Techniques for the characterization of physical gels - Physical Gels from Biological and Synthetic Polymers - page 35

to hold a large amount of solvent (99%) is strongly related to their microstructure. The
size of the pores, or

' mesh ' size, has a direct in uence on the capillary forces which are

especially strong for the smallest pores, of size 1 100 nm (from the Laplace law). Direct

observation of the structure is of primary importance. However, images of gel networks
are dif

-

cult to obtain. In this chapter we try to explain the reasons for this, and to brie y

describe the techniques based on TEM imaging used so far.
The dif

culties in obtaining good-quality electron micrographs, in particular of aque-
ous physical gels, are similar to those encountered for most biological specimens
(Hermansson and Langton, 1994 ). First is the lack of contrast: many such polymers
consist of elements with low atomic numbers (C, H, O, N), so do not absorb the incident
beam, a basic requirement for observing amorphous structures. However, labelling
techniques can be used which make such specimens visible by a selective '

with solutions containing heavy atoms (such as osmium, tungsten or uranium). The
staining methods usually include chemical

' staining

fixing of the specimen and embedding with

epoxy resins. Another dif culty arises from the high degree of hydration: while the

electron beam in the microscope is under vacuum, water must not be in the liquid state. In
such gels, the high degree of hydration and the weakness of the bonds holding the
network make all manipulations very delicate and so likely to disrupt the structure. As an
alternative to chemical fixing, which is traditionally used for biological specimens,

cryotechniques, based on a physical

fixing method, can be used. The physical network

is -

seems very natural and easy to perform, a certain number of precautions must be
followed (Ayache et al., 2010 ).
When a liquid is cooled below its freezing temperature, crystallization involves

' locked ' by very fast freezing of the sample. Although the idea - freezing water

rst
a nucleation step, then a nuclei growth step which allows the crystals to extend
throughout the liquid. Liquid water has a particular structure such that water molecules
can establish hydrogen bonds between each other, and this structure is labile, because
of thermal agitation. In ice, below 0°C and at atmospheric pressure, the crystal structure
is hexagonal, allowing the maximum number of hydrogen bonds to form (ice has an
open structure, with a lower density than water at 0°C). The development of this
hexagonal ice can cause very severe damage to the local structure of the gel network,
which destroys the original architecture. Crystal size is a function of the cooling rate
and of the freezing temperature, so the lower the temperature, the slower the diffusion
of liquid molecules towards the crystal surfaces and the smaller the crystals. The
number of crystal nuclei is also important, but when they are small enough that they
do not show any diffraction pattern under the electron beam in TEM experiments,
water is assumed to be in a vitreous state. This state can be reached at temperatures
below 196°C (liquid nitrogen temperature). In pure water, the vitreous state is reached

at cooling rates of around 10 9 °C min − 1 . In aqueous solutions containing species such
as ions, macromolecules or small organic molecules, the presence of these solutes
stabilizes the system and reduces the diffusion of water molecules during freezing, so
the vitreous state can be accessed with (still very high) cooling rates of 10 6 °C min − 1 ,
and at higher temperatures, say around

−

− 148°C for a biological system with a solute

concentration of 300 mM.