CHAPTER 2 - SAMPLE PREPARATION
Lightsheet Z.1
Sample Mounting for LSFM
Carl Zeiss
02/2013
000000-1790-528
13
There are two principal methods of embedding an object. The first is to directly mix the object with the
agarose then pump it into the sample embedding cylinder. This is a convenient way of embedding very
small objects such as pollen grains (Swoger et al., 2007); yeast (Taxis et al., 2006) or cell clusters
(Pampaloni et al., 2007) or even large objects like fish embryos. The action of pumping in the sample
with the agarose results in a self-alignment of the specimen within the tube (Fig. 7/
A
,
E
and
F
).
The second method is to fill the sample embedding container with the gelling agent, then to place the
object within the gel using a needle or forceps (Fig. 7/
A
,
B
,
C
and
D
). This approach is more suitable for
those samples that cannot be easily aligned using the first technique.
In some cases, it may still be challenging to align the specimen in the most suitable way for imaging. The
orientation of the sample must then be optimized, so that interesting details are facing the surface of the
agarose cylinder with as little material as possible in the optical path. One solution is to fill a syringe with
agarose and allow it to cool until it solidifies. The agarose is then pushed out of the syringe
(Fig. 8/
A
and
B
). A small V-shaped groove can be cut into the gel and the sample then positioned in the
V-groove.
Fig. 7
Basic principles of sample embedding.
A cylinder with a suitable plunger is used as a mounting device (A). The 1 % low melting-point
agarose is melted, then brought to 37 °C, then pumped into the cylinder. (B) The object is then
introduced to the agarose with a needle or forceps. (C) Once solidified, the embedded sample can be
pushed out and imaged (D). Alternatively, the object, devoid of water, or other solution, is added to a
solution of 1 % low melting-point agarose at just above gelling temperature (typically 40 °C) and
sucked into the cylinder (E) and then allowed to polymerize. The embedded sample can then be
pushed out and imaged (F).
Summary of Contents for Lightsheet Z.1
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