Zeiss Lightsheet Z.1, Operating Manual

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CHAPTER 2 - SAMPLE PREPARATION
Lightsheet Z.1 Sample Mounting for LSFM Carl Zeiss
02/2013 000000-1790-528 15
The Lightsheet Z.1 has a maximum Field of View of approx. 2.5 mm (depending on zoom settings).
This and the dimensions of the sample chamber might limit the size of the sample.
Interestingly, such a hook can also be tailored to mount small objects embedded in agarose. The drop of
agarose is more stable as it is closely held by the hook. This is very important when imaging at very high
magnification (100x). The hanging method has been successfully used for imaging single Saccharomyces
cerevisae
(Taxis et al., 2006) and holding fish fins during laser nanosurgery (Engelbrecht et al., 2007).
2.2.3 Enclosed Samples
The Lightsheet Z.1 is optimized for gel embedding samples The sample chamber must be filled with
a watery solution (refractive index of 1.33) at all times, to ensure optimal image quality.
This mounting technique can also be used, but will require some initial adaptations to the sample
holder.
The last important technique of holding samples to be mentioned in this section is to create a container
that can hold the object in front of the objective lens. This technique is particularly suitable for specimens
that should not be embedded (for example due to temperature, physical constraints etc.) or that need to
be constantly maintained in a specific buffer (for example in vitro assays, or living cells). The container
must be suitable for LSFM imaging. It must be basically transparent and be suitable for the object but also
for the imaging chamber and the sample holder. It can be hooked or clipped using specific holders.
There are two main methods of generating such containers, using gelling agent to shape out a container
(Fig. 10/
A
) or using polymers such as PTFE (Polytetrafluorethylen, Teflon) or FPE (Fluorinated Ethylene
Propylene) to make it (Fig. 10/ B and C ).
Fig. 9 Different ways of hanging a sample.
Samples can be either hooked or deposited on a bent glass capillary (A, D), glued to a rod or
capillary (B), or clamped on a syringe tip using the plunger and the syringe body as a holder (C).