Chapter 28. Bacterial and biofilm iridescence part 2. Refraction and diffraction

The rainbow iridescence of the aquarium biofilm and the colonies of rainbow coloured bacteria I observed within it had got me thinking about rainbows, probably because they are so familiar. I imagined that the coloured tetrads might be acting like drops of water in the air, refracting the light due to their having a different refractive index from water they're floating on, and so causing the dispersion of the visible wavelengths of light. But after some reading I discovered two problems. Firstly, the difference in refractive index between a bacterial cell (1.38) and water (1.33) is much less than between the atmosphere (1.0) and a drop of water (1.33). Would that be enough to cause dispersion? Even allowing for the EPS which is reported here to have an RI of 1.4, it seems unlikely. Second, a rainbow can only be seen in the sky opposite the sun and from an angle of 42 degrees from the direction opposite the sun. In other words, rainbows are only visible under epi-illumination at a fixed angle. When looking down a microscope I'm staring directly at the sun and the biofilm dispersion at the aquarium surface was visible under trans-illumination and a range of angles of epi-illumination so rainbows don't make sense as a model. I decided to stop thinking about them, what I'm trying to understand is obviously very different.

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.

To my mind this looked oily, which fitted with TFI being responsible, but the more I read the less likely that seemed. The first problem is the thickness of the biofilm. Even if it is one cell thick that's still almost 1 micron, and when I looked at a sample of the above biofilm under the microscope it looked like it had topography.

Aquarium iridescent biofilm x100. I didn't see a single coloured tetrad in this sample.

The second problem is refractive index again but in a slightly different sense. When light waves meet a boundary between to mediums with the same refractive index they pass straight through. So I'm not sure there would be enough reflection from the lower boundary of the biofilm to cause TFI. The third problem is that TFI seems to be all about reflected light so I'm not sure it would be visible by trans-illumination.

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?

The problem was, when I put a coverslip on an iridescent biofilm sample, I lost the iridescence, so I'm not sure if this image is an accurate representation of the biofilm structure. I needed a way to examine iridescent samples at high magnification but without a coverslip and completely by accident I found one.

Aquarium biofilm iridescence. Fresh sample trans (top left) and epi (top right) illumination. Dried sample trans (bottom left) and epi (botton right) illumination.

Just as you would expect, the fresh biofilm sample is iridescent under trans (top left panel) a epi (top right panel) illumination. What I noticed after not cleaning my slides one evening, is that the same is true after the sample has dried onto the slide over 24 hours. This is interesting because it suggests that the biofilm structure, in terms of it's ability to diffract light, is the same after drying. The only other possibility is that the iridescence of the fresh sample is due to diffraction and the iridescence of the dry sample is due to TFI. This is more plausible than for a fresh sample because the dry biofilm might be thin enough and have a sufficiently different refractive index to glass (~1.5) to cause it. But it doesn't explain the iridescence under trans-illumination (I think) and I can see a full spectrum of colours in the dry sample iridescence which is more in keeping with diffraction. Because the sample had dried I could examine it with higher power objectives without a coverslip.

Iridescent aquarium biofilm dried onto microscope slide x400 (digitally zoomed x3). The maze-like patterns formed by the bacteria apparently act as a diffraction grid. The circular objects range in size from 2.5 to 10 micrometres and are probably dried amoebae.

A strong candidate for a diffraction grating was revealed I think. The lawn of bacilli, which appeared to be unstructured at x100 magnification, actually formed complex maze-like patterns at x400. It is possible to view the dried film x1000 but it is not possible to use lens oil! As a result x1000 images should suffer a loss of resolution and be effected by spherical aberration.

Bacterial diffraction grating x1000 (x2 digital zoom)

I think there is a loss of reolution around the edge of the image but I get that with any of my objectives. The way the bacteria might be forming slots through which light waves would be diffracted is clear. Remarkably, the film appears to be one cell thick which I would never have guessed from looking at the biofilm under epi-illumination.

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.