Enhanced imaging of lipid rich nanoparticles embedded in methylcellulose films for transmission electron microscopy using mixtures of heavy metals

Abstract

Synthetic and naturally occurring lipid-rich nanoparticles are of wide ranging importance in biomedicine. They include liposomes, bicelles, nanodiscs, exosomes and virus particles. The quantitative study of these particles requires methods for high-resolution visualization of the whole population. One powerful imaging method is cryo-EM of vitrified samples, but this is technically demanding, requires specialized equipment, provides low contrast and does not reveal all particles present in a population. Another approach is classical negative stain-EM, which is more accessible but is difficult to standardize for larger lipidic structures, which are prone to artifacts of structure collapse and contrast variability. A third method uses embedment in methylcellulose films containing uranyl acetate as a contrasting agent. Methylcellulose embedment has been widely used for contrasting and supporting cryosections but only sporadically for visualizing lipid rich vesicular structures such as endosomes and exosomes. Here we present a simple methylcellulose-based method for routine and comprehensive visualization of synthetic lipid rich nanoparticles preparations, such as liposomes, bicelles and nanodiscs. It combines a novel double-staining mixture of uranyl acetate (UA) and tungsten-based electron stains (namely phosphotungstic acid (PTA) or sodium silicotungstate (STA)) with methylcellulose embedment. While the methylcellulose supports the delicate lipid structures during drying, the addition of PTA or STA to UA provides significant enhancement in lipid structure display and contrast as compared to UA alone. This double staining method should aid routine structural evaluation and quantification of lipid rich nanoparticles structures.

Keywords: Bicelles; Lipids; Liposomes; Membranes; Methylcellulose; Nanodiscs; Negative stain; Phospholipids; Phosphotungstic acid; Sodium silicotungstate; Uranyl acetate.

Fig. 1

Liposomes. Liposomes in methylcellulose films containing uranyl acetate (A), phosphotungstic acid (B) and uranyl acetate/phosphotungstic acid mixture (C). Note the difference between the clarity of display of the membrane structures visualized with UA and UA/PTA (A and C respectively; larger structures, arrows and smaller structures, arrowheads). Inset in C shows characteristic precipitates generated in the mixed UA/PTA stain. The clumping of vesicles that occurs with PTA (arrow in B) alone is not apparent when the UA/PTA mixture is used. Bars 100 nm.

Fig. 2

Comparison of single UA stain with mixed double stains on liposomes adsorbed to EM support films. Single UA (A and B); UA/PTA (C and D) and UA/STA (E and F). (A, C and E) are low magnification overviews and (B D and F) higher magnification. Note the precipitates observed with UA/PTA and their absence with UA/STA (E and F). Bars 100 nm.

Fig. 3

Analysis of contrast. Randomly selected particles from randomly taken micrographs were analyzed using imageJ software. Dm4 files were displayed and grey scale values measured along linear intercepts placed orthogonal to, and across, the most prominent membrane profile of the sampled vesicle. Care was taken to include stretches of surrounding film, which occupied up to one half of the total intercept length. Only precipitate-free particles were included in the analysis. (A) – range of grey scale, (B) − coefficient of variation (CV) of grey scale values and, (C) – mean of grey scale values. Error bars represent the standard error of the mean, N = 13 in each case.

Fig. 4

Bicelles. Bicelles were adsorbed to EM support films at 22°C and stained using (A) UA alone (classic negative stain), (B) UA in MC, (C) PTA in MC and (D) UA/PTA mixture in MC. The smaller structures are difficult to discern in (A) and (B) and undergo clumping in (C). Smaller round profiles (arrowhead) and the edges of the wormlike extended structures (arrows) are best displayed in the mixed UA/PTA stains, (D). Bars, 100 nm.

Fig. 5

Nanodiscs. Nanodiscs in MC films containing heavy metal stains. UA staining alone (A) provides higher background density compared to PTA (B) and UA/PTA (C). The perimeter of the nanodisc is best displayed in the mixed stain. Clumping occurred when PTA is used alone (B) and is more marked for particles oriented end-on (inset in B). Consistent with support of these structures, increasing the MC content (μl) of the staining mixture increases the fraction of nanodiscs with “end-on” orientation (N = 35, 45, 49, 38, 37, 60 and 70 for each successive category; total, 334) (D). Bars 50 nm.

Fig. 6

Double staining in sequence. Uranyl acetate and PTA in MC were applied sequentially as described in the methods after absorption of liposomes (A and B), bicelles (C and D) and nanodiscs (E and F). Left hand panels (A, C and E) illustrate results obtained from uranyl acetate followed by PTA staining and right hand panels (B, D and F) results obtained from PTA followed by uranyl acetate. Uranyl acetate followed by PTA produced similar contrast to that obtained using uranyl acetate alone. Using PTA prior to UA produced similar contrast to that obtained with PTA alone, although aggregation of the particles was less evident. Bars for A and B (100 nm); for C and D (100 nm) and for E and F (50 nm).