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susceptible to both the in vacuo drying and cooling conditions normally
required.
For this reason, FFEM studies of lipid and other LLC systems were initially
dogged by concerns regarding the preservation of mesophase structure after
freezing, and this led to the development of “fast-quenching” cryofi xation
procedures (e.g., using liquid ethane), which offer a reduced risk of ice crystal
formation or other sample perturbation during the freezing process (Gulik-
Krzywicki, 1994). Simultaneous studies using X-ray scattering and electron
microscopy were critical in validating such approaches (Costello and Gulik-
Krzywicki, 1976 ; Gulik - Krzywicki and Costello, 1978 ).
Combining cryogenic preparation techniques with conventional TEM has
led to the development of cryo-TEM. Here, a thin fi lm of sample is produced
on a 200-mesh copper TEM grid coated with perforated carbon fi lm, which
then is plunged into a cooling medium, such as liquid ethane just above its
freezing point. The fi lm very rapidly vitrifi es, without ice or sample crystalliza-
tion. Frozen grids are stored in liquid nitrogen until required. The grid with
the vitrifi ed fi lm is then transferred to the microscope and examined at liquid
nitrogen temperature in the transmission mode. The structures captured in the
vitrifi ed fi lm are thus observed without dehydration, and hence amphiphile
assemblies can be imaged directly in their aqueous state. Cryo-TEM has con-
tributed signifi cantly during the last 30 years to understanding the variety of
self-assembled nanostructures formed by amphiphilic molecules in dilute
aqueous solutions. The majority of these studies has focused on dispersed LLC
systems and are described in Section 4.3.2, since bulk viscous/gel phases are
not amenable to the sample preparation approaches described above (Moren
et al., 2000 ).
4.3 DISPERSED LYOTROPIC LIQUID CRYSTALLINE
CHARACTERIZATION
The earliest description of dispersed lamellar phase systems (vesicles and
liposomes) involved electron microscopy techniques in the 1960s (Bangham
and Horne, 1964). Since then, the preparation of LLCs in dispersed form has
been explored extensively from both fundamental scientifi c and technological
viewpoints (Spicer, 2005a). Dispersed lamellar phases in the form of unilamel-
lar and multilamellar vesicles and liposomes have structures reminiscent of
biological membranes, and hence have received considerable attention (Genty
et al., 2003).
In 1994 Landh proposed the use of amphiphlic triblock copolymers to col-
loidally stabilize submicron dispersions of nonlamellar LLCs of monoglycer-
ides, now known as cubosomes and hexasomes (Amar-Yuli et al., 2007). While
most of the techniques mentioned in the previous section are broadly appli-
cable to the characterization of dispersed LLCs, their advent has also spawned
new characterization approaches (Sagalowicz et al., 2006b). In this section, we
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