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CHAPTER 2 - SAMPLE PREPARATION
Carl Zeiss
Specific Examples of Sample Preparation
Lightsheet Z.1
28
000000-1790-528
02/2013
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Specific Examples of Sample Preparation
In order to make this sample preparation section as useful as possible the following pages describe
mounting techniques for specific samples, in particular describing the equipment needed, step by step
protocols and illustrations based on our own laboratory
3
experiences.
Fluorescent beads are used for later Landmark Registration processing of acquired multiview data,
and should be included during embedding of samples of interest. Prepare agarose as described in
section
Preparation of Fluorescent Beads
3.1
Preparation of Fluorescent Beads
Samples with fluorescent beads are often used to characterize the imaging properties of a microscope
such as the LSFM. Using a reproducible sample is an important tool to calibrate the instrument. This
protocol describes how to handle fluorescent beads and to prepare optimal concentrations to image with
an LSFM.
Equipment and reagents
−
Fluorescent beads
−
1 % Low Melting Point (LMP) Agarose in deionised water
−
Capillary (Size 4, Blue, #701910, BRAND GmbH)
−
Sonicator
−
Heating block- Vortex
Method
1.
Vortex the bead solution to make a homogeneous dispersion.
2.
Dilute a small volume of the bead dispersion in deionized or distilled water to a concentration 100x
higher than the one desired for the specimen. Depending on the size of the beads and the
magnification required it is first necessary to calculate the bead-agarose ratio (see below).
3.
Sonicate the dilution for 5 minutes at maximum power.
4.
Prepare a liquid agarose solution of a chosen concentration (0.5 % - 1 %) and cool it down to just
above the gelling point (usually 38-40 °C).
5.
Mix diluted fluorescent beads with the agarose in ratio 1:100 and vortex the mixture. Use a pipette
or a capillary (by sucking in and out the liquid agarose several times) to mix the bead solution and
the agarose thoroughly.
6.
Insert an appropriate plunger and Teflon tip.
7.
Push the plunger through the capillary, so the front end of the plunger is sticking out of the
capillary by a bit before entering the liquid agarose and sucking the agarose in. This will avoid air
bubble formation at the plunger.
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Flood P.M., Kelly R., Gutiérrez-Heredia L. and E.G. Reynaud
School of Biology and Environmental Science, University College Dublin, Belfield, Dublin 4, Dublin, Ireland
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