Zeiss Lightsheet Z.1, Руководство по эксплуатации

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CHAPTER 2 - SAMPLE PREPARATION
Carl Zeiss Sample Mounting for LSFM Lightsheet Z.1
6 000000-1790-528 02/2013
2 Sample Mounting for LSFM
For technical description of the sample chamber, incubation hardware and sample holder of the
Lightsheet Z.1, you should refer to
CHAPTER 1 HARDWARE
.
The Lightsheet Z.1 system is optimized for gel embedded samples. The sample chamber must be
filled with a watery solution (refractive index of 1.33) at all times, to ensure optimal image quality.
The Lightsheet Z.1 is not designed for the use with clearing reagents. The Lightsheet Z.1 is built for
aqueous media. Also the sample chamber and the system cavity for the sample chamber and
sample holder are not compatible with aggressive chemicals.
2.1 The perfect Sample for LSFM
Prior to observing a sample using a microscope, a preparation step is usually necessary. The sample
properties and the microscope characteristics provide guidelines and also limitations to sample
preparation and imaging. The classic method of mounting an object for microscopy requires the use of a
slide and coverslip that in turn limits access to the sample from one side (Fig. 1/
A and B ). The sample is
not touching the objective lens and the number of refractive barriers is at least two (coverslip, mounting
medium) and can increase further if immersion oil is needed. The depth of the field of view is dependent
on the type of objective lens and the sample properties, and will deteriorate with the thickness of the
sample.
Light Sheet-based Fluorescence Microscopy (LSFM) utilizes illumination along an axis that is perpendicular
to the detection axis (Fig. 1/
C
). Moreover, it usually allows sample rotation to record multiple image
stacks that are acquired independently along different directions. To allow the high level of sample
mobility required for such "Multiview" imaging, the sample is inserted in a sample holder from above.
The sample holder is hanging in and linked to an x, y, z, and alpha positioning motor stage, allowing
complete three dimensional translations and free rotation around an axis parallel to gravity. This
configuration leaves the illumination and the detection paths completely open but requires the
preparation of a sample that can be held from above or below in a medium-filled chamber. Such
geometry goes hand in hand with the convenient use of water dipping or air objective lenses. As
mentioned already in the introduction, another important aspect of sample preparation is the
transparency of the specimen, especially when imaging large objects. Ideally, the light sheet penetrates as
deeply as possible into the sample. Any obstacle or opaque medium will limit the light sheet penetration
depth, generating shadows that will be read out as artifacts on the final image.