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.
4% (v/v) glutaraldehyde in 12.5 mM cacodylate*2 (pH 6.9)
EtOH (20, 40, 60, 80, 100%)
Clearing and mounting
2:1 mixture of benzyl benzoate and benzyl alcohol
(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.
*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)