Visualization of meiotic events in intact living anthers by means of two-photon microscopy

In this paper we describe the application of two-photon microscopy (2PM) to the study of meiosis in plants. Fresh, unfixed anthers of Agapanthus umbelatus were briefly incubated on a minimal medium containing the DNA fluorophore DAPI. DAPI incorporation took place in about 30 min and nuclei and other DNA-containing organelles kept their fluorescence for more than 24 h. Using 2PM it was possible to optically section the whole, unfixed anthers to a depth of approximately 200 mum. This was up to the mid sagital section and into the sporogenic tissue. Several meiotic figures were observed with unparalleled resolution. Sequences of nuclear dynamics and division were occasionally observed in the surrounding tissues and epidermal layer of cells. However we could not optimize the procedures up to the level of observing the dynamics of division on the meiotic nuclei as well. We hypothesize that either (1) meiotic cells are sensitive to the reasonably high excitation levels of infrared light needed to attain such penetration in the tissue, or (2) that our incubation procedures are not sufficiently non-invasive for meiosis to remain unperturbed. To the best of our knowledge this is the first report on direct observation of living meiotic cells in plants and establishes the potential of 2PM for intact organ research. (C) 2001 Elsevier Science Ltd. All rights reserved.