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Natural Science Forum / Physics / Optics / January 2007More on A.pellucida
Microscope Group, I wish to present a light microscope image (LM) of A. pellucida, with a determination of the distance between the striae. The re- sult is compared with those obtained on various A. pellucida frustules imaged by field emission microscopy (FESEM). Dr. G. Gaugler, USA did this work and his SEM images are presented at his site, www.photoweb.net/micro/ . My LM image is at www.mikroskopie-forum.de/read.php?2,25394,25394#msg-25394 . Pertinent details are as follows. The sample for the FESEM images was specially prepared by Mr. Klaus Kemp, UK. It con- sisted of nine A.pellucida frustules placed on top of a coverglass in free air, no mounting medium was used. Dr. Gaugler used Palladium to sputter the sample before doing SEM microscopy. The frustule used for the LM picture is on one of Mr. Kemp's test slides. It is mounted the conventional way in Zrax and there is one A. pellucida diatom together with samples of other species on the test slide. All of the samples, SEM and LM show "inner views" of the diatom, that is views with the concave side poining up. Mr. Klaus Kemp's site is at www.diatoms.co.uk/ . The LM picture was obtained using an Olympus BX51 micros- cope using a planAPO objective 100x/1.35 using their universal condenser with top oil lens and double immersion. DIC was used to enhance contrast and the image was projected with a 2.5 Olympus eyepiece upon the CCD of a Pentax *ist DS digital, single lens reflex camera. The vertical dimension of the CCD is 1.57 cm. JPG format was used with 2000 pixels along the vertical. Illumination was by electronic flash and image processing was limited to adjustment by Auto Levels, follow- ed by Grayscale in Adobe Photoshop. No other processing was done. The image horizontal extent was shortened, the vertical dimension preserved at 1980 pixels from the full 2000 and that is what is shown at www.mikroskopie-forum.de/read.php?2,25394,25394#msg-25394 . While I have been able to resolve the striae I was unable to re- solve the striae into punctae (dots) using my light microscope. Since I do not have a stage micrometer I determined the distance between the striae as follows: I used the magnification of the objective of 100x and that of the photoeyepiece of 2.5x to calculate a total magnification of 250 x. Counting the number of striae in 150 pixels as 18, one obtains 150/18 = 8.33 pixels per stria. Each pixel represents 1.57cm/2000 = 7.85 micrometers on the CCD and with a magnification of 250 this corresponds for 8 pixels to 8.33 x 7.85/250 = 0.26 micrometers for the distance between striae. Dr. Gary Gaugler used his Zeiss Supra 55VP FESEM instrument to image and measure important distance parameters on the samples for SEM microscopy. One needs to look at the images at his site www.photoweb.net/micro/ to appreciate the signifi- cance of the results. An overview showing a bird's eye view of the samples is given under the thumb nail Apellucida-specimens. Currently there are details for five of the nine A. pellucida dia- toms posted. This allows a comparison of variability between the parameters for individuals belonging to the same species. It will be seen that the variability of distances between striae and between the pores of a stria (dots) amounts to as much as 9 %. This is probably more than has generally been expected. Please refer to Apellucida-50kx-2.jpg, Apellucida-50kx-D.jpg, Apellucida-50kx-E.jpg, Apellucida-50kx-F.jpg and Apellucida-50kx-I.jpg for images and measurements. For these diatoms the following results apply (all in micrometers): -2 distance between striae 0.289 distance between dots 0.229 -D 0.272 0.176 -E 0.301 0.283 -F 0.284 0.181 -G 0.275 0.161. For LM from above: LM 0.26 ........................... Even within the same diatom distances between pores may vary. This is best appreciated at high magnifications. Also note the thickness measurements of the frustule's bottom (Apellucida- 100kx-3). These were made at 25 degrees inclination. If a frustule is inclined, the bottom layer may obstruct a fair portion of the pore and have an effect upon the LM picture. The measured thickness of the bottom was about 0.09 micrometers at the center and less at the ends. It will also be noted that some pores are plugged up, which might make LM observation of details more difficult on some diatoms. Particularly diatom E shows many plugged pores. Now the question may be raised whether separation of the pores within a stria, can be done by LM. I do not believe that I can do it with the setup described above. I might be possible to do better with a projection magnification of 5x or more. However Peter Hoebel at www.mikroskopie-forum.de/read.php?2,25384,25384#msg-25384 presents such a LM picture with dots separated for A. pellucida. He had used a near UV LED and averaged 1000 images to obtain the separate dots. Details can be found at the above link. Regards G.R. You shouldn't use DIC for ultimate resolution. Ren?. > Now the question may be raised whether separation of the pores > within a stria, can be done by LM. I do not believe that I can do it > with the setup described above. I might be possible to do better > with a projection magnification of 5x or more. Rene, You are of course correct, in principle. The problem with A. pellucida however is not only the one posed by resolution, but also and very importantly, by contrast. With insufficient contrast one is unable to see the object and with insufficient resolution one will not see details. One must find a compromise. In my case the DIC image was far superior to bright field or dark field. The imaging of an object like A. pellucida ist not only a question of resolution, but of the objects innate contrast and of the modulation transfer function of the entire setup. The latter involves more than the NA of the objective. Why dont you point us to your images and describe the details under which they were taken! G.R. You shouldn't use DIC for ultimate resolution. René. > Now the question may be raised whether separation of the pores > within a stria, can be done by LM. I do not believe that I can do it > with the setup described above. I might be possible to do better > with a projection magnification of 5x or more. Discussion of KKemp material and imaging of Ap has been going