Note: Some years ago, a giant diatom named Coscinodiscus wailesii was declared a nuisance in European waters. Until then, it was just a large but unassuming planktonic inhabitant of the Indian and Pacific Oceans. First noticed in the English Channel in 1977 where it may have been dumped by visiting ships emptying ballast, the diatoms rapidly grew in number. Soon, they formed coagulating sludges that fouled equipment aboard fishing trawlers. The equipment proved very difficult to clean. When the sticky clouds of diatoms grew large enough, they would sink dragging huge quantities of other plankton to the bottom. Perhaps in an effort to make lemonade out of these waterborne lemons, or perhaps because of their peculiar periodic structure, researchers began investigating C. wailesiis' potential as photonic crystals. It had long been known that many members of the animal kingdom incorporated photonic crystals in their surface structures. Now, scientists wanted to explore the plant kingdom—starting with diatoms. (Not long ago, diatoms were unceremoniously booted out of the plant kingdom. They are now considered protists.) Some felt that if photonic crystals were also found in plants, it probably was a rare anomaly due to evolutionary chance. Perhaps it 's neither chance nor rare.
Diatoms as photonic crystals
Mention photonic crystals to physicists and their eyes light up! These crystals can be described as dielectric structures with periodically modulated refractive indices. Also known as photonic band gap (PBG) materials, laboratories everywhere are scrambling to design, manufacture, and find novel uses for these crystals. They are already being applied in electrodynamics, photon-state squeezing, micro-fabricated lasers, simulated opals, and as waveguides in the infrared and microwave regions. It has been said that photonic crystals are to electromagnetic waves as semiconductors are to electron waves. Interestingly, some remarkable examples of photonic crystals can be found in most any body of water. What follows is less about techniques and more about observations.
Diatoms and regular periodic structure
In my limited collection of mounted diatom microscope slides, I have one produced many years ago for National Scientific Instrument Co. The company was located in a building on Chambers Street in downtown New York. The company's store was loaded to the brim with microscopes, practically antique optical accessories, handheld direct-vision spectroscopes, and lots of other scientific goodies no longer available. I bought this slide from the company just prior to my leaving New York City for warmer climes. Shortly after I left New York, National Scientific went out of business. Coincidence? Anyhow, the slide is a strewn assortment of five different species of diatoms. It was intended to be used to test the quality of microscope optics. It's well known that mounted diatoms often exhibit coloration under the right lighting. Just simple diffraction it was thought. I noticed this coloration in some of the diatoms on my test slide when I examined it under dark-field illumination, but I set the slide aside as being of little consequence.
Recently, I was reading several articles describing some newer photonic crystals capable of propagating visible light. The crystals do this well, except for light that falls into the forbidden range of the crystals' band gap. In that case, the band gap light is either reflected or trapped. If trapped, the light's energy is dumped into shorter wavelengths that are reradiated. Examples in nature, such as butterfly wings and beetle cuticles, were given. Then I remembered the diatom coloration. Some of the colors were vivid and reminded me of the intense interference effects seen in soapy liquids. Scanning electron micrographs of insect parts containing photonic crystal structures often resemble the appearance of some diatoms under very high magnification. (Of course, the striking periodicity of diatoms hasn't been lost on the materials scientist. Researchers are working on ways to get diatoms to construct periodic structures built according to blueprints supplied by science. This is being done with the intent of manufacturing photonic crystals faster, better, and cheaper.)
Once again, out came the diatom test slide. First, I set up my Leitz with simple dark-field illumination and examined the slide under a 16X objective. The Pleurosigma angulatum diatoms exhibited a bluish color similar to that of smoke rising from a burning cigarette. The other diatom species on the slide did not appear to exhibit any characteristic colors. However, no matter how haphazardly the Pleurosigma diatoms were positioned on the slide—some were slightly rotated around their long axes relative to others on the slide—they all exhibited the same color. Also, there were occasional overlaps of the diatoms, and interesting moiré effects could sometimes be seen. Let's look at that first.
