CONFOCAL MICROSCOPY AND IMAGE-PROCESSING IN THE STUDY OF PLANT NUCLEAR-STRUCTURE

We describe measurements of the point spread function (PSF) for a confocal microscope and compare them with the PSF for a conventional (wide-field) fluorescence microscope. In situ hybridization with probes to telomere and ribosomal rDNA sequences, combined with three-dimensional (3-D) microscopy, has been used to study interphase nuclei in root tissue of Pisum sativum and Vicia faba. Nearly all the telomeres in both species are located at the nuclear envelope, and are highly clustered in the Vicia tissues, suggesting specific binding interactions. rDNA labelling in P. sativum shows four brightly staining knobs, corresponding to condensed regions of the rDNA genes from the two pairs of nucleolar organizer genes in this species, arranged approximately tetrahedrally around each nucleolus. Deconvolution using the measured PSFs can be used to improve these images, revealing a fibrous substructure in the perinucleolar knobs, and a large amount of interconnecting internal structure, which we suggest represents rDNA both in the fibrillar centres and also more diffuse, widely dispersed rDNA. Finally we show that accurate conventional data coupled with deconvolution can produce 3-D reconstructions comparable to those obtainable with confocal microscopy, but that the clearest images are obtained by applying deconvolution to the confocal data.