (3D) reconstructions of single macromolecular particles without averaging, using individual-particle electron tomography (IPET). This improved protocol provides a robust, efficient, and reliable method for imaging macromolecules in their lipid-binding states, offering significant advances in understanding macromolecule–lipid interactions.