on for years on the yahoo microscope discussion group, you can find my images overthere. It is similar to your result, but with an old microscope with 15W Tungsten/420nm bluefilter, annular illumination with a modified BF condenser and a 70 year old achromatic Beck 1.3 NA objective. Many others have done the same exercise with better results (then mine) by playing with oblique illumination. Indeed, lack of contrast is the bottleneck, caused by 'thin frustules' (as KKemp calls it), ie little silicification between pores in the striae. The (inter)striae themselves are heavily silicified bars, therefore reasonably easy to image compared to the pores. René. Rene, Well, thank you for your reply and the reference to the Yahoo group. I am aware of the long history surrounding the imaging of A. pellucida and the many attempts, more or less success- ful to resolve striae or dots. What has been missing so far was a comparison of a good light microscopy image with superb and quantitatively precise SEM images. These were obtained by Dr. G. Gaugler and presented in the current thread. These images are unique in several respects: They show measurement of important parameters on the frustules of A. pellucida, not only on one, but on several specimens. This allows the comparison of these values on samples of the SAME origin and of the same size. It also shows a light microscope image of A. pel- lucida from the SAME source and of comparable size. I think this makes for very interesting reading for those inter- ested in the subject and is not meant to be imply that others have not obtained good pictures of A. pellucida. In fact I do point to a very detailed image of A. pellucida by a link. GR. Discussion of KKemp material and imaging of Ap has been going on for years on the yahoo microscope discussion group, you can find my images overthere. It is similar to your result, but with an old microscope with 15W Tungsten/420nm bluefilter, annular illumination with a modified BF condenser and a 70 year old achromatic Beck 1.3 NA objective. Many others have done the same exercise with better results (then mine) by playing with oblique illumination. Indeed, lack of contrast is the bottleneck, caused by 'thin frustules' (as KKemp calls it), ie little silicification between pores in the striae. The (inter)striae themselves are heavily silicified bars, therefore reasonably easy to image compared to the pores. René. As Gary clearly showed with his images, one should use SEM for AP. Regarding resolving AP with LM, what has been achieved at the beginning of last century is all we will be able to resolve using LM until someone comes up with a greatly improved LM setup. (Well, I am not considering recent progress in looking at self-luminous particles at the moment.) The good old times proofs once again to be a great source for knowledge ;-) But, as usual, you are correct. DIC is not the best choice for AP. COL is the champ. DF is recommended. With lambda = 420nm, one should be able to resolve AP with almost any reasonable scope and a decent DF condenser. (Of course, both oiled to the slide.) Another question, do you use a water immersion lens? If yes, do you have the one that works with a coverglass or without? If I remember correctly, you use a LOMO at home and a Zeiss at work. - I am looking for one for my Ortholux. Cheers, Gregor You shouldn't use DIC for ultimate resolution. René. > Now the question may be raised whether separation of the pores > within a stria, can be done by LM. I do not believe that I can do it > with the setup described above. I might be possible to do better > with a projection magnification of 5x or more. Hi Gregor, I have used a CZJ for this stuff. Yes, I also have the Lomo water immersions, particularly the 70/1.23 apo is ok for diatom work with COL. I managed to get it working for DF with the CZJ aplanatic 1.4 condenser, by inserting the DF stop just below the frontlens (not in the filterholder!). Haven't tried it with Ap though. Yes, it is the one with correction collar 0.11-0.22. It's an oldy, contrast in BF is not great, I prefer to use the achro 85/1.0 WI for normal work. I know Lomo made a new batch of the 70x in 2004, not sure whether they managed to improve the contrast, but for a couple of 100$ (new!) they are great value for money. René. gto schreef: > As Gary clearly showed with his images, one should use SEM for AP. Regarding > resolving AP with LM, what has been achieved at the beginning of last [quoted text clipped - 25 lines] > > with the setup described above. I might be possible to do better > > with a projection magnification of 5x or more. > As Gary clearly showed with his images, one should use SEM for AP. > Regarding resolving AP with LM, what has been achieved at the beginning of [quoted text clipped - 16 lines] > > Gregor DF reduces resolution over bright field or DIC. With a DF system the NA of the objective and condenser is quite low in comparision to bright field and DIC. NA is resolution, resolution is NA. Thats why DF is yesterday's news. Thanks, Kevin Cunningham SMS > You shouldn't use DIC for ultimate resolution. > [quoted text clipped - 4 lines] >> with the setup described above. I might be possible to do better >> with a projection magnification of 5x or more. Is this your intuition or where did you get this from?? R. > DF reduces resolution over bright field or DIC. With a DF system the NA of > the objective and condenser is quite low in comparision to bright field and [quoted text clipped - 4 lines] > Kevin Cunningham > SMS > Is this your intuition or where did you get this from?? > R. Dr. Abbe, 1872. Zernicke's paper and talk he delivered in 1938 at the Carl Zeiss Wercke, Jena. Publication by Dr. Nomarski. Publication by Dr. Francon. Training from Zeiss, Nikon, Olympus and Leica. Working in the field since the late '70's. In that period I sold 2 DF condensers and I accidentally sold 6. Thats in 35 years. I've never sold a high power DF objective to a researcher. I've had the honor of working at the Rockefeller Inst., Harvard, Yale, Stanford, US-C, UG-A, Down State Med., Emory, etc. Rene, you do a lot of good for this group, your questions are terrific and your responses are on point, usually. This is not one of the usual times. Kevin Cunningham SMS >> DF reduces resolution over bright field or DIC. With a DF system the NA >> of [quoted text clipped - 7 lines] >> Kevin