BUFFALO, NEW YORK

State University of New York

LONDON, ONTARIO, CANADA1

University of Western Ontario

The use of confocal microscopy to study the developmental morphology of shoot apical meristems: a procedure to prepare the specimens --V. R. Bommineni1 and P. C. Cheng Confocal light microscopy associated with modern image processing systems can provide information about the spatial arrangement and orientation of cells, their nuclei, and their division patterns within a developing organ, such as a shoot apical meristem. Data obtained from confocal microscopy may also be used to obtain information about changes in the metric relationships of cells during development (such as the initiation of leaf primordia) or following exposure to different metabolic conditions.

We have developed a procedure to prepare the cells in the apical meristem to permit high resolution confocal microscopy.

1. Dissect the meristems and fix in 3:1 (EtOH: acetic acid) fixative for overnight.

2. The tissue is then stained in Feulgen for 20 min. (follow the maize root tip procedure described by Chen, C. C., Can. J. Genet. Cytol. 11: 752-754, 1969).

3. Then the tissue is washed in bleaching solution (which contains 1ml concentrated HCl, 0.4g potassium metabisulfite or sodium metabisulfite and up to 100ml of distilled water) for several hours to overnight with three changes.

4. Proceed with gradual ethanol dehydration and clear first with 1:1 absolute ethanol: methyl salicylate for 1-2 hours.

5. Clear the specimens in methyl salicylate and store them in refrigerator until use.

6. Mount the specimen in a specially made specimen holder or in a small chamber on a slide (made of a ring of vacuum grease on a microscope slide), add cover slip and gently press the cover glass so that the specimen just touches the cover slip (do not crush!).

7. A modified BioRad MRC-500 laser scanning confocal system attached to a Leitz Orthoplan microscope equipped with a Nikon Fluor 40X objective is used. BioRad GHS filter pack (Excitation filter 514 DF 10 and dichroic beam splitter DR 540LP) is used. Therefore, the 514nm green line of the Ar ion laser is used as the excitation wavelength. An OG550 barrier filter (560nm long wavelength pass) is used for the detector. The resulting optical sections can be projected at different viewing angles to obtain 3D stereo-pairs on a computer.

A stereo viewer is required to view Figure 1 and 2 which illustrates reconstructed three dimensional views of the apical meristem prepared from a 72 hr imbibed mature seed of waxy (wx wx) genotype. Figure 1 illustrates a 20um thick median longitudinal optical section, whereas Figure 2 illustrates 20um deep cells from a top view of the meristem. Note the clear view of the L1 tunica layer (protoderm) in Figure 1 and the systematic and organized pattern of cells in Figure 2.

This and the previous work was partially supported by grants from the US DoE (DEAS0888DP10782) (DEFGO 389SF18012), Acrux Co., and the Whitaker Foundation, Program for Biomedical Engineering to PCC. The Advanced Microscopy Laboratory (AML) is a joint facility of the School of Medicine and Biomedical Sciences and the School of Engineering and Applied Sciences, State University of New York at Buffalo.


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