Roots are relatively thin and have low background fluorescence, making them highly suitable for analysis with the laserscanning confocal microscope. Confocal microscopy is an excellent tool for imaging at cellular resolution.
For many applications, it is necessary to visualize tissue anatomy.
We counterstain with propidium iodide (PI), a nucleic acid binding dye that is excluded from the membranes of healthy cells. The result is red fluorescence surrounding each cell in the root.
PI Staining
1000x concentrated stock: 10 mg/ml PI in water (avoid light)
1x working solution: 10 mg /ml PI in water
Propidium Iodide Sigma P-4170
Dip whole seedlings or cut roots in PI solution for approximately one minute. Successful staining can be achieved by incubations as short as 30 sec and as long as 3 hours. We have noticed, however, that leaving the seedlings in stain appears to render cell membranes more vulnerable to permeation (perhaps due to osmotic stress).
Membrane permeation allows PI to enter cells and bind in the nucleus, causing bright punctate fluorescence that obscures anatomy and other fluorphores.
After staining, roots can be mounted under a coverslip, either in staining solution or distilled water. We recommend a quick dip in distilled water, followed by mounting in water, simply to avoid spreading the toxic and mutagenic PI around the microscope. PI staining is not compatible with glycerol.
Considerations
It is important to ensure that (1) roots are handled as little as possible throughout the procedure, (2) excessive pressure on coverslips is avoided, and (3) tissues are not allowed to dessicate. Keeping roots healthy is the best way to guarantee good images.
Imaging
We use a Leica TCS SP2 system with an Argon line. John Runions has put together an excellent tutorial for the confocal microscope that is accessible from Jim Hasseloff website at the Department of Plant Sciences, University of Cambridge
www.weed3D.com
http://www.plantsci.cam.ac.uk/Haseloff/JohnRunions/Confocal_instructions/home.html
