From frithjof@PI.NETTue Apr 29 14:43:32 1997 Date: Wed, 21 Aug 1996 08:40:24 PDT From: "F.A.S. Sterrenburg" To: Multiple recipients of list DIATOM-L Subject: Re: Pleurosigma angulatum I have no experience with darkfield REFLECTION microscopy, but I do have very comprehensive data on the colour of Pleurosigma and Gyrosigma valves in darkfield transillumination. The first data were given in: Sterrenburg, F.A.S. (1991). Studies on the genera Gyrosigma and Pleurosigma. Light microscopical criteria for Taxonomy. Diatom Research 6,2 367-389. This paper also described how to standardize darkfield illumination. The database now includes about 40 Gyrosigma and 40 Pleurosigma species, thousands of valves in total. Because not too many diatomists are familiar with darkfield, and the colour phenomenon is often not believed until people see it, here is a summary. GENERAL USEFULNESS OF DARKFIELD Even the very simplest form of darkfield, with a central stop in the filter ring of the condenser, is a superb aid if you want to scan diatom slides quickly. It's very 'eye-friendly' particularly for long working sessions and even at the lowest power (say 100x) you notice a lot more than with brightfield. Tiny and very delicate frustules, which may be easily overlooked otherwise, stand out clearly. Also, many diatoms show colours in darkfield, by which they stand out even more. This is not only the case for several large Centrics, but also for Cocconeis ssp. and Anomoeoneis ssp., for instance. SIGMOIDS: SPECIAL CASE Gyrosigma and Pleurosigma species form a special case in this respect. Each species of these genera shows a colour that varies somewhat in intensity (depending on the degree of silicification of the individuals) but remains strictly within a narrow range of hues. For Pleurosigma angulatum, for instance, the colour is intense (for the large growth forms = angulatum and quadratum) to faint (for the small growth forms = "aestuarii") ultramarine. This permits immediate separation from P. strigosum, which has been confused with angulatum on a large scale and has even been mooted as a "variety" of angulatum. Strigosum is NEVER ultramarine, but light blue, a sort of frosty blue. Angulatum is NEVER 'frosty blue'. So at 100x you can infallibly separate both species when the sample is a mix - which is quite frequently the case because both species have the same ecology. There are many more such examples of frequently confused Pleuro and Gyro species that can be separated immediately in darkfield at the lowest power. I use the colour phenomenon as the first test in any identification of Gyros or Pleuros, at just 100x or 200x. In combination with the general morphology, this may settle the identification in over half the cases - higher powers and SEM then just confirm it. DIFFRACTION GRATINGS The colour in darkfield is a diffraction phenomenon. Of all diatoms, Pleurosigma and Gyrosigma are perhaps the most perfect example of a diffraction grating, because the stria RATIOS are highly constant. If we limit ourselves to Pleuros here, the periodicity of the transverse and oblique areolar systems is so closely linked that the whole valve shows a continous network of lines whose intersection angle varies by only a few degrees. In Gyros there is a similar situation and in both genera, this constancy of stria RATIO is a major taxonomic criterion. INDICATION OF ULTRASTRUCTURE Periodicity is not the only factor involved, however. It may sound a "tall tale", but colour in darkfield at 100x may actually tell you that something is going on at a structural level FAR BEYOND optical microscopy. Pleurosigma angulatum and P. strigosum are a classic illustration of this. They have approximately equally fine striation, but their colour in darkfield is totally different. The reason becomes clear only in the SEM: the internal areolar foramina of angulatum are simple, those of strigosum are double (or: an oval with a bar across, if you prefer). This, of course, is one of the reasons why strigosum cannot be a variety of angulatum - there is a major discontinuity involved. REPRODUCIBLE? Stuart Stidolph and I have tested the reproducibility of colour in darkfield independently (we live as far apart as it is possible to do on Earth). We used the same materials of dozens of different Gyro and Pleuro species, following the description of the standardised DIY darkfield set-up in the paper mentioned above to the letter. With a great variety of condensers and objectives - different manufacture, different optical correction and VERY different price - the colours are fully reproducible. The prerequisite is that the conditions of this "diffraction grating experiment" - viz. the NA of the illuminating cone of light - be standardised. If you do some minor physics with diatoms, you need to 'calibrate' your measurement equipment! Difficulties people have reported to us were invariably due to imperfect microscopy technique (incorrect illumination, attempt to use a phase annulus as a central stop). Further to the note by Arthur Strange: I suppose you use incident DARKFIELD illumination, since you mention colours? This is also an excellent method. It greatly increases contrast - that's why you can resolve Amphipleura pellucida. See my earlier notes on contrast vs. resolution on the Diatom List. Incident BRIGHTFIELD illumination is also possible with diatoms and earlier this century interesting experiments with silvering were carried out... From Arthur_Strange@MAGIC.CATue Apr 29 14:43:32 1997 Date: Tue, 20 Aug 1996 15:14:51 -0400 From: Arthur Strange To: Multiple recipients of list DIATOM-L Subject: Pleurosigma