Cunningham >> SMS Kevin, yes, I might have been too sharp in this discussion, might refllect my state of mind these months. Anyway, like for like, a 40x BF or in DF shouldn't suffer in resolution. I achieved DF with a 70x objective with the full aperture of 1.23 by relatively simple means (a patched aplanatic 1.4 condenser). The view in DF is *very* detailed, see my yahoo photo album for an example. DF is just not very practical for most specimen, especially not in high NA. And, I agree with Gregor, even for diatoms I prefer COL over DF. René. > DF reduces resolution over bright field or DIC. With a DF system the NA > of the objective and condenser is quite low in comparision to bright field > and DIC. NA is resolution, resolution is NA. Thats why DF is yesterday's > news. Kevin: Yes, DF reduces resolution over BF and DIC due to the closed iris located inside the oil immersion objective. We both know that it must be closed to around 1.10 but not more than 1.20 in order to obtain decent darkfield illumination. The condenser for DF does not lower resolution since any decent DF condenser using oil immersion provides a light cone that illuminates between NA 1.20 to 1.40. Hence the resolution decrease is purely due to the objective's iris (or stop). However, the problem to distinguish certain, tiny diatom structures, is not just a lack of resolution; it is the availability of sufficient contrast. In DF, I can detect particles that are way below the resolution limit of BF and DIC. But these particles appear in DF larger than their physical dimensions. It is clear from diffraction theory of light that when the size of a particle or feature falls below the minimal particle size that can be resolved with a given optical setup, the size of the image of said particle is no longer smaller than the minimal particle size, but its contribution to the image contrast decreases as the diameter of the subresolution particle gets smaller. DF is recommended for this application in circles that study diatoms intensively using LM, while DIC isn't due to its image artifacts that make particle/feature interpretations difficult. Amateur microscopists tend to use DIC and PC to produce pretty pictures. Personally, I prefer COL for diatoms, which happens to decrease contrast when compared to DF but, of course, offers the full resolution of the objective/condenser combination. Cheers, Gregor You shouldn't use DIC for ultimate resolution. René. > Now the question may be raised whether separation of the pores > within a stria, can be done by LM. I do not believe that I can do it > with the setup described above. I might be possible to do better > with a projection magnification of 5x or more. Let me disagree with Rene. Actually if you look at the light path diagrams and the formal and informal papers on DIC it actually increases resolution by a tiny amount. This is caused by the two light path phenominon that creates DIC. Thanks, Kevin Cunningham SMS Not my experience. Have also never seen A.p. resolved to dots with DIC, and especially diatoms are suposed to be ideal. Check the MTF's http://www.olympusmicro.com/primer/java/mtf/contrasttechniques/index.html What (in)formal papers are you talking about? René. Kevin Cunningham schreef: > You shouldn't use DIC for ultimate resolution. > [quoted text clipped - 14 lines] > Kevin Cunningham > SMS Not my experience. Have also never seen A.p. resolved to dots with DIC, and especially diatoms are suposed to be ideal. Check the MTF's http://www.olympusmicro.com/primer/java/mtf/contrasttechniques/index.html What (in)formal papers are you talking about? René. ______________________________________________________-- Rene, An informal publication can be a talk or a letter. Kevin Cunningham SMS Kevin Cunningham schreef: > You shouldn't use DIC for ultimate resolution. > [quoted text clipped - 15 lines] > Kevin Cunningham > SMS Hi Thanks for sharing your A. pellucida results and as you remark the comparison of SEM and LM photos of the same material is valuable. As an aside, I think the work of Spitta to me as a hobbyist is jaw dropping if not humbling. His book 'Microscopy' 1909 2nd edition has very well resolved photos of A. pellucida to 'dots' with blue light and convincingly resolved to 'dots' with green light, all of high contrast. These photos are presented in conjunction with his chapter devoted to using this and other diatom species and Abbe test plate as test objects. His earlier book 'Photomicrography' 1899 has two text pages on photographing A. pellucida and a beautiful photo of this diatom resolved to striae. Although it's his photo of another test diatom P. angulatum in 'Microscopy' that blows me away, not a tricky one to resolve but it's a beautifully crisp photo of this diatom with internal detail. with regards David Hallo, Thank so much for for your constructive contribution and the reference to Edmund J. Spittas work. I have looked at Google Books and found "Photomicrography" of 1899 I downloaded it and will look at the photos. I could not locate the other work "Microscopy" of 1909. The existence of both was unknown to me. Regarding the ability of some observers to resolve A. pellucida into dots while others were not able to do so, may have two causes. One is that A. pellucida might occur with fine or coarse structure. There is strong variability of the parameters for samples from the same site. There may be even stronger variability for samples from different locations. This aspect is something which Dr. Gaugler will attempt to clarify in the near future by using some special samples from Mr. K. Kemp. The other cause may have to do with what is nowadays called the modulation transfer function. Since modern images are taken on 36 mm film or on CCD or CMOS sensors, the size of the a resolved element thrown upon the film or sensor compared to the film grain and pixel size is a most important parameter. It is desirable to have as large as possible a magnification upon the sensitive surface as is practical and combine this with as small as practical a grain size on film or of a pixel. The latter of course needs to have a certain size to achieve a good S/N ratio. Nowadays with 36 mm film and small CCD or CMOS sizes there are limits. In the old days when microscopists used very large photographic plates and large projection distances these limits were more favorable. These two causes may have a bearing while microscopists of over one hundred years ago, were able to resolve A. pelluci- da into dots. Greetings GR. > Hi > [quoted text clipped - 19 lines] > > David No Spam, from my memory the first image I have is in Neuhauss, Lehrbuch der Mikrophotographie 1907. I would have to find it again, but it was made with 'Cadmium light', 275nm I believe was mentioned. Search for Neuhauss in the yahoo microscope archives and you will find additional info on earlier and later work on A.p. It is > desirable to have as large as possible a magnification upon > the sensitive surface as is practical and combine this with as [quoted text clipped - 6 lines] > very large photographic plates and large projection distances > these limits were more favorable. The more empty magnification, the lower the contrast. 