The method consists of imaging developing pollen grains as they form within intact, immature Arabidopsis thaliana anthers. Using two-photon excitation in the infrared wavelength range, the intrinsic fluorescence (autofluorescence) of developing pollen grains and surrounding sporophytic tissues of the anther wall, including the tapetum, middle layer, endothecium and epidermis, can be visualized in the three-dimensional space of an intact anther. In contrast to conventional confocal microscopy, the application of red-shifted light by two-photon microscopy improves depth penetration into specimens, while the scattering of light and subsequent phototoxicity is minimized, making this a superior method for imaging the developing pollen grains and tapetal cells enclosed within anthers. The technique described was optimized for the detection of autofluorescent components of the pollen wall, including sporopollenin and the pollen coat, and provided spatial and developmental data on the autofluorescent metabolites in anthers of wild-type and pollen wall mutant plants (Quilichini et al., 2014). The use of two-photon imaging of live, intact anthers holds potential for future studies aimed at understanding the spatial relationship between gametophytic and sporophytic tissues during pollen development and the distribution of metabolites or fluorescently-tagged proteins within developing anthers.
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