Improvement of Soft Matter transmission Electron Microscopy; Application and Interpretation Pitfalls
Authors: Franken Linda , Boekema Egbert , Stuart Marc 
Published: December 2016

Transmission Electron microscopy (TEM) is an increasingly popular tool for the characterization of soft, supramolecular materials. TEM can give direct structural information and can be used to complement indirect techniques like dynamic light scattering, NMR and spectroscopic methods. Although the most common TEM preparation techniques are well documented, many are erroneously used or the images misinterpreted. A survey of around 200 recent and highly cited publications indicates that artefacts are a wide‐spread problem in the field of soft matter electron microscopy.

Subjecting doxorubicin loaded liposomes, “Doxil
®
”, to the four most common TEM techniques, drying, drying followed by staining, negative staining and cryo‐electron microscopy, reveals the possibilities and impossibilities of each of them. Upon drying (figure 1a,b), the structure of the liposomes is completely altered. Because of the lack of heavy elements, without staining the remaining structures are only visible at extreme defocus values (Figure 1a). Staining after drying improves the contrast, but the structure of the liposomes was lost (Figure 1b). Negative staining preserves the structure of the liposomes but only the outside is visible (figure 1c). Cryo‐electron microscopy reveals both the liposome and the doxorubicin crystal inside the liposomes. Furthermore, the bilayer of the phospholipid vesicle is clearly visible and its dimensions can be measured (figure 1d).

Unaware of the lack of structural preservation, many studies use drying of their soft materials without staining to prove self‐assembly into vesicles or micelles. Most supramolecular self‐assembled structures are composed of light elements which hardly scatter electrons, nevertheless high contrast images are presented. Most likely these are the result of the drying process where also dissolved materials will form particles. A better solution to image soft materials is negative staining, where the structures are surrounded by a “glassy” dried heavy‐metal stain that preserves the structures and gives the objects a high contrast halo. Although better than just drying, negative staining suffers from limited resolution by the grain size of the stain, and the obtained information originates from the surface of the object as the stain obscures the internal structure. However, various mistakes are made including positive contrast after negative staining and measuring the stain layer as proof of bilayer thickness. The best method is cryo‐electron microscopy where the sample is vitrified in a thin layer of its own solvent. The structure is well preserved and a near atomic resolution can be achieved. The lack of heavy metal stain is compensated by a very good phase contrast upon defocusing. Expertise is needed to distinguish between sample and artefact. Lack thereof results in ice contamination interpreted as part of the sample.
A systematic search on “self‐assembly” or “vesicle” in combination with “electron microscopy” in the recent, highly cited, literature brings to light that almost half of the evaluated papers contains erroneous or non‐interpretable electron microscopy pictures or images that were misinterpreted. In most cases soft, supra molecular, materials were dried without any staining on the grid and present dense round objects. Apparently peer review fails at this point. Lack of TEM expertise amongst reviewers and previously published incorrect literature seem to be the main cause. Regarding the number of mistakes in application and interpretation, there is room for improvement in both the reviewing process and the application of different TEM techniques and their corresponding interpretation.