MULTIPHOTON LASER SCANNING MICROSCOPY
Carl Zeiss
Using the LSM 510 NLO direct coupled system
LSM 510 META NLO
9-24
B 45-0021 e
03/06
Artifacts can also occur in fixed tissue. Local
heating effects can cause bubbles to form in the
mounting media or induce localized
photochemistry in some mounting medias. It is
important to test all mounting medias before use
for multiphoton imaging. Fig. 9-10 shows an
image where a large artifact is produced in an
image using multiphoton excitation. In this
example, Epon-araldite resin, generally used for
EM embedding, was used. This is particularly bad
for multiphoton imaging as it quickly becomes
heated using focused NIR light.
Similar effects can been seen if oil and water are
mixed, such as on the top of a coverslip when
different immersion objectives are used on the
same sample. Similar to the way water sizzles in a
frying pan, steam will be produced from the
focused heating of the oil and image artifacts
result. Usually, these types are artifacts are
dynamic as the water evaporates or the oil breaks
down and the artifact may grow as you image for
a longer time. It is important to clean the coverslip
of your samples well with ethanol to dry off the
water before mounting in oil or to clean off the oil
before mounting in water.
Fig. 9-10
Bubbles have formed in the
mounting media of a fixed sample
of a moth embryo. The embryo was
labeled with DiA, but the specific
signal is obscured by the artifact
produced. Sample courtesy of Patty
Jansma, University of Arizona.
