Zeiss Lightsheet Z.1, Руководство по эксплуатации

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CHAPTER 2 - SAMPLE PREPARATION
Carl Zeiss Sample Mounting for LSFM Lightsheet Z.1
26 000000-1790-528 02/2013
2.5.1 Choosing a Fluorescent Label
The choice of label depends upon the available equipment on your LSFM set-up (lasers, filters) and the
availability of certain fluorescent protein variants, fluorochromes conjugated to required antibodies for
use in multiple labeling schemes. In general, the laser lines available dictate which fluorophores or
fluorescent proteins can be used. Recent advances in biochemistry have created new families of
fluorophores with very favorable signal-to-noise and quantum efficiency (QE) properties. Similarly, many
laboratories have developed a wide variety of fluorescent proteins that span the spectra from GFP
2
to
Plum.
2.6 Antifading Agents
Fluorescently labeled cells and tissues exhibit a characteristic photobleaching curve in response to
excitation by the light. Much of the photobleaching can be attributed to the generation of free radicals.
The use of free radical scavengers has been shown to decrease the rate of photobleaching. Common
scavengers include n-propyl gallate, p-phenylenediamine and DABCO (1,4-diazobicyclo-(2,2,2)-octane).
Live systems have been reported to reduce photobleaching in the presence of vitamin C or Trolox. As the
LSFM technology reduces greatly the phototoxicity and photobleaching effects during imaging, we never
encounter samples that require the use of antifading agents so far. However, some applications may
require the use of radical scavengers during long time imaging of GFP expressing samples as repeated
exposure may lead to a regular increase of the free radical contents, which might affect its behavior over
time.
2.7 Cleaning, Labelling and Storing Samples
One important point about samples is their handling. In the case of LSFM, all the samples are three
dimensional objects that are mounted to be imaged in a chamber containing water based medium. They
must then be maintained in a moist environment. Once prepared and prior to imaging, the samples can
be held in a filled beaker or Falcon tube filled with the appropriate medium, e.g. water, PBS (Fig. 15).
One simple solution we have developed in the laboratory is to use a beaker filled with the right buffer.
The samples are maintained by using plasticine on the beaker border. An alternative is to cover the top of
the beaker with an aluminum foil and accommodate the sample holders such as the 1 ml syringe by
drilling a hole in the foil. This handling technique limits evaporation. More advanced holders can be
designed and manufactured according to need. You will find a couple of examples that we have made in
our laboratory 2 to handle various size of sample embedding containers. They include a water tank that
keeps the samples moisture at all time. They are stable and can be easily move from the laboratory to the
microscope as well as stored in the fridge.
2
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