Zeiss Lightsheet Z.1, Operating Manual

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CHAPTER 2 - SAMPLE PREPARATION
Lightsheet Z.1 Introduction Carl Zeiss
02/2013 000000-1790-528 3
1 Introduction
This section describes theoretical and practical aspects of sample preparation for Light Sheet-based
Fluorescence Microscopy (LSFM). We present general rules for sample handling and mounting, as well as
guidelines with respect to the best preparative technique to use, taking into account sample type,
structure and properties. Step by step protocols and recommended materials for Lightsheet Z.1 samples
are included. These protocols cover sample preparation ranging from micrometer-sized fluorescent beads
to millimeter-sized insects, providing detailed information relating to preparation and observation
techniques. Finally, this section identifies the main artifacts and problems that can result from the
preparation techniques.
A microscope generally performs best on suitable samples, and when the samples are optimally prepared
for the imaging method and microscope type. In Light Sheet Fluorescence Microscopy (LSFM), the sample
is commonly mounted in a liquid filled chamber and can be rotated easily. It is scanned through a sheet
of light which illuminates the focal plane of a perpendicularly mounted objective lens. The resulting
image of an optical section is observed through the objective and is usually detected on a camera-based
detector. Since the geometry of the optical beam paths and the optics differ significantly from the
conventional inverted and upright compound microscopes, the sample mounting protocols also differ
significantly.
If the sample is perfectly transparent, like a block of 1 % agarose with beads inside, the light sheet can
penetrate deeply and does not change its properties and shape along the illumination axis. Also, the
fluorescent signal can reach the detector unperturbed by scattering effects in the specimen or hydrogel.
However, if the sample is slightly opaque and diffracts or scatters the light sheet heavily (lipids, lipid
vesicles or dense collagen fiber arrays that scatter light strongly) then the well-defined shape and
thickness of the sectioning light sheet degrades along the illumination axis. In a second effect, the
detected image from a well illuminated sample might still be degraded by such a poorly transparent
sample. These effects can contribute independently to the final image quality of a LSFM and can be
minimized or worked around by careful sample positioning in the microscope as well as by an optimized
sample preparation protocol.
Ultimately, a fully opaque sample that can completely block the penetration of light and a light sheet
(insect cuticular structures, bones…) will limit the imaging capabilities of Light Sheet Fluorescence
Microscopes and the Lightsheet Z.1 to its surface.
Furthermore, the image quality in Fluorescence Microscopy in general – and in LSFM in particular - is not
only determined by sample transparency that can be optimized by choosing a suitable model (transparent
fish like Medaka), suitable growth conditions (no phenol red in the growth media to avoid
autofluorescence) or, potentially, a clearing treatment (not suitable for the Lightsheet Z.1). It is also
important to have a homogenous signal from a homogeneously labelled sample. Antibodies, for example,
are rather large molecules that cannot penetrate deeply into tissue so it is difficult to image a complete
juvenile fish after antibody as only the first 50 µm to 100 µm will be labelled, the interior showing
reduced signal levels due to the limited diffusion of the antibody.
Samples must be carefully considered when using LSFMs such as the Lightsheet Z.1 as well as the label or
dye used must be carefully chosen. In planning an experiment, it should be kept in mind that most
labelling and imaging protocols have been developed for thin specimens and therefore many aspects are
not adapted to imaging larger samples such as embryos, tissue slices or complete mosquitoes.
Many organisms have been imaged using Light Sheet Fluorescence Microscopy (Table 1) and you may
want to read further specific papers to clarify sample preparation issues.