36mm fim and 3Mp cameras are more then adequate. The problem with A.p. for test specimen is it's variability in silification (or so I believe) and possibly variation in RI of mounting media. Even slight differences in refraction index between batches will have tremendous impact on contrast. This all makes a diatom test slide a personal subjective test. Last comment, the use of contrast enhancement techniques like oblique/single sideband/DIC/phase contrast makes it even more subjective. Only brightfield will show the quality of an optical system. As you say, the resolution is there, no problem, but without contrast (by the optical system OR the test specimen), you cannot see it. Oh yes, and be careful with taking sizes from SEM, in respect of tilted valves, also SEM calibration. I wouldn't trust my own SEM pics from A.p. within 10%. I'm sure Gary's FESEM would be better in that respect. Ren?. Hello again Rene, You wrote: > ..from my memory the first image I have is in Neuhauss, Lehrbuch > der Mikrophotographie 1907. I would have to find it again, but it was > made with 'Cadmium light', 275nm I believe was mentioned. > Search for Neuhauss in the yahoo microscope archives While working on the prototype archive website (some time back!), I often wondered if the image shown here: http://204.3.164.54/images/Pic_NH_Ap.htm is actually Neuhauss' A.pellucida. Best regards, Keith Rene, You stated: >The more empty magnification, the lower the contrast. 36mm fim and 3Mp cameras are more then adequate.> This statement is incorrect. Contrast is affected by the ratio of image detail relative to grain size or pixel size. That is: for a given size Y of the resolved element on a given sensitive surface, the contrast is enhanced by increasing Y by a factor n to n x Y, where n is larger than 1. At the same time the area under the image of the resolved element increases by (n x Y)**2, while the noise increases by n x Y. This results in an improve- ment of S/N by a factor of n x Y. This result would even be correct up to a certain factor of n at which resolution would be satisfactory for the average eye, that is into the range of empty magnification. For those who believe more in experiments than in theory I have done the experiment: I produced an image of A. pellucida by projecting the intermediate image of this diatom upon the CCD of my *ist Pentax (n=1) and was UNable to resolve the striae. I then projected the intermediate image through a 2.5 projection lens (n=2.5)and could resolve the striae perfectly well as shown at http://www.mikroskopie-forum.de/read.php?2,25394,25394#msg-25394 . To forestall any questions: the intermediate image in my Olympus scope is fully corrected and does not require additon of a projection lens to take full advantage of the resolution the objective is capable of. It would be nice, would it not be, if you presented the dotted image from Neuhauss' book? I have tried and I can't get a hold of this work and it would certainly be of interest to many to be able to see it. GR. >Last comment, the use of contrast enhancement techniques like >oblique/single sideband/DIC/phase contrast makes it even more [quoted text clipped - 8 lines] > >René. A coated specimen will increase LM contrast. Done right, you won't see the coating. Au is not good. Au/Pd, Ir, Pt or Pd are IMO better. The surfaces are coated thicker than the pores due to plasma flux variations (more difficult to get into the holes the same as the flat surfaces). The tilted specimens were optimal for thickness measurement. These specimens were the internal (concave) view rather than the external (convex) view. AP is like a W-shaped specimen with the middle of each side flat on the cover slip. So, if one considers one side, it is like a U where the bottom of the U is flat on the cover slip. This allows for contrast variation between the slip and specimen. Tricky but doable. I use Geller MRS-5 microscopy calibration standard but only up to 500KX. The standard has patterns at 80nm (+- 1nm) on up to 3um. Most of my work is between 5KX and 300KX. Since calibration correction is electronic, it is easy to check and tweak if necessary. Typical calibration limits are around +- 1% of FS at high mag and about 2-4% at low mag (due to inability to put the cursors exactly on the edges). So, this is mostly an operator problem. gg Kiss French. Drink California. Hi Gary, I've tried goldsputtering only, in different thickness on sectors of the same coverglass. A thin layer increased visibility of the striae in BF, no sign of the pores. In annular illumination (COL), pores were visible in the UNcoated part of the slide, not in the coated parts. Not sure why, but I can think of a couple of reasons to do with annular illumination. It was an experiment after I saw metallized slides of Ap, I think aluminium shadowcasts. That created an image that resembled phasecontrast. The striae were visible in BF, much better then my goldsputtered specimen, no doubt because of the shadowing technique used. No sign of the pores, alas. René.. >Hi Gary, > [quoted text clipped - 11 lines] > >René.. Au is good for low mag images only. Even when ideally laid down, and not thick, it forms a spider web of Au rather than an amorphous layer. The next option is Au/Pd which if done right will work up to about 200KX. At this point, the coating becomes quite visible as mounds. High vacuum low current coating with Pt, Ir or Pd produces extremely fine grain results that are not seen up to at least 600KX. Probably the best coating is Cr plasma deposition. However, Cr oxidizes rapidly