Zeiss Lightsheet Z.1, Operating Manual

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CHAPTER 2 - SAMPLE PREPARATION
Lightsheet Z.1 Sample Mounting for LSFM Carl Zeiss
02/2013 000000-1790-528 23
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 preparation. 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 molecules 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 refractive 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 agarose gel with the same stability. It has a higher index of refraction but less scattering compared
to agarose. Gelrite has a consistent batch-to-batch quality due to a stringent control of the fermentation
process. Only half the amount of Gelrite is required for the same purpose. It hydrates rapidly and gel
setting can be easily controlled. The stability of the gel depends on the concentration of divalent cations
(Mg
2+
, Ca
2+
) therefore a gel made with Gelrite and pure water is unstable compared to a PBS- (buffer)
based gel. Polymerisation is faster compared to agarose, which might be advantageous for some
applications. The temperatures for gelling and remelting are similar to that of agarose.
Additional list of gelling agents
−
Galactan
−
Agar
−
Gelatin
−
Carrageenan
−
Alginate
−
Phytagel™
−
Agargel™
−
Transfergel™