B17: Initiation to Cryo-Transmission Electron Microscopy (CryoTEM) : Application to the study of liposomes
Rachel Auzely-Velty et Isabelle Pignot-Paintrand
Centre de Recherches sur les Macromolécules Végétales, UPR CNRS 5301
Université Joseph Fourier, B.P. 53 – 38041 Grenoble Cedex 9 – France
Keywords : Plunge freezing, cryo-TEM, lipid vesicles
Transmission electron microscopy technique allows one to obtain high spatial resolution
images of nanomaterials. This technique is in particular used to characterize liposomes
which play a significant role as drug delivery vehicles. These systems are formed by selfassembly of natural or synthetic lipids, leading to a concentric lipid bilayer surrounding
aqueous compartments. The in vivo application of these drug delivery systems widely
depends on their physico-chemical and technological characteristics such as the structure,
shape, size distribution, surface modification and drug interaction. To describe the
morphology of liposomes, there are two well-established electron microscopy techniques, the
negative staining and the direct imaging by cryo-TEM. The advantage of the latter method is
the possibility to observe the sample in native conditions since no chemical treatment or
desiccation is applied. Indeed, in cryo-TEM preparation, the sample is incorporated into a
vitreous ice thin film.
The objectives of this practical are to compare these two techniques and appreciate the
advantage of the cryo-TEM for the study of lipid vesicles.
100nm
Drawing of vesicle (Dr. A. Pampel, Universität Leipzig)
Direct imaging Cryo-TEM of liposome
The practical schedule is as follows: 1) synthesis of large
unilamellar vesicles by reverse-phase evaporation and/or
extrusion of large multilamellar vesicles, 2) negative staining
of the samples and observation by TEM, 3) Vitrification of the
samples in liquid ethane using a fast freezing workstation,
transfer of the vitrified sample to a liquid-nitrogen-cooled TEM
holder and imaging by cryo-TEM.
EMCPC Leica fast freezing workstation