angulatum [The following text is in the "ISO-8859-1" character set] [Your display is set for the "US-ASCII" character set] [Some characters may be displayed incorrectly] Hi Phil; Re: “striking prismatic color pattern in Pleurosigma” Colours that can be observed in diatom frustules is generally of two types, brilliant iridescent and milky opalescent. The colours can be seen using incident-light illumination under two conditions; With dry mounted specimens (with coverslip). Mounted specimens with oil immersion objectives. >From my own observations the type of colour varies from genus to genus, Pleurosigma angulatum is particularly colourful. Many of the centric diatoms are bright blue on the perimeter and bright golden yellow in the centre. Observation using incident-light can increase the resolution of detail. For example, Amphipleura pellucida when observed in brightfield illumination it is difficult to resolve the striae which are 0.25µm apart, however, incident-light shows the striae quite clearly. The colour of A. pellucida specimens varies from pale yellow to coral pink. You can find more information on incident-light microscopy in my paper: Strange, A., 1994. “Visibility and Resolution of Covered Specimens with Incident-Light illumination”, Journal of Biological Photography, Vol. 62, No. 3, 91-102. Regards, Arthur B I O A R T S Photography for Science and Education I O Arthur Strange A astrange@mail.magic.ca R T 3420 Eglinton Avenue East, Unit 503 S Scarborough, Ontario, Canada. M1J 2H9 From pbrody@LAMP0.ARL.MILTue Apr 29 14:43:32 1997 Date: Mon, 19 Aug 1996 15:01:28 -0400 From: Phil Brody To: Multiple recipients of list DIATOM-L Subject: Pleurosigma angulatum I am viewing the diatom Pleurosigma angulatum (the frustule) on a North Carolina Biological Supply Co. Diatom Test Plate, 8 forms using (1) Transmision Nomarski DIC, (2) darkfield reflection (3) a special laser interference microscope. The interest is in the microscopy; the diatom is a test object. Using transmission DIC with a 1.32 NA oil, the fine striae are nicely resolved. According to the information sheet which comes with the slide they have a periodicity of 19 striae per 10 microns and this agrees with the periodicty as observed. Using a 0.60 NA air objective, the striae are barely resolved but this is, I suppose, because of the lower numerical aperture. Using the darkfield reflection objective,.which has a low numerical aperture, the striae are, as expected, not resolved. However, there is a complex gross pattern, symmetric with the frustule form and within it, seen in this image but not seen in the DIC images. The pattern is composed of prismatic colors, as if being produced by reflection gratings dispersing the light. Of the eight different diatoms on the test slide only Pleurosigma shows this prismatic color pattern in such a striking manner ( only some color can be seen in some of the others) although all have striae periodicities similar to that of Pleurosigma. I have checked two different test slides and both show the pattern (only for Pleurosigma). The pattern appears only in reflection; It is not seen in bright field transmission or, as I have described, in transmisssion DIC images. The pattern or rather something homologous to it does shows up in the laser interference micrcoscope image (in this the striae are also resolved; the pattern is not a pattern of the pores), The image from the laser microscope does , however, include a reflection contrast mechanism. The question I ask is: what is the source of the striking prismatic color pattern in Pleurosigma that I obtain using darkfield reflection? Both the test slide and dark field reflection micrscopy are commonly available so certainly this image with its strong prismatic pattern has been seen before. Does anyone know or have a suggestion? Thanks Phil Brody From frithjof@PI.NETTue Apr 29 14:43:32 1997 Date: Wed, 21 Aug 1996 21:06:03 PDT From: "F.A.S. Sterrenburg" To: Multiple recipients of list DIATOM-L Subject: Re: Pleurosigma angulatum For SEM (and LM!) images of Pleurosigma angulatum, including the characters separating it from at first sight similar - but wholly different - species plus its synonymization with "P. quadratum" and "P. aestuarii" see: Sterrenburg, F.A.S. (1991). Studies on the Genera Gyrosigma and Pleurosigma (Bacillariophyceae). The typus generis of Pleurosigma, some presumed varieties and imitative species. Botanica marina vol. 34, pp 561-573. Unfortunately, my offprints are long "sold out". P. strigosum together with other confused entities including P. latum and P. acutum is in the pipeline, but if anybody would like to have a pre-publication print of SEM pix of these beasts, just yell and I'll run off an extra copy when I can force myself to withdraw into the darkroom and muck about with the horrible chore of printing... By the way, it's perhaps not easily believed (but really true) that it is possible to obtain clear and separate images of the very fine "slits" that constitute the EXTERNAL areolar openings and the roundish much larger INTERNAL areolar openings in many Gyros and Pleuros with the humble LM (!). If anybody is interested I can tell the little bit of trickery that's required. Incident light at the highest power should give different images depending on whether the inside, or the outside, of the valve is "up". I have not seen this myself (I have no incident light set-up capable of handling an oil-immersion objective) and would like to hear whether this is indeed the case. Apparently you've all returned from your various vacationary escapades, silence has long reigned on the Diatom List!