Metalizing slides of diatoms to boost contrast is a technique which has been around for over 100 years, and variations on the technique still live on today. Probably the most well known practitioner was Horace Dall (an optical genius and someone I’ve written about before on this site), however the slide maker John Dale is also known for making high quality aluminium coated diatom slides. Today’s post share a few images from one of his slides which recently arrived.
All images were done using my modified Olympus BHB microscope, using 450nm LED light. a 63x Leitz Pl Apo NA 1.40 objective was used with oil immersion. The condenser was an Olympus Aplanat Achromat, oil immersion, set to normal brightfield (more on that later, as these images do not look like standard brightfield ones). 2.5x Nikon CF PL photoeyepiece. Monochrome converted Nikon d850 camera. Images have been reduced in resolution for sharing here, which is a pity given the amazing resolution slide slide offered, but if I don’t do that, my available storage rapidly disappears. Most are single images, but a couple are stacks.
If we go in close to the image above, inside the black dots, are two light ones. These are about 308nm apart (thank you ImageJ)!!
Again, looking at the image above in ImageJ gives a spacing of about 525nm between the black dots.
And, as always, the slide.
The images above were collected using a brightfield setup, but they look more like dark field ones. What is going on here? A thin layer of a few nm of aluminium has been deposited over the diatoms. This effectively makes flat areas such as the bare glass opaque. As the diatoms have holes in them when the aluminium is deposited parts of the diatom go opaque but the light can still get through the holes. What you end up with when imaged from below is a high contrast image with a dark background.
A fascinating technique and just about doable at home if you have the right kit and expertise. As always thanks for reading, and if you’d like to know more about my work, I can be reached here.
Always good to see an article getting published. This time a piece in the Balsam Post, which is the newsletter of the Postal Microscopical Society. The article looks at a range of diatom slides made by Northern Biological Supplies. These have different sized features, and I used circular oblique illumination in combination 450nm to look at them on my modified Olympus BHB microscope.
As a teaser, here’s one of the images from the article – cross polarized, circular oblique lighting of Amphiupleura pellucida, showing the puncta, spaced about 250nm apart.
While I spend a lot of time working on my dermatology projects, it is nice to occasionally take a break and look at other samples using the equipment I’ve built. This work took and fairly standard set of diatom slides and did imaging using a setup which is adaptable to most microscopes, so unlike my UV research is open to everyone to try. I also donated a set of the slides used to the Postal Microscopical Society so they can send them out to members to try it for themselves.
Organizations such as the Postal Microscopical Society and the Quekett Microscopical Club should not be overlooked when it comes to research. While not all the members will be actively involved in scientific research the collective knowledge of members is enormous, especially when it comes to historical work. I can do nothing but recommend anyone interested in microscopy look to join these groups. Giving something back by writing the occasional article is my way of saying thank you, and provides work which will hopefully be of help to others in the future.
As always, thanks for reading, and if you’d like to know more about my work, please feel free to contact me.
I’m a huge fan of imaging diatoms on my microscope as they provide such interesting, challenging and varied structures as well as being beautiful to look at. As said on the diatoms.org website, diatoms are ‘algae that live in houses made of glass’. The silica (glass) structures that they produce are what I, and many others, enjoy imaging. Today I’ll share a few of the ones I have imaged recently on my microscope. I tend to use 450nm LED light for most of my visible light work, as the shorter wavelength gives and improvement in resolution for a given setup (see Abbe equation for resolution). In addition to showing the diatoms I’ll also share the slides that they came from. Slide making is an amazing art in itself and the makers deserve to be recognized for their talent. Note I have reduce the resolution of the images to make them easier to share here. Most are actually much higher resolution than I can share on this site.
First is Auliscus stockhardtii by WA Firth.
And the slide.
This one comes from Allen’s Farm in Oamaru, New Zealand. Not only is the structure amazing, but is has survived for over 30 million years before being collected and mounted.
Next is Triceratium megastomum again by WA Firth. This one is from South America.
This was originally a bright field image, but I have inverted it to make the photo above. Here’s the slide.
Up next is what I think is an Actinoptychus grovei from a strew slide by Klaus Kemp.
Here’s the slide.
Strew slides offer a lot of different material to image. Some of it is fragments, however there are often some complete specimens as well. It is a bit more of a lottery though – you may get lucky or you may not. My experience has generally been good when sticking to older slides by well known makers. This one again was a fossil one from Oamaru (Flawes Creek) so will be around 30 million years old.
This is from another Klaus Kemp strew slide (from Klamath Falls, Oregon), and I don’t know the name of it unfortunately.
This one was tiny, and I nearly missed it when looking over the slide. And the slide.
Final one for today. Aulacodiscus grunowii Cleve by ECP Bone.
The image was captured as bright field, but I have inverted it as I like the look of the inverted image. Another fossil sample, this time from Castel Hungary. Here’s the slide.
Microscopy allows us to see the beauty in these tine things which to the naked eye look just like specs of dust. if we can even see them at all. I use a relatively basic microscope – an Olympus BHB which I’ve modfiied slightly to allow me to use it for UV imaging as well – but the key is in setting your kit up correctly, using the right lighting and decent objectives. ‘Decent’ does not necessarily mean new, and I typically use Leitz objectives from around the 1970s for a lot of my visible light work.
Anyway, I hope you have enjoyed these, and I will share more in the future as I have a few slides to work through. Thanks for reading and if you’d like to know more about my work I can be reached here.
A relatively quick update today. It is always nice to see my work getting recognized. Another of my diatom microscope images has been selected as the September 2023 cover image for the site amateurmicrography.net. It an Actinoptychus heliopelta diatom taken using 450nm light on my Olympus BHB microscope. Here’s the image.
The Amateur Micrography page links through to the Photomacrography forum which has some of the best macro and micro imagers from around the world. It is full of useful advice on setups and techniques, and is well worth checking out if you have a minute.
A quick update for this post, looking at some work I’ve been doing with UV microscopy recently. I’ve been imaging a strew slide by Neville Bradpiece made in 1998 which has a wide range of diatoms on it from Toome Bridge, Northern Ireland. Today’s post looks at some Pinnularia diatoms, and imaging of very small