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CHAPTER 2 - SAMPLE PREPARATION
Lightsheet Z.1
Sample Mounting for LSFM
Carl Zeiss
02/2013
000000-1790-528
7
Fig. 1
Relations between the sample and the objective in microscopy.
Samples are traditionally isolated from the objective by a glass coverslip (A and B) limiting access to
one side only. (C). In LSFM, the illumination is positioned at 90° compared to the detection axis and
can be set up in a sideways geometry ("horizontal microscope").
In LSFM, the sample is usually imaged in a water-based buffer. Generally, it can be kept dry and imaged
in air but this has extensive limitations like diffraction due to the significant jump in refractive index from
air to the sample material. This has several consequences for sample preparation. First, the refractive
index of the mounting medium should be close to that of the sample buffer. The mounting medium
should not scatter the illumination or the detection light. Second, the mounting medium should not
dissolve in water. Third, its diffusive properties should be close to those of water/medium. Fourth, the
medium should be non-toxic for live samples. Fifth, the medium should be flexible to allow the sample to
develop. Finally, it should not change its mechanical properties during a period of observation (72 hrs and
more).
The following part of this section will deal with sample as a general term but we have devised them in
four main classes (Fig. 2) and you can check their size relationship (Fig. 2) and keep that in mind as
different samples of different sizes will mean different sample preparation approaches and handling.
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