Zeiss Lightsheet Z.1, Руководство по эксплуатации

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CHAPTER 2 - SAMPLE PREPARATION
Carl Zeiss Sample Mounting for LSFM Lightsheet Z.1
14 000000-1790-528 02/2013
The gel can be cut into various shapes depending on the needs (cylinder, hole). Afterwards the gel with
the specimen is pulled back into the syringe and is covered with more molten agarose. The agarose is
allowed to cool and solidify, this time period can be shortened by cooling the whole sample. For example
the housing of the sample can be rinsed with cold water, although care must be taken to ensure that the
polymer does not come into contact with the water, otherwise the cooling agarose would become
diluted and lose its stability necessary for holding the sample. After polymerization, the sample is ready
for imaging.
2.2.2 Hanging Samples
The Lightsheet Z.1 is optimized for gel embedding samples The sample chamber must be filled with
a watery solution (refractive index of 1.33) at all times, to ensure optimal image quality.
This mounting technique can also be used, but will require some initial adaptations to the sample
holder.
An intuitive way of imaging an object is to simply take it as it is and place it in front of an objective. In an
LSFM, this can be done by hanging the object in front of the objective where the axis of rotation and
gravity are parallel. This can be achieved using a simple hook made of glass, stainless steel or plastic
(Fig. 9/
A
). This mounting technique can be used for large samples such as organs (for example the brain)
or complete organisms (insect, fish). One main drawback is the fact that the hook will partially damage
the object and may also interfere with the field of view.
Fig. 8 Aligning an embedded sample.
The sample can be aligned in a particular orientation to allow the details of interest to be close to the
outer surface of the agarose. The solidified agarose is pushed out the syringe a few millimeters and a
small v-groove is cut into the cylinder to take up the sample (A). The sample is placed into the v-groove
(B). The sample on the agarose is pulled back into the syringe and more agarose is added (C). After the
cylinder has completely solidified the sample is pushed out of the syringe allowing free sight on to the
sample (D). The same approach can be used to carve a central tunnel in the middle of the agarose to
align the sample along the agarose tube axis.