Optimized negative-staining electron microscopy for lipoprotein studies

Abstract

Background Negative-staining (NS), a rapid, simple and conventional technique of electron microscopy (EM), has been commonly used to initially study the morphology and structure of proteins for half a century. Certain NS protocols however can cause artifacts, especially for structurally flexible or lipid-related proteins, such as lipoproteins. Lipoproteins were often observed in the form of rouleau as lipoprotein particles appeared to be stacked together by conventional NS protocols. The flexible components of lipoproteins, i.e. lipids and amphipathic apolipoproteins, resulted in the lipoprotein structure being sensitive to the NS sample preparation parameters, such as operational procedures, salt concentrations, and the staining reagents. Scope of review The most popular NS protocols that have been used to examine lipoprotein morphology and structure were reviewed. Major conclusions The comparisons show that an optimized NS (OpNS) protocol can eliminate the rouleau artifacts of lipoproteins, and that the lipoproteins are similar in size and shape as statistically measured from two EM methods, OpNS and cryo-electron microscopy (cryo-EM). OpNS is a high-throughput, high-contrast and high-resolution (near 1 nm, but rarely better than 1 nm) method which has been used to discover the mechanics of a small protein, 53 kDa cholesterol ester transfer protein (CETP), and the structure of an individual particle of a single protein by individual-particle electron tomography (IPET), i.e. a 14 Å-resolution IgG antibody three-dimensional map. General significance It is suggested that OpNS can be used as a general protocol to study the structure of proteins, especially highly dynamic proteins with equilibrium-fluctuating structures.

Visualization of the protein corona: towards a biomolecular understanding of nanoparticle-cell-interactions

“…uranyl acetate) providing high contrast samples, suitable for conventional electron tomography. 18,19 In order to assure that the corona morphology is not affected by the embedding into the trehalose film, additional cryo-TEM examinations have been performed. This showed no noticeable structural difference between cryo-TEM and trehalose embedding preparation (Fig.…”

Localized delivery of low-density lipoprotein docosahexaenoic acid nanoparticles to the rat brain using focused ultrasound

“…Excess sample was removed with filter paper, and repeated cycles of washing with deionized water and staining with 1% saturated aqueous uranyl acetate was performed on the grid as descried by Zhang et al . [33] The final stain was then drained off with filter paper, and the grid was air dried before digital images were taken.…”

The Effects of Electron Beam Exposure Time on Transmission Electron Microscopy Imaging of Negatively Stained Biological Samples

“…The advantages of negative staining over high resolution electron microscopy (EM) approaches, such as cryo-EM, are its convenience, simplicity, rapid applicability to biological objects ranging from individual proteins to large macromolecular assemblies, and ability to detect structural homologies (Anderson et al, 1989;Boekema et al, 2009;Zhang et al, 2013); hence, negative staining is used widely to detect the structural features of bio-materials and small proteins. To obtain the best contrast between stain and protein, the electron beam exposure time, which is related to the thickness of the support film, is an important parameter to consider.…”