Birefringence (Fluoresence!) of Anthraquinone Inclusions in Lobster Mushroom

Images made through a microscope. All subject types.

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ldflan
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Birefringence (Fluoresence!) of Anthraquinone Inclusions in Lobster Mushroom

Post by ldflan »

Lightly edited in view of Rik's observation below.

Images to accompany text posted here:

https://www.yogile.com/dch4a5mzfyi/51m/ ... =d5c26cfec

According to Robert Weaver’s article on the subject here (http://zeiss-campus.magnet.fsu.edu/refe ... 5-2003.pdf), anisotropic or birefringent materials include certain crystal systems, certain substances placed under mechanical strain or stress, fibers or sheets containing oriented polymers (e.g. plant cell walls, fungal hyphae, hair, spindle fibers), liquid crystals, and optically active (that is, chiral) gases, liquids and solutions.

In the case of a single isolated birefringent crystal imaged on a petrographic or polarizing microscope, the specimen will display extinction points on rotation, and potentially different colors depending on orientation with respect to the analyzer. The colors displayed will also depend on the degree of relative retardation of the extraordinary and ordinary rays, which in turn depends on both the structure and the thickness of the specimen. (This is why petrographic specimens are always ground to the same thickness.) These samples will also show a maltese cross or similar interference pattern.

One doesn’t encounter too many true crystals in biological specimens.

One that has been discussed here on the forum is druse or calcium oxalate, frequently encountered in plants. Druse crystals in plants show white birefringence, no extinction point, and no interference patterns. Viewed between crossed polars with a first order (red) wave retarder inserted, either in white light or monochromatic green light, druse crystals will appear apple green, pretty much regardless of size or thickness of the druse crystal. However, when the crystals are small enough (in many plants, druse crystals are not static, but wax and wane in size, likely in a metabolic process recently identified and labelled “alarm photosynthesis), one can detect a spectrum of colors across the smallest crystals. This suggests that the white birefringence is a consequence of druse inclusions being cryptocrystalline in nature, that is, composed of a mass of crystals oriented in many different directions at once.

Starch grains are usually described in the literature as quasi-crystalline. This is likely the result of starch (being of two types – long and stringy or short and highly branched) being laid down in an alternating and highly ordered fashion by the hila, essentially creating a kind of crystal. The exact pattern is unclear, but from my review of the literature, I suspect something like a gyroid pattern is involved as there are plainly holes in the crystal structure of starch grains that allow entry of other molecules and enzymes. Viewed between crossed polarizers, starch grains show a spectrum of colors across the grain, presumably depending mostly on thickness at any given point. They also show the classic maltese cross pattern.

Birefringent oriented polymers are much more common in living systems, of course. The case of birefringence of oriented fibers in biological specimens is readily seen in lignified plant cell walls or in the chitinous cell walls of some fungal hyphae. Lignified plant cell walls will often show variable colors depending on orientation with respect to the analyzer (blue/yellow), but no extinction point. Fungal hyphae will present either as white birefringence, or in some cases colored birefringence that is independent of orientation, and presumably the product of either retardation of the extraordinary ray or (far less likely, I think) the actual presence of a colored component between the birefringent hyphal wall and the viewer.

The question of visualizing effects of chiral molecules on light via the microscope is intriguing. The other day I stumbled on a phenomenon that is new to me, and that I thought for a short while may represent an example of this last category of birefringence that one might encounter in a biological source (i.e., optically active substances). Sadly though, in fact, as Rik points out below, the phenomenon is almost certainly a naturally occurring and highly fluorescent substance - closely related to alazarin red, probably.

On a walk in the woods the other day I found a “Lobster Mushroom.” This is a fairly common large white basidiocarp called Russula brevipes that has been colonized by a parasitic fungus (often referred to as a “mold” in the literature) called Hypomyces lactiflorum. Among other things, the parasite converts an inedible white mushroom into a bright orange-red, highly deformed version of the Russula that is a prized edible. To be honest it’s just too weird looking, and I have never been able to bring myself to eat one.

The orange coloration of the Hypomyces lactiflorum parasite is attributed in the literature to the presence of some variety of anthraquinone, and the mushrooms are frequently used to dye wool (color outcomes largely depending on pH of the dye prepared from the Lobster Mushrooms). Many forms of anthraquinone (maybe all of them?) are optically active or chiral.