and thus, tends to be useful only for a session. gg Kiss French. Drink California. I was incorrect re identification of the A.pellucida image referenced in a previous post, i.e. it may have been by Neuhauss. On going back over some old notes, it appears that this image is from W.B.Carpenter's book "The Microscope and its Revelations" published in 1891. It is accredited to Prof. Van Heurck, a rather well known microscopist! This image most probably originated with Martin Mauch at : www.baertierchen.de Regardless, the definition of the dots within the right-hand section of this mount is startling - in fact it's hard to believe. It would be real interesting if anyone can dig out information on the equipment and illumination technique used by Van Heurck. My notes indicate a Zeiss objective (presumably 100x) with NA 1.4 - a very nice piece of glass (hand ground) back in the 1890s. -Keith Not entirely sure, might have been the 1.6 NA (!!) exotic immersion one, something like monobroomnaphtalene immersion directly on an uncovered specimen. Will dig out the Neuhauss book tonight. René. > I was incorrect re identification of the A.pellucida image referenced in a > previous post, i.e. it may have been by Neuhauss. [quoted text clipped - 15 lines] > > -Keith I have found some more information in an article by Trivelli and Lincke (1931): Fraenkel and Pfeiffer showed photomicrographs of the Ap dots first in 1889 (according to Neuhauss). In 1890 van Heurck and 1893 Zettnow succeeded also with use of the new Zeiss apo 1.6NA, as said before a monobrome naphtalene immersion lens. Zettnow was able to count dots to 52/10um. He used a filter of cuppramonium solution and iodine (ie violet). The diatom was mounted in mercury iodide, and as said before the 1.6NA lens was a monobrome naphtalene immersion lens. Neuhauss in 1907 was slightly contradictonary as he mentioned van Heurck and Zettnow used the same lens, but a Realgar mounted slide and blue light (violet would be absorbed by Realgar). I guess both will work. Watch this remark on van Heurck's images (Neuhauss p 264, translated): van Heurck tried to resolve the striae (Langstreifung) and pores (Querstreifung) (1886). However, he masked in many images along the edge of the diatom. Therefore, it was impossible to differentiate the 'Querstreifung' from diffraction lines [that are optical artefacts from the edge of the valve]. Neuhauss reproduced a truly magnificent image from Ap, made with 275nm UV-light in 1907. As far as I can make out this image originate from the Zeiss labs where the new UV monochromat was developed, a glycerin immersion 1.25NA. If Keith contacts me I can send him a couple of images from the page of this book so that everybody could see it on his website, as I have no webspace to show it on. René. > Neuhauss reproduced a truly magnificent image from Ap, made with 275nm > UV-light in 1907. > .. > If Keith contacts me I can send him a couple of images from the page of > this book > so that everybody could see it on his website... Courtesy of Rene, here they are: http://www.kscitech.com/Pic_04.1.htm http://www.kscitech.com/Pic_04.2.htm Enjoy!! >> Neuhauss reproduced a truly magnificent image from Ap, made with 275nm >> UV-light in 1907. [quoted text clipped - 8 lines] > >Enjoy!! If I had images like these with my SEM I would be very upset. The wavelength of an electron beam is less than .5A and is obviously superior to LM. Duh. I know this. The same problems occur with SEM as with LM....astigmatism and distortion. So, LM and SEM have limits. With LM, the variables are known. With SEM, the variables are mostly confined to the column. Thus, much of perfection is lost in trying to get a perfect beam. Not possible. But Zeiss and others make huge investments in this area. gg Kiss French. Drink California. Dear Keith, Yes! The image is by Dr. Henri van Heurck. The image and des- criptive details can be seen at http://www.baertierchen.de/archiv.html , which is the site of the Baertierchen microscopist Martin Mach of Munich, Germany. Mr. Martin Mach gives Willliam B. Carpenter "The Microscope" 1891, as the source of this image of A. pellucida. Actually William B. Carpenter's "The Microscope and its Revelations" is a series of books with this title which contain important work in the fields of microscopy. The mentioned volume is edited by W.H. Dallinger, since W.Carpenter had died previous to the publication of this volume. The book mentioned by Mr. Mach can be found at the Google book site under http://books.google.com/books?vid=OCLC00999815&id=YIYAAAAAMAAJ&pg=RA1-PR10&l pg=RA1-PR10&dq=William+carpenter++microscopy+1891#PRA1-PR21,M1 and may be downloaded as a pdf file without charge. I shall briefly describe what I found giving the page numbers which the reader can enter on the bottom of the pdf reader. This will save time because the search function does not work on this type of pdf file. Choose p. 13 on the counter on the lowest line of the Adobe pdf reader and you will see the Frontispiece. Fig. 5 of the frontispiece shows: "Amphipleura pellucida x 1860 diams., by apochromatic 1/8 1.4 N.A. illuminated by a very oblique pencil in one azimuth along the valve." Choose p. 25 and you will get to read about Plate XI which contains three images of A. pellucida at magnifications of 2000x, 3000x and 2600x. The description goes into very interesting details of what Dr. van Heurck thought was the structure of the valves of this diatom. The text is too extensive to reproduce here. Unfortunately I could not find Plate XI in the Google pdf file. Only Plate I seems to have been scanned. There are additional descriptions and remarks regarding the microscopy of A. pellucida. You will find them on pp. 90, 106, 116, 260 and 551, again using the search function at the bottom of the Adobe reader. Page 551 makes very interesting statements regarding the great varia- bility of the structure of A. pellucida which can present with easily resolved valve details or details which are extremely difficult to resolve. Again I must refer to the original text. These statements, if supported by modern data, may have an important bearing on the ability to resolve this diatom into "dots" using light microscopy. Dr. Gary Gaugler will most likely explore the variability of A. pellucida using his FESEM. Mr. Klaus Kemp (UK) will supply samples. Finally I wish to point out that my image of A. pellucida (see the link in my original post) was obtained using a magnification of 250x (100x from the objective, 2.5x from the projection lens), while Dr. von Heurck used magnifications of 1800x to 3000x. GR. > I was incorrect re identification of the A.pellucida image referenced in a > previous post, i.e. it may have been by Neuhauss. [quoted text clipped - 16 lines] > > -Keith Hi NoSpam, can you tell me where exactly this is located on the Baertierchen site?? Also, you seem to have been lucky to be able to download Spitta's and Carpenter/Dallinger's book, I could only get snippets which were useless. Would you mind sending them to www.yousendit.com, so that we could download them from there? In return some more comments on your messages: You stated: >The more empty magnification, the lower the contrast. 