Moiré magic
When moiré patterns are superimposed, there can be an effect resembling magnification due to coinciding structures. The effect works best when identical patterns are used. To demonstrate moiré magnification, I prepared a pair of identical Pleurosigma shell patterns from a photomicrograph. The moving pattern was placed as a layer in Photoshop and shifted around for each frame.The animation shows what happens when two diatoms overlap. Note the mobile hexagonal patterns that form. I find that stepping back several feet from the screen helps in seeing the magnification effect.
Superposition of diatoms
The photos at right were taken using a Leitz NPL Fluotar 16X 0.45 NA objective under dark-field illumination. The images were captured with a Mintron 12V1E-EX b&w CCD video camera connected to an Argus-20 image processor. The Pleurosigma diatoms appeared blue under the microscope. Adobe Photoshop was used for post-processing. The magnification for both images is 200X.
Compare the above moiré animation with the these two photos taken of the diatom test slide. These pictures show two instances of the chance overlaying of Pleurosigma angulatum frustules or shells. The image at upper right displays a particularly favorable superposition. The diatom's periodic structure is clearly visible where the diatoms meet. The moiré magnification here is about 2000X. The image at lower right shows a different configuration of an overlap with little true structure showing. This illustrates the importance of proper positioning for moiré magnification to work well.
Typical spacing between pores in Pleurosigma angulatum has been listed as ranging from 0.52 to 0.65 microns. The pattern of holes is described as forming a hexagonal array, and this can be seen in the animation shown above. The porous shell is composed of silicon dioxide.
Also visible in the photos are several football-shaped Frustulia rhomboides diatoms.
Oil immersion view
The photo at right shows a very highly magnified view of Pleurosigma angulatum using bright field illumination. It was taken using a Leitz Plan Apo 100X 1.32 NA oil immersion lens. A deep-blue filter (436 nm) was used to improve resolution.
The same image capturing system was used as in the above photos. Similar views of a different Pleurosigma organism on the test slide were used in generating the patterns for the animation shown above.
The periodic structure of the diatom is the most critical property responsible for its possible behavior as a photonic crystal. Two more essential ingredients are the silica frustules serving as one dielectric medium along with air- or water-filled pores as the other medium.
The image clearly demonstrates the hexagonal pattern, as well as the validity of moiré magnification, in the above photos. Its magnification is at least 5000X based on the pore-to-pore spacing stated above. (Except for the SEM micrograph seen below, magnification figures given on this page are estimated final values. They take into account the optical magnification produced by the objective and projecting eyepiece, the magnification introduced by the CCD chip, and a best guess of the magnification resulting from the monitor size and resolution that the viewer is using.)
So, even without having a scanning electron microscope on hand, it is possible to examine many diatom structures to hunt for potential PBG properties. But not all diatoms are as cooperative as Pleurosigma.
A look at some more diatoms
Here are two more diatom species from that same test slide. Both of these photos were taken using the same image capturing setup as for Pleurosigma. Some image processing was applied to accentuate periodic structure but in different ways.
Frustulia: I first reversed the image (a negative image sometimes enables subtle details to be seen clearly) and overlaid that image with a partially transparent positive version. The positive image overlay was then displaced by several pixels both horizontally and vertically, thereby producing a bas relief that emphasized the pore pattern.
Surirella: I photographed it using a Leitz 63X 1.40 NA Phaco 4 oil immersion lens and matching phase contrast condenser. All that was needed was some minor tweaking of brightness and contrast.
Unlike Pleurosigma, both Frustulia (formerly called Navicula) and Surirella pore structures appear to be laid out in a square or orthogonal pattern—not hexagonal. Unfortunately, I could not find any superpositions of these diatoms on the slide that displayed moiré magnification.
Diatoms are to be found in rivers, streams, ponds, lakes, oceans, and, occasionally, even puddles. They are usually alive when found and stuffed to the pores with chloroplasts. Diatoms need to be thoroughly cleaned to allow examination of their shell structures. It's much easier to buy diatomaceous earth or prewashed diatom mixes rich in the organisms you want. Books on introductory microscope techniques, especially the earlier books, give instructions on how to prepare diatoms for mounting and examination.
Better yet, there are several small companies run by very talented individuals that specialize in supplying prepared diatom slides. Fortunately, the least expensive slides are typically those with diatom strews. And those are exactly what one needs to hunt for moiré effects and diatom colors.
