Cryo transmission electron microscopy of liposomes and related structures

Abstract

Cryo-transmission electron microscopy, c-TEM, has during the last 10 years contributed significantly to the understanding of the numerous, and often complex, structures formed by amphiphilic molecules in dilute aqueous solutions. In particular, the method has evolved as an important tool for the investigation of liposomes. In this review, we discuss and show examples of how the technique has been utilised to gain new information on the form and structure of liposomes, as well as on the morphological changes taking place upon encapsulation of drugs or interactions with surfactants, DNA and other polyelectrolytes. Several examples where c-TEM has been successfully employed to visualise related amphiphilic structures, such as thread-like micelles and particles of reversed phases, are also presented. In addition, we discuss recent developments concerning sample preparation and interpretation of images, as well as possible artefacts and their origin.

Introduction

With cTEM or sometimes cryoTEM, we mean transmission electron microscopy of thin vitrified aqueous films, kept at liquid nitrogen temperature [1], [2]. The technique has opened the possibility for direct imaging of structures formed by amphiphilic molecules in aqueous environments [3], [4]. These delicate and dynamic structures require an aqueous environment to form and persist, and do not withstand the drying or the staining and fixation steps of conventional EM. In the cTEM method the solution is applied to a microscopy grid in such a way that a very thin aqueous film is formed, which then is plunged into a cooling medium, such as ethane just above its freezing point, where the film very rapidly vitrifies, without crystallisation. The grid with the vitrified film is then transferred to the microscope, and examined at liquid nitrogen temperature in transmission mode. The structures which in this way are captured in the vitrified film, are thus observed without dehydration, and are so quickly vitrified that normally no important reorganisation takes place.

The size of the self assembled fluid structures is in a range well suited for electron microscopy. The contrast is a limiting factor, however. It derives only from the difference in electron density between the atoms of the amphiphile and the surrounding water, and it is necessary to under-focus the image to improve the contrast. In the end, it turns out that a dimension of about 4–5 nm is the smallest that can be resolved. Ordinary surfactant micelles are thus seen only as dots, and it is difficult to claim anything about the size of an object unless it is clearly larger than 5 nm. However, in a few reports the bilayer membrane has been resolved, and its thickness measured [5].

There are also limitations on the upper size of an object, set by the thickness of the film. In practice it is limited to about 500 nm; otherwise the scattering of electrons by water gets too large, and the cooling rate during vitrification too slow. Liposomes (or vesicles; in this review the two words will be regarded as synonyms) of various kinds and in different stages of transformation, are particularly well suited for investigation with the technique, and also other bilayer structures. The lipid bilayer, with a width of about 4 nm, is just at the resolution limit, which means that no information is obtained about the inner architecture of the bilayer. Normally liposomes have a radius of more than twice that size, and one can clearly see the rounded shape and the inner aqueous compartment, as well as various morphological details. Recently, micro-vesicles have been identified in some systems, with a mean radius of only 7 nm, as determined from SANS studies [6]. These vesicles were imaged only as dark dots by cTEM, without a discernible inner compartment.