Further reading of the wikipedia page on iridescence led me to other ways light waves can be interfered with in order to split them into different visible colours. These effects are often caused by multiple reflections from two or more semi-transparent surfaces. They're responsible for many examples of iridescence in biology. One example is thin film interference (TFI), and I think because the description includes the word 'film' I was convinced I had found the answer. In TFI some wavelengths of light are cancelled out and some accentuated when reflected from the surface and lower boundary of a thin film. The colours affected dependent on the thickness of the film, which must be in the range of the wavelength of visible light so ~0.4-0.75 microns. It's the reason for the iridescence of oil on wet tarmac and it was while I was reading about it that I took the following photo.
|Iridescent aquarium biofilm formed on the top of a powerhead.|
|Aquarium iridescent biofilm x100. I didn't see a single coloured tetrad in this sample.|
It turned out the answer had been staring me in the face all along, if only I had read the caption to the photo of an iridescent aquarium biofilm in the wikipedia page on iridescence. The phenomenon responsible is diffraction not refraction/reflection. In the context of biofilms it seems the idea is that bacteria can grow in ordered patterns and form a diffraction grating. The spaces between the bacteria form slits, which have to be small enough to be within the wavelength of visible light, so as to split the light into it's constituent colours. Importantly "Diffraction will produce the entire spectrum of colors as the viewing angle changes, whereas thin-film interference usually produces a much narrower range". That fits with the full spectrum effects I observed in chapter 27 but not so well with the photo above which is dominated by red blue and green. I occurred to me that diffraction might also be responsible for the apparent iridescence of the coloured terads. In that case they would have to have some kind of repetitive structure in their cell walls, perhaps a protein, in order to diffract the light. Towards the end of this article there's a very interesting discussion of the possibility.
I couldn't see any order or pattern to the growth of the bacteria in the biofilms I examined at x100. It looked totally random to me no matter how much I digitally zoomed in. When I put a coverslip on this sample and used a higher power objective, I did perhaps see some evidence of structure. The edges of the image are out of focus but in the areas where the bacteria seem to be forming slits, the slits do seem thinner that the width of a bacteria, which puts them in the range of the wavelength of visible light.
|Aquarium iridescent biofilm x1000. Could the gaps between the bacteria act as a diffraction grating?|
|Aquarium biofilm iridescence. Fresh sample trans (top left) and epi (top right) illumination. Dried sample trans (bottom left) and epi (botton right) illumination.|
|Bacterial diffraction grating x1000 (x2 digital zoom)|
|Aquarium surface film x100 epi-illumination.|
I think both images also reveal that any small gaps in the biofilm are populated by other rod shaped bacteria which don't like to be crowded. Perhaps they and the tetrads produce an antibiotic which is why they're always in clearings in the film.
However, in terms of whether the coloured tetrads were iridescent, the x400 image was deeply troubling. True, the coloured tetrads were still coloured (which would be expected if some kind of nanostructure in their cell wall was diffracting light) but then so were many of the objects in the image. The bacteria in the gaps in the diffraction grating appeared purple and the amoebae green. As far as I know amoebae aren't green and I can't start claiming to have discovered iridescent bacteria willy nilly. More concerning still, was that neither object was coloured at x1000 demonstrating that my optics/imaging are capable of giving a misleading representation of colour in certain circumstances. All this doesn't look good for the 'iridescent' tetrads.
I needed to pause, do some more reading about chromatic aberration and also examine how my microscope in particular is affected by chromatic aberration.