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A few points must be taken into account when choosing a
particular mount:
• Compatibility. This is a crucial issue. The mount must be
compatible with the object you want to image (chemistry,
temperature etc.), but it must also be compatible with the
stage holder.
• Stability (mechanical, optical, chemical).
• Tightness. In the case of embedded samples, once the
• gel has solidified, the cylinder of gelling agent is pushed
through the capillary out of the distal end by a plunger
fitting into the capillary. The system must be air tight to
avoid air entry leading to a displacement of the gel rod.
The plunger can be sealed with a drop of wax, acrylamide
or nail polish, i.e. anything that prevents the plunger and
hence the agarose containing the sample from moving.
• Cost.
2.3.3 Sample Holder
Once the specimen is prepared and properly labeled, it is ready
to be imaged. While in conventional imaging there is a suitable
platform on which to place the glass slide or the chamber, in
LSFM the object must be held from above via the sample hold-
er. Depending on the size of the sample there are two different
types of sample holders available: sample holder for capillaries
and syringes (Fig. 14/C-F and I). Always use the minimal cylinder
diameter necessary for your specimen size to avoid excessive
amounts of agarose. The largest sample holder has been de-
signed to accommodate a 1ml syringe that can be inserted
from the top with a plunger that can be operated once the
sample holder is mounted on the stage. Once inserted, the
syringe is perfectly fitted to the sample holder as the two flaps
used for injection fit the upper part of the holder. In this way
the object support is well maintained, an essential issue for
imaging and multiview imaging as the object is moved through
the light sheet by the stage. Capillaries have been extensively
used to image small embedded objects, as hooks for very large
objects, and as support for enclosed objects, so the capillary
has become commonly used for LSFM sample embedding.
Capillaries are made of glass. They can break. They
will slide when wet. Please handle them with care and
dispose of them properly.
2.3.4 Gels and Polymers
Gelling agents are commonly used for preparing semi-solid
or solid tissue culture media. Gels provide support to tissues
growing in static conditions. The gelling agent usually has
several properties. In particular, it does not react with media
constituents, is not digested by enzymes, and remains stable
at all incubation temperatures. Gelling agents are very versatile
and useful tools in LSFM as they allow easier sample prepa-
ration. This section will present in more detail the properties,
advantages and disadvantages of two well-described gelling
agents and provides an additional list of gelling agents.
Agarose
Agarose is a complex carbohydrate polymer material, generally
extracted from seaweed. It is used in chromatography and
electrophoresis as a medium through which a substance can
be analyzed by separating it into its components. The mole-
cules are extremely water-soluble due to their large number of
hydroxy groups, and solutions tend to be low-melting point
aqueous gels. A wide range of different agaroses, of varying
molecular weights and properties are commercially available.
These include low melting types, (for example, Agarose Type
VII, low melting temperature: gelling temperature below 30 °C,
melting temperature above 65 °C) which can be used if the
sample is sensitive to high temperatures. Interestingly, the re-
fractive index of the low melting type is lower than that of
normal agarose. However, to obtain the same strength, a
higher concentration needs to be used. With a concentration
of 1 % (w/w) the low melting point agarose has the same
stability as a 0.5 % agarose (normal). The refractive index at
this concentration is still lower than that of normal agarose,
minimizing distortions when imaging. In our laboratory,
we preferentially work with agarose as it is easy to handle,
has good optical properties and is not expensive.
Gelrite
Gelrite gellan gum is a self-gelling hydrocolloid that forms
rigid, brittle, transparent gels in the presence of soluble salts.
Chemically, it is a polysaccharide comprised of uronic acid,
rhamnose, and glucose. It is produced by the bacterial strain
S-60 of Pseudomonas elodea. Gelrite is a trademark of Merck
and Co, Inc (Rahway, NJ), Kelco Division, USA. One advantage
of Gelrite is the lower scattering of light compared to an aga-
rose gel with the same stability. It has a higher index of refrac-
tion but less scattering compared to agarose. Gelrite has a
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