3.8@
Observation of archegonia and embryos by confocal laser scanning
microscopy
Confocal
laser scanning microscopy (CLSM) provides us a powerful tool to observe thick,
intact specimens because of the use of spatial filtering to eliminate of image
degrading out-of-focus information and its ability to collect serial optical
sections from thick specimens.
In the moss Physcomitrella
patens,
antheridia and archegonia are formed on the gametophore shoot apices and they
differentiate sperm and egg cells, respectively.@ Fertilization occurs in the egg cell within the archegonium and
resulting embryo develops there.@
Observation of egg cells or embryos is very difficult because relatively
thick archegonium tissue interferes it; therefore we observed them with
CLSM.@ This method is applicable to
observations of buds or gametophores.
Usually
imaging specimens with fluorescent probes are needed for the observation with
CLSM.@ In our
method, glutaraldehyde is used for the fixation and induced fluorescence is
detected.@ This autofluorescence is
highly stable and little care is needed for its photobleaching.@ We used a UV laser for signal detection, but
a visible laser (such as a green helium/neon laser) is also available.
@
Fixative*1@
4%
(v/v) glutaraldehyde in 12.5 mM cacodylate*2 (pH 6.9)
Dehydration
EtOH
(20, 40, 60, 80, 100%)
Clearing
and mounting
2:1
mixture of benzyl benzoate and benzyl alcohol
Procedure
(1) Dissect
appropriate gametophore shoot apices with some leaves under stereomicroscope
and transfer them into the fixative immediately.
(2) Apply the
vacuum for 30 minutes and then release slowly.
(3) Fix materials
overnight at 4ºC
(4) Dehydrate
materials with graded ethanol.
(5) Clear
materials*3 and mount them in the
same solution.*4
(6)
Observation.*5 (We used Leica TCS SP2 confocal system equipped with a UV laser.
Fluorescence between 400 and 700 nm was detected under excitation with 351- and
364-nm excitation beams.)
*1
We added DAPI in the fixative (1µg/ml).@
This somewhat strengthened signals, but observation is possible without
DAPI.
*2
Toxic
*3
It was impossible to obtain images without clearing.
*4
Nail polish is not necessary for sealing.@
Appropriate amount of mounting solution and leaves, which work as a
spacer, are important.
*5
Nuclei and cell walls have strong signals.@
Egg cells fluoresced brightly from the whole cells.
Christensen
et al.: Sex. Plant Reprod. 10 49-64
(1997)
Tanahashi
et al.: Development 132 1727-1736
(2005)