36mm fim and >3Mp cameras are more then adequate. I stand with this conclusion. A 100x 1.4NA lens with 20mm secundary image (such as seen with a 10x eyepiece with fieldnumber 20) only needs 2000 pixels along the middle line for all detail for all to be resolved (ie 2 pixels for 0.2 um). If you fix a 36mm frame inside the field of view, you will need less pixels, but add some to compensate for the Bayer filter in the digicam, then you will have enough with 3Mp. I agree with you, increase pixelSIZE (but not total amount of pixels) and with corresponding extra magnification will reduce noise, but that is irrelevant in this discussion. > I produced an image of A. pellucida by projecting the >intermediate image of this diatom upon the CCD of my *ist Pentax (n=1) >and was UNable to resolve the striae. I then projected the intermediate >image through a 2.5 projection lens (n=2.5)and could resolve the striae >perfectly well as shown at >http://www.mikroskopie-forum.de/read.php?2,25394,25394#msg-25394 . I'm not sure exactly where your exp did go wrong. By 'direct projection' you mean you simply took out the projection lens/eyepiece and adjusted focus? That would increase the effective tubelength a couple of cm from the original 160mm it was designed for. This would create spherical abberation, ie unsharpness. Your CCD should be at the place where the secundary image is placed (where your eyepiece 'picks it up'), somewhere a cm or so below the rim of the tube). > Finally I wish to point out that my image of A. pellucida (see > the link in my original post) was obtained using a magnification > of 250x (100x from the objective, 2.5x from the projection lens), > while Dr. von Heurck used magnifications of 1800x to 3000x. That would make your specimen a very big one ;-) René. ps, keep it up, you are learning a lot. Rene, I am beginning to have doubts regarding your competence. Your recommendation that I should keep it up because I will be learning a lot is entirely misplaced as the following lines will clearly show. From your recent post: >"rene" <renevanwezel@hotmail.com> wrote in message >news:1169727731.244566.22640@k78g2000cwa.googlegroups.com... >Hi NoSpam, can you tell me where exactly this is located on the >Baertierchen site?? The site where you can find the article by Martin Mach showing the van Heurck A. pellucida picture is under http://www.baertierchen.de/archiv.html where there is the telling title: Februar 2004: Feine Strukturen. From your recent post: >Also, you seem to have been lucky to be able to download Spitta's and >Carpenter/Dallinger's book, I could only get snippets which were >useless. Would you mind sending them to www.yousendit.com, so that >we could download them from there? The Carpenter/Dallinger book can be found exactly where I said it can be found. Now you will know that long, that is several line long links, are not properly handled in newsnet messages. It is therefore unproductive to just click on the address as given. What you need to do is copy the entire link to a text editor, like Notepad. You do this by "copy" the link in sections, line by line, and reconstructing the address by "paste" in your text editor. Then you copy the entire address so obtained into your browser and VOILA, there is the book. (To be entirely sure I repeated this process just now and it works, like it always does.) I do not know how else I can make it easy to find this source. Believe me, I spent a lot more time finding it and then the page numbers, than it would have taken you to follow the described process! So go ahead, download the book, look at the pages I quoted and you will be able to read the text and see the picture. You are quoting from a prior text of yours: >>The more empty magnification, the lower the contrast. 36mm fim and >>3Mp cameras are more then adequate. Your addition to that text is: >I stand with this conclusion. A 100x 1.4NA lens with 20mm secundary >image (such as seen with a 10x eyepiece with fieldnumber 20) only needs [quoted text clipped - 5 lines] >and with corresponding extra magnification will reduce noise, but that >is irrelevant in this discussion. You seem confused by the issue of contrast. I will say it one more, final time: contrast increases when the target's image spatial frquencies move to the left on the MTF curve. The only way for this to happen at a fixed pixel or grain size is to decrease the spatial frequency, that is to increase the size of the image. What may lead you astray is the custom of presenting the MTF curve for targets with 100 % contrast. The x% which are deemed sufficient for detection are then at a certain position on the hori- zontal axis. BUT, when the contrast of the target is less than 100%, this point moves to the left and for small contrast it moves a lot to the left. Your quote from a prior post of mine: >>I produced an image of A. pellucida by projecting the >>intermediate image of this diatom upon the CCD of my *ist Pentax (n=1) >>and was UNable to resolve the striae. I then projected the intermediate >>image through a 2.5 projection lens (n=2.5)and could resolve the striae >>perfectly well as shown at >>http://www.mikroskopie-forum.de/read.php?2,25394,25394#msg-25394 . Your response to that quote: >I'm not sure exactly where your exp did go wrong. By 'direct >projection' you mean you simply took out the projection lens/eyepiece [quoted text clipped - 3 lines] >place where the secundary image is placed (where your eyepiece 'picks >it up'), somewhere a cm or so below the rim of the tube). You say you are not sure where my experiment