The literature says that anthraquinones dissolve readily in organic solvents. I can tell you that the anthraquinones responsible for Hypomyces lactiflorum’s orange/red color do dissolve readily in ETOH above 50%, and very readily in FPA fix (FAA with propionic acid instead of acetic acid). So, visualizing any microscopic structures containing the anthraquinone colorant, then, generally needs to be done without involving reagents that will dissolve it away.

To that end, I mounted several vibratome slices from the edge of the Lobster Mushroom cap in lactophenol cotton blue, or in Fluormount G with DAPI. In these specimens, the anthaquinones are seen to be contained in millions of small inclusions scattered through the outer layer of the parasite. The orange/red anthraquinone inclusions are not clearly crystalline, though some do appear to be what I would describe as “spikey.” Not all of them are spikey, though that may be a product of sample handling.

Between crossed polarizers, the anthraquinone inclusions are strongly birefringent and show up as a bright scarlet color. The red color of the inclusions is essentially the same whether viewed using white light or monochromatic green. The color is the same regardless of the size / thickness of the inclusion. The color exhibits no extinction point on rotation, and no interference pattern. Alazarin red or "madder," a common anthraquinone dyestuff, fluoresces red from green excitation, so that is the likely cause.

The anthraquinone inclusions lose their ordering into nodules when the mushroom dries. Dried scrapings of the pigmented tissue show the colored component spread out in or on hyphal threads, and no longer contained in a discrete nodule. A weaker red-orange glow remains, however, so long as the material is dry. When I attempted to mount the dried scrapings gum Arabic, the red glow vanished, leaving only the normal birefringence of the dried hyphae. I interpret this as indicating that the nodules burst on drying, and that the chemical component became dispersed in the liquid gum arabic such that it was no longer concentrated enough to display visible birefringence or fluourescence. (Photos of this to be added later).

The other possibility that occurs to me is that perhaps in the case of these nodules, the light comes from birefringent chitinous cell walls, but it is shining through a film of red anthraquinone like a colored Christmas lightbulb or something. That seems unlikely based on the intensity of the light compared to nearby cell walls.

A couple of other observations. First, the parasite develops some thick hypal threads where it intersects with host mushroom tissues. These show a yellow / green birefringence, independent of orientation with respect to the analyzer. In other words, they behave very much as does calcium oxalate / druse inclusions under polarized light. I am wondering whether a spiral ordering of polymers would have this effect? This may also be a flourescence effect as well.

The second observation is that this “mold” can either sense gravity or “knows” when it is growing on the host gill tissue. The parasite forms spore-bearing ascocarps only on the lower surface of the Russula, just as though it were a regular gill-bearing mushroom. These structures do not survive paraffin embedding well, and are better seen with fresh samples.

If you've got possibly better explanations for what I am seeing with these anthraquinone inclusions, by all means please speak up! PLM is extremely useful for biological specimens, but interpreting the results is hard!

All you in the northern hemisphere - get out and enjoy the glorious mushrooms of fall! Best time of the year...

Cheers -

Leonard
Last edited by ldflan on Tue Oct 12, 2021 9:28 pm, edited 1 time in total.

rjlittlefield
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Re: Birefringence of Anthraquinone Inclusions in Lobster Mushroom

Post by rjlittlefield »

The red color of the inclusions is essentially the same whether viewed using white light or monochromatic green.
Do I understand correctly that you illuminate with green, but then observe red?

If so, then I suspect that you're seeing fluorescence, red emission from a green excitation. Any non-absorbed excitation will be blocked by the crossed polars, so you always see red against a black background.

--Rik

ldflan
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Re: Birefringence of Anthraquinone Inclusions in Lobster Mushroom

Post by ldflan »

Hi Rik. Yes, you are almost certainly right. Duh. Alizarin excitation is green, emission red. I'll check it tomorrow to be sure.

Leonard

houstontx
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Re: Birefringence of Anthraquinone Inclusions in Lobster Mushroom

Post by houstontx »

All that the rain brings...and MORE! Awesome post & pics thank you

rjlittlefield
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Re: Birefringence of Anthraquinone Inclusions in Lobster Mushroom

Post by rjlittlefield »

ldflan wrote:
Tue Oct 12, 2021 8:20 pm
Hi Rik. Yes, you are almost certainly right. Duh. Alizarin excitation is green, emission red. I'll check it tomorrow to be sure.
You know, cryptocrystalline birefringence was sounding good to me too, until I tripped over that bit about the "monochrome green". I'm glad you made that observation!

--Rik

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