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CHAPTER 2 - SAMPLE PREPARATION
Lightsheet Z.1
Specific Examples of Sample Preparation
Carl Zeiss
02/2013
000000-1790-528
33
5.
Once the root is visible within the bottom part of the beaker, a normal plunger is inserted in the
top part of the syringe, where the open part of the beaker is present.
6.
Push down the beaker into the syringe until the root can be seen outside of the syringe cylinder
7.
The beaker is mounted in the sample chamber filled with distilled water or plant growth media at
room temperature.
As the plant depends on light to grow long-term imaging must take into account the illumination
of the leaves between imaging sessions.
3.5
Imaging Cell Cysts in an Extracellular Matrix Gel
Live imaging of cells has been a major tool in cell biology. For this, cells must be maintained in optimal
conditions during the complete time of the experiment. The incubation options for the Lighsheet Z.1 are
described in another section of this manual (
CHAPTER 1 HARDWARE
). However, cells must be mounted
in a way that allows them to hang in front of the objective from above. This protocol describes one way
of imaging MDCK cells that naturally form cysts when grown in an extracellular matrix.
Equipment and reagents
−
MDCK cells grown in an extracellular matrix (Matrigel, ExtraCell etc.)
−
1.5 % Low Melting Point (LMP) Agarose in PBS
−
Modified plunger
−
Sealing device
−
Slitted capillary
−
1 ml syringe
−
Capillary holder
−
Heating block
−
Polytetrafluorethylene foil or FPE tube
Method
1.
An agarose beaker or a polymer foil chamber is prepared as previously described in the section
2.
MDCK cysts are grown in an extracellular matrix gel.
3.
Cells can be stained at this stage with live markers (nuclear, mitochondrial, lysosomal etc.) before
mounting.
4.
Cells within the gel are transferred into the chosen chamber (agarose, polymer) and mounted in a
37 °C and CO
2
chamber on the LSFM using a cut tip to limit shearing damage.
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