went wrong. Well, it just simply did not go wrong! The image was taken by placing the CCD of the mentioned camera to exactly where the intermediate image is located and taking the picture. The picture itself was only 100x (the magnification of the objective) x 70 micrometers( the length of the diatom), or 0.7 cm tall. This is smaller than the vertical dimension of the CCD leading to a loss of contrast and inability to see the striae by the mechanism I have described above. It may well be that you did not read my notes well. In these notes it is stated that I am using an Olympus BX51, a microscope with infinity optics and a fully corrected intermediate image. This intermediate image is accessible above the dovetail connection on the trinocular leading to the camera port. You quote a prior post of mine: >> Finally I wish to point out that my image of A. pellucida (see >> the link in my original post) was obtained using a magnification >> of 250x (100x from the objective, 2.5x from the projection lens), >> while Dr. von Heurck used magnifications of 1800x to 3000x. Your response to it: >That would make your specimen a very big one ;-) Please see the arithmetic I have gone through above, showing that my diatom was of regular dimension and not a "very big one". From your recent post: >ps, keep it up, you are learning a lot. Rene, please refer to the first paragraph in my response. G.R. GR, I still find you lacking on all points except for the Baertierchen info. >You seem confused by the issue of contrast. I will say it > one more, final time: contrast increases when the target's > image spatial frquencies move to the left on the MTF curve. > The only way for this to happen at a fixed pixel or grain size is > to decrease the spatial frequency, that is to increase the size of > the image. So what you are suggesting is that if we enlarge the diatom, then we are able to see more. Duh. René. Hello, Rene and GR: I am getting headache from reading this part of the thread. I am convinced that both of you know how to succeed in photomicrography, but this part of the thread entered the state of verbal jousting and looping. I can only hope that both agree on the following basic summary: 1) Smallest resolvable distance 'd' (diameter of Airy disk) is given by d = 0.61*lambda/NA(obj) assuming we are using a NA(cond) of 1.4 and hence get no limitation due to insufficient illumination. 2) The size of the smallest resolvable distance 'D' in the INTERMEDIATE image (also referred to as real image) is given by D = d * magnification of objective 3) To resolve a feature of the size 'D', one needs a pixel size of D/2 (Nyquist theorem [1]) to avoid undersampling. But remember, this is the ACTIVE pixel size and not the PHYSICAL pixel size in case of a Bayer filter (see point 4). Let's call this the required pixel size 'r'. 4) The required pixel size for a Bayer filter depends now on the wavelength. If blue light is used the active pixel size is 2 times the physical pixel size. For green, it is square-root 2 times the physical pixel size. Let's call a = D/2 to be the active pixel size. 5) To be able to resolve the entire image, the active pixel size must be smaller or equal to the required pixel size: a <= r Now let's look at an example: For a Nikon D70 (I don't know the Pentax that GR is using), the active pixel size 'a' is a HUGE 12um when we use primarily blue light (no green). This is much too big to resolve the fine structure with the CCD of a D70 located in the intermediate image plane (direct projection). For an objective with NA 1.40 with lambda = 425nm, d = 0.185 and D = 18.5 um. This gives r = 9.25 um, which is smaller than the 12um provided by the D70 for blue light. When using a 2x projection ocular (or relay lens) the image information is spread out onto a larger area and hence we can multiply 18.5 um by two = 37 um. Now, r = 18.5um, which is easily captured by the image sensor's pixels with a PHYSICAL size of 6um and using blue light. Now that would explain one aspect of the fact that GR cannot resolve AP when using direct projection but by using a relay lens. Another important aspect is, of course, the fact that using the appropriate relay lens, one gets a complete correction for aberrations when using older gears. Cheers, Gregor [1] http://en.wikipedia.org/wiki/Nyquist-Shannon_sampling_theorem GR, I still find you lacking on all points except for the Baertierchen info. >You seem confused by the issue of contrast. I will say it > one more, final time: contrast increases when the target's > image spatial frquencies move to the left on the MTF curve. > The only way for this to happen at a fixed pixel or grain size is > to decrease the spatial frequency, that is to increase the size of > the image. So what you are suggesting is that if we enlarge the diatom, then we are able to see more. Duh. René. Please change point 4 into: 4) The pixel size for a Bayer filter depends now on the wavelength. If blue light is used, the active pixel size would be 2 times the physical pixel size. For green, it is square-root of 2 times the physical pixel size. Let's call the active pixel size 'a'. Hallo, I have now had time to follow up on the very valuable suggestion to look at Edmund J. Spitta's book on "Photo- micrography" of 1899 and search it for A. pellucida de- scription and image. The book can be found at http://books.google.com/books?vid=OCLC03895698&id=bNYKAAAAIAAJ&pg=RA5-PA1&lp g=RA5-PA1&dq=spitta+edmund and downloaded from that page in pdf format without charge. As the search function for this type of Adobe pdf page does not function I am giving the pages to enter in order to go directly to A. pellucida description and figure. The description of how the image in this work has been taken starts on page 163. How others have done this work is also described. The table of contents for Plate 4 in which one image of A. pellucida is presented is on page 199, while the Plate itself with Fig. 4 showing A. pellucida is found on page 201. This picture shows striae. No picture showing dots is pre- sented. The author discusses the possibility that dots if obtained may be an artifact. The details for the picture showing A. pellucida with striae are as follows: Distance between striae :100,000/inch (that is 0.254 microns). Zeiss Apo 1.40; Oculoart 18; Oblique light; Crescent shaped Diaphragm. I hope you will find this information of