Zeiss Lightsheet Z.1, Руководство по эксплуатации

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CHAPTER 2 - SAMPLE PREPARATION
Carl Zeiss Sample Mounting for LSFM Lightsheet Z.1
12 000000-1790-528 02/2013
The important points to consider are that the materials used do not interfere with the gel, the object to
image or the sample preparation (chemical compatibility, melting point, transparency etc.), it must be
easily prepared or easily purchased, it must be compatible to the LSFM sample holder as well as the x, y, z
stage, it must fit to the sample chamber and should not cause damage to the objective lens once rotated
or moved. It is also reasonable to consider reusable sample holders to limit waste. We have found that
home-made sample embedding container using 1 ml syringes (BD Biosciences, Braun, Terumo or your
local laboratory plastic ware supplier), 1 ml plastic pipettes (see your local laboratory plastic ware supplier
such as Falcon or VWR), and glass capillaries (Brand, Sutter Instruments or check your local glassware
specialist, see also section
4.3 Suggested Additional Sources of Information ) (Fig. 6) are particularly
effective. The plungers usually come with the cylinders or can be made using metal rods, plastic or metal
wires of an appropriate diameter.
Once the sample embedding container is prepared, the sample preparation can begin. The first step
consists of preparing the supporting agent at a suitable concentration and temperature. The gelling
agent is usually a 0,7 to 1 % solution of low melting agarose in water or PBS, depending of the sample
to be embedded (fixed, living, sensitivity to osmotic pressure etc.). If the sample needs to be maintained
in a drop of solution or contains water or buffer it is advisable to use a higher concentration of agarose
to obtain a final concentration of 1 % once the sample is embedded. The use of low melting-point
agarose is recommended (Roth, n° 6351.1) as its melting temperature is only approx. 60 °C and it can be
maintained liquid at just above 37 °C prior to embedding.
Fig. 6 Preparing a sample embedding container with a capillary.
A glass capillary can be used. For the Lightsheet Z.1 they come from the manufacturer (Brand) in just
the right length (A). Other capillaries can be cut to an appropriate length (B), the agarose with the
object can be easily pumped in using the suitable plungers with Teflon tips (C). If no such plunger is
available it can be made, for example, from a piece of electrical wire (D). To avoid leakage, such a
plunger can be sealed with nail polish (E) once the sample is pushed out. The sample can then be
imaged (F).