Screening diatoms and a new illumination technique I've developed
I wanted a screening method to help evaluate diatoms for possible PBG behavior. I experimented with a number of different approaches. Working with standard dark-field illumination produced strong coloration in a number of diatoms, as expected. Illuminating the microscope slide near any of its four edges with a high-intensity fiber-optic illuminator helped bring out prominent coloration in many other diatoms as well. (See photo of opal below.) Also, edge illumination allows using a monochromator to tune the color of light. To do that, I use a fiber-optic cable to couple the high-intensity illuminator to a spectrometer configured as a monochromator. A second fiber-optic cable is run from the monochromator to the specimen slide sitting on the microscope stage. In this way, I can see how different wavelengths interact with different regions of the diatoms.
As I mentioned above, the luminous ring diameter should be greater than the existing substage light diameter, but not too much greater. That's where experimentation in matching the ring light diameter to the dark-field condenser optics is required. I also tried different objective lenses with different numerical aperture (NA) values until I found a setup that worked well. The NA of the condenser should be higher than that of the objectives to be used. For higher power objectives that have too high an NA for the obliquely excited dark-field effect to work, I insert a small aperture disc or funnel stop at the rear of the lens. (An objective lens with a built-in iris is ideal. Simply vary the opening until the desired color effects are visible.) Don't forget to remove the aperture before attempting high-resolution photomicrography. Your microscope setup and mine probably differ substantially, so I can't be more specific. However, centering the condenser, objectives, and ring light is very important.
The edge illumination technique described earlier works nicely and can produce some really colorful images. But an obliquely excited dark-field condenser produces superior results that are repeatable. For best results with my microscope, I usually restrict examination to 20X objectives and below. However, even a 40X objective will still show color effects, especially if a reducing aperture of some type is used. Again, the numerical aperture of the dark-field condenser is key.
Finally, the main problem, as always, is in trying to capture what the eye so plainly sees. The composite photo at right is an attempt. You are looking at mounted, clear-as-glass silica skeletons of diatoms and radiolarians colored only by diffraction effects. Through the eyepieces, images are crisp and drenched in color. Blues are electric and intense. Pastels and saturated colors abound. Reds and purples are rich and royal. Naturally, there are those diatoms that stubbornly refuse to reveal diffractive colors and only reflect incident light. To the eye, there are no washed out areas in the centric diatoms as the eye/brain easily folds the brightness range into clearly discernible oranges, reds, and yellows.
After checking out the diatom colorations with my chosen method, I select a likely candidate. I then examine the "diatom of interest" under high magnification to look at its pore structure. This is done in an attempt to correlate the pore configuration with the observed color play. At the moment, that's as far as I've taken my technique.
Questions, questions, questions...
All this leads to a bunch of questions about diatoms. For example:
What is the relationship between the diatom pore spacing, the pore layout (e.g., orthogonal, hexagonal), the pore diameters, and the diffracted wavelengths observed under the microscope?
I noticed that a number of diatoms I looked at using edge lighting seemed restricted to glowing within certain parts of the visible spectrum. This was especially noticeable for Pleurosigma angulatum and Frustulia rhomboides. Both favored blue or blue-green. However, Pleurosigma sometimes glowed red or orange. The characteristic play of lights has long been been used to help confirm the identify of a particular diatom species. These color observations are not new discoveries, but they should be reexamined in a whole new light!
Are diatoms, in general, photonic crystals? If so, why?
Nature is not wasteful. Butterflies, beetles, weevils, and seed shrimp have survival, territorial, or mating motivations that can explain their need for photonic crystals. Look carefully at the photo of Surirella above. Note the area devoid of pores (at left). Does this flat surface represent a planar defect, or is it just a random defect? Photonic crystals do their best work at defects (points, lines, or planar) within their structures. That is where they truly shine! Might diatoms use their structures to channel certain wavelengths of light more efficiently? After all, these organisms are a tremendous source of nutrients and oxygen on this planet. It seems that any mechanism that can improve their efficiency in carrying out photosynthesis would be highly beneficial, especially in competing crowds of phytoplankton. But then there's the question of why there are different photonic structures in diatoms if their sole purpose is light propagation. Chlorophyll functions best at certain wavelengths. Presumably, each unique structure is associated with a different band gap and a different forbidden wavelength of light.
Permanently mounted diatoms on a slide are not diatoms sitting in water. The diatom pores may be partially or completely filled with mounting medium. The refractive index (RI) of water is 1.33. An excellent diatom mounting medium, such as Pleurax, has an RI greater than 1.7, while the RI of silica is 1.46. When possible, any thorough evaluation of mounted diatoms should be followed up with an examination of the same species of diatoms in water.
Are there differences between the photonic crystal properties of fresh water and marine diatoms? And what about fossil diatoms?
What about diatoms that do not exhibit the color effects associated with truly periodic structures? How do they behave in their environment versus the periodic diatoms?
Could the illumination methods discussed here prove helpful in screening laboratory produced photonic crystals?
So, are diatoms photonic crystals?
Diatom frustules can be thought of as inverted opals with their hollow pores replacing opals' silica spheres and their silica valves replacing opals' silica solution-filled gaps.
Below, at left, is a photo displaying a precious opal's characteristic play of colors. The colors are the result of Bragg reflection/diffraction from periodic layers of silica microspheres in the opal. At right is a photomontage of edge-lit diatoms. There are no color pigments remaining in the thoroughly washed silica frustules. Not all diatoms demonstrate this play of colors, but that's also true of opals. Potch opal has a less regular structure than the colorful variety and displays a milky opalescence.
Because of the fiery display of colors exhibited by some diatoms, past observers have referred to them as miniature opals. And opals are examples of photonic crystals. Opaline structures that exhibit PBG properties can be synthesized in the lab. Those with suitable periodic configurations designed to function in the visible spectrum also demonstrate the opal's play of colors. Perhaps diatoms are just living, breathing water opals after all!
It does seem strange that the inherent beauty possessed by diatoms only becomes obvious when studying their lifeless skeletons. Living diatoms are also very interesting. In fact, from the moment the microscope was invented, both amateur and professional researchers have been drawn to study diatoms. And with the observed PBG behavior of C. wailesii, as discussed above, perhaps new lines of research will be opened.
Living in a glass house
Still retaining its membership as phytoplankton, diatoms are unicellular algae belonging to the phylum Bacillariophyta. Broadly divided into planktonic (free floating) and benthic (living at water/submerged surface interfaces) there are, more or less, 100,000 species extant. They are predominantly nonmotile, though some species can be seen oozing around. Their glassy skeletons are built up from silicic acid monomers. They are constructed in a pill-box fashion with one valve fitting comfortably into the other. This is accomplished in different ways with different species. And diatoms are not the only siliceous organisms in the ocean.
Virtually paralleling the shabby treatment given its glass-encased cousin but tossed out from the animal kingdom instead, radiolaria are still considered zooplankton. Like diatoms, radiolaria have pore-filled, symmetrical, silica-based skeletons. But they are composed of a single geometrical structure rather than two pieces fitting together. Radiolaria are amoeboid protists belonging to the phylum Sarcodina.
Much like diatoms, radiolarians are nonmotile, wafted about by ocean currents. However, unlike diatoms, radiolarians are primarily planktonic. They are found in enormous quantities in deposits of radiolarian ooze. Generally speaking, radiolarian pores are larger and not nearly as tightly arranged as those present in diatoms. At right is a scanning electron micrograph showing a radiolarian in the foreground with a diatom behind it.
(The original photo of specimens obtained from the Scripps Institute was taken at 1280X. It was recorded back in the days when I actually owned a vintage SEM.)
Accident or design?
As you can see from the photo, Coscinodiscus wailesii truly is a giant among diatoms! Perhaps this organism started it all, but now the question remains whether it pretty much ends there or whether, in fact, diatoms have optical structures instrumental in their survival. In other words, are diatoms' photonic crystal structures exceedingly rare and unnecessarily ornate serving only to separate their inner world from the surrounding hostile environment, or are they exceedingly common and designed to serve as selective light conduits?
In summary, it's clear that the iridescent color play of diatoms is due to diffraction...but, undoubtedly, it is not so simple. Up next, a look at the least expensive nanomotors currently available!