interest. GR. > Hi > [quoted text clipped - 19 lines] > > David Great thread! Resolving AP ain't that tough (with enough NA). Doing so with sufficient contrast to see it, is! DIC has best resolution in one direction (orthogonal to the plane of polarisation I think). I don't know if that's slightly greater than the equivalent brightfield or not (as someone suggested). I have seen the pores of AP appear and disappear at 90 degree intervals as the frustrule is rotated. Reckon Mr Kemp might get a few orders for AP 'cos of this thread ;-) Might grab some mounted with no coverslip to try a few epi techniques. Hadn't thought of that before. NoSpam mentioned "plugged pores" which triggered a thought. Biological sections are stained to increase contrast so could the pores (only) of AP be plugged with an opaque material? They'd stand out in bright field then! Or embed (cast?) them on a slide with that material and treat like a rock section; done before? Cheers Beats > Microscope Group, > [quoted text clipped - 105 lines] > Regards > G.R. Steve, You touch on important points. First is the effect of the direction of the diatom's axis with respect to the direction of polarization upon achievable resolution in DIC observation. In my case the diatom was as right angles to the direction of the polarizer and parallel to the direction of the analyzer and at 45 degrees to the so- called shear axis. The image and pertinent text is at: http://www.mikroskopie-forum.de/read.php?2,25394,25394#msg-25394 . I did try at one time to rotate the specimen, but this lead to loss of focus and loss of good immersion oil contact. Regarding the pores I have the following to say. At http://www.mikroskopie-forum.de/read.php?2,25440,25440#msg-25440 I reproduce TEM-images from the article "Fine structure of the Diatom Amphopleura Pellucida" by E.F. Stoermer and H.S. Pankratz from the Amer. Jour. Bot. 51(9): 986 - 990, 1964. This article describes the punctae (pores or dots or pearls) as a series of rounded openings of 60 millimicrons in diameter which in turn are covered by porous plates with even finer pores of some 6 to 8 milli- microns in diameter. I hypothesize that if these porous plates are not removed by the cleaning process, they may obstruct the imaging of the punctae (pores,dots, pearls) by light microscopy. The tiny pores in the porous plates may not even be visible on SEM if the sputtering process obscures them. Then they may also obscure the punctae on SEM. Obscured pores are visible in some of Mr. K. Kemp's SEM samples. It may be important to verify the existence and nature of these porous plates. I recommend a study of the mentioned article or at least of the pictures in the above post of mine. One picture shows one horizontal row of porous plates, while the other image shows the rectangular arrangement of the punctae covered by porous plates. Note that the 60 millimicrons quoted for the diameter of the punctae by Stoermer and Pankratz does approximately, but not precisely agree with Dr. Gaugler's determinations in the image http://photoweb.net/micro/Apellucida-I-50kx-1.jpg . Other images from Dr. Gaugler's FESEM are shown at http://photoweb.net/micro/ . I hope the forgoing is of interest to you and the group. G.R. > Great thread! > [quoted text clipped - 129 lines] > > Regards > > G.R. The TEM images are very interesting. Given the irregularities of the pores, the absolute, perfect regularity of the finer pores is surprising - almost suspicious... > Steve, > [quoted text clipped - 175 lines] > > > Regards > > > G.R.- Hide quoted text -- Show quoted text -- Hide quoted text -- Show quoted text -- Hide quoted text -- Show quoted text -- Hide quoted text -- Show quoted text - Thought so. I think the nature of those smaller pores is that they are a halftone pattern. They're the same size in both images!!! > The TEM images are very interesting. Given the irregularities of the > pores, the absolute, perfect regularity of the finer pores is [quoted text clipped - 179 lines] > > > > Regards > > > > G.R.- Hide quoted text -- Show quoted text -- Hide quoted text -- Show quoted text -- Hide quoted text -- Show quoted text -- Hide quoted text -- Show quoted text -- Hide quoted text -- Show quoted text - Steve, Keep in mind that the image is a digital camera copy of an image transmitted from a digital storage device for rare journals to a printer. One would have to see the original article to be sure whether the images are an artifact. They were not thought to be an artifact by the authors of the article. G.R. > Thought so. I think the nature of those smaller pores is that they are > a halftone pattern. They're the same size in both images!!! [quoted text clipped - 182 lines] > > > > > Regards > > > > > G.R.- Hide quoted text -- Show quoted text -- Hide quoted text -- Show quoted text -- Hide quoted text -- Show quoted text -- Hide quoted text -- Show quoted text -- Hide quoted text -- Show quoted text - Hi Steve, no it's real, see for example my micscape article on Pseudonitzschia. Of course, this membrane like material covering the pores is so thin, it wouldn't be visible in any LM image. It's on molecular basis, the poresize is set by the assembling machine, therefore very regular. Rene. > Thought so. I think the nature of those smaller pores is that they are > a halftone pattern. They're the same size in both images!!! I believe that I have found a definitive reason why A. pellucida is so difficult to image using LM. When I looked at the inside (concave) side of the diatom, the pores are about 100nm x 145nm at a pitch of 285nm. With deep UV, one should be able to resolve one pore from another. However, resolving the pore itself would not be possible. Now, with new specimens from Klaus Kemp of the outside (convex) of diatoms, I find new info. These pores are about 30nm x 100nm and are 90 degrees rotated from the inside pores. Basically, the pores look like: http://www.photoweb.net/micro/Apellucida-pore.pdf Thus, if one was trying to see the large pores, the light is restricted by the underlying small pores. If one was trying to resolve the small pores, that already is a no go. http://www.photoweb.net/micro/Apellucida-100kx-1.jpg already showed this. Look at the lower right area and one can see the 90 degree difference of inside and outside pores. gg Kiss French. Drink California. |
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