Zeiss Lightsheet Z.1, Operating Manual

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CHAPTER 2 - SAMPLE PREPARATION
Carl Zeiss Sample Mounting for LSFM Lightsheet Z.1
10 000000-1790-528 02/2013
2.2.1 Embedded Samples
Embedding objects in plastic materials is a routine procedure widely used in the preparation of samples
for electron microscopy. In the case of sample preparation for LSFM however, the immobilization of
hydrated biological materials must not impair biological activity. It is necessary to keep the object we wish
to observe in a perfect condition.
In the case of LSFM, it is also necessary to contain the sample in such a way that it can be positioned and
rotated in front of the objective. Furthermore, transparency of the mounting medium is essential to allow
imaging. A basic technique of mounting objects for the LSFM is to shape them into a cylinder of gel (for
example agarose, see also section
2.3.4 Gels and Polymers
) that can then be mounted on a dedicated
holder. The Lightsheet Z.1 package provides four capillaries sizes adapted to the sample holder to embed
objects of various sizes. The special sample holder of the Lightsheet Z.1 adapts to hold these capillaries
for precise positioning (translation and rotation) of the cylinder-shaped object for observation through
the detection optics. The used gel such as agarose behaves like mechanically stabilized water, supporting
the object. It can be easily molded and the gel chosen should have an optical (refraction) index close to
that of water. The object can be any size, as the gel can be molded accordingly (Fig. 4/
A to D ). The
various gelling agents and polymers that can be used are discussed in greater detail in section
2.3
Materials and Equipment (see below).
The preparation of embedded samples requires a container suitable for molding the gel. The simplest
approach is to use any cylinder with a tight-fitting plunger to pump the molten gel into it and let it
polymerize inside before pushing it out. The cylinder can be a syringe, a capillary or even a pipette.
Fig. 4 Embedded samples.
Large samples, such as an adult Drosophila melanogaster , can be embedded in a large gel tube or a
cut 1 ml syringe (A), intermediate size samples, such a Medaka or Zebrafish embryo can be prepared
by using either a cut 1 ml plastic syringe (see also Fig. 5) or a glass capillary (C and D) and small
samples such as
Drosophila melanogaster embryos or early stage cell clusters can be prepared using a
smaller capillary (D).