Zeiss Lightsheet Z.1, Руководство по эксплуатации

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CHAPTER 2 - SAMPLE PREPARATION
Lightsheet Z.1 Tips, Troubleshooting and Additional Information Carl Zeiss
02/2013 000000-1790-528 39
High background signal within the sample.
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In the case of immunofluorescence, you should decrease antibody concentration or incubation time
but this may decrease the overall signal. You can also use blocking steps during the
immunofluorescence (eg. BSA, FCS…). If preparing tissue section, you should increase the
stringency of the washing steps.
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The imaging chamber may be dirty as well as the media inside. This contamination affects the
quality of the light sheet and scattering occurs.
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You can apply deconvolution to your stacks afterwards.
The sample is moving.
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If you are imaging life samples, it may be simply due to life. The sample is moving so you may want
to increase the anesthetic concentration or the agarose concentration to restrain any movement.
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The mounting is unstable. This occurs if the chosen material is not properly maintained (bad
tweezers, leaking plunger, badly polymerized agarose…). For embedded sample preparation, you
can improve the stability by limiting the amount of agarose emerging out of the syringe or the
capillary, as the longer is the agarose tube the more unstable it will be. You can also tighten the
plunger by sealing it with nail polish to avoid air leakage that will lead to gliding of the agarose
tube.
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The system table might not be a float. Check that it is connected to the pneumatic supply and the
air-dampening is active.
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The cables are not properly secured with the cable holders at the system table.
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Other instruments that produce vibrations, not completely dampened by the system table, are in
close proximity (e.g. fridges, centrifuges, etc.).
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The stage is not properly fitted or damaged and prone to vibrations. This includes the sample
holder and the imaging chamber support.
Optical aberrations
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As in any optical technique, the LSFM has advantages and disadvantages. Some optical aberrations
are more generals and can be dealt with easily. Do not hesitate to ask experts in the field, check
the literature as well as Internet resources that may provide a more detailed solution to the problem
you have encountered.
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A few optical aberrations are, however, typical for LSFM, as the optical axis are at a 90 degrees
angles. Lines and stripes occur as any objects blocking the light sheet will reduce the light intensity
leading to a discrepancy along the illuminated plane. This is often a problem with big samples,
highly scattering samples or sample which have absorbing structures at the surface of the specimen
volume. Rotating the sample to give a better path for the light sheet should be considered first.
Dual Side illumination and/or Pivot scanning of the light sheet can often eliminate these effects
(available in the Lightsheet Z.1) Second, the sample could be orientated differently during
mounting or partially dissected to limit obstacles. The concentration of the objects can also be a
problem especially with samples with optical properties (beads, tubes, glass capillaries…). For
example, if you image large number of cell clusters, you may have a few of them in the light sheet
path. By reducing the amount of objects, you will automatically reduce the lines and stripes.
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The use of image processing methods such as deconvolution may help to get rid of those
aberrations.