This past week, I vanquished the dark zone but at a cost. Readers of a previous post will remember my main struggle with the project thus far is to get the cellulose-stain, fast scarlet, to stain roots reliably. To recapitulate: Dissolving the dye in water gives lousy staining; using a pH 6 or 7 buffer brightens up the staining nicely, except for a dark area right around where the maturation zone begins; the dye can be coaxed into that area with neither detergent nor following removal of some pectin. 

What I tried next was a different stain: Calcofluor white. This stain and I go way back. During the first year of my Ph.D., I TA’d the light microscopy course taught by my advisor, Paul Green. In the exam, which was a smorgasbord of practical exercises with apparatus and explanations of micrographs (i.e., pictures taken thru a microscope), a question featured a pair of pictures from a then-recent publication of fluorescence from Calcofluor staining a filamentous stalk: One picture was taken with the exciting light polarized parallel to the stalk and the second one with the light perpendicular. The exam question was to explain why the pictures looked different and the answer was that Calcofluor stains cellulose an oriented manner, so that the fluorescence intensity depends on the polarization state of the exciting light. 

For a while, the answer to this exam question (fluorescence from Calcofluor is polarized) featured in plans for my dissertation. Paul Green was studying cellulose reinforcement during the production of organs at the shoot apex (a process termed phyllotaxis). To determine cellulose orientation, he was using conventional polarized light microscopy (by “conventional”, I mean with transmitted light, using the birefringence of cellulose). However for this method, he needed single cell layers and getting these from the shoot apex required feats of dissecting dexterity.

I got animated for a while by realizing that the same determination could be made from polarized calcofluor fluorescence on intact shoots—no dissection. But among the difficulties of transforming this shiny toy into a working model was that I would need a dissecting microscope equipped with fluorescence. Today, thanks to the advent of GFP and its friends, such microscopes are as common as rain in Britain; in 1981, not so much. 

Last week, I stained some roots in Calcofluor: staining was bright and clear from the tip to the stump. No hint of an dark zone. Staining in water was fine and in pH 7 buffer was even brighter but also some dye was taken up into the cells (nuclei were stained). Uniform staining confirms my argument that the dark zone contains cellulose and hence the failure of that region to be stained by fast scarlet arises from an interfering substance. I think this pestiferous substance is hydrophobic because fast scarlet dissolves readily in water but Calcofluor dissolves in water only sparingly. 

Time to ditch scarlet and embrace the Calcofluor? Not so fast! Calcofluor is routinely excited by ultraviolet light but 405 nm (the edge of blue) will do. Yes! The confocal microscope where I plan to do this work has a 405 nm laser line, so that is ok. Alas, 405 nm light might damage the liquid crystal gadget needed for establishing the polarized exciting light. Might, might not; the gadget is expensive, I need to find out the odds. Is irony a sufficient word to describe polarized fluorescence from Calcofluor once again tantalizing me after 40 odd years? Do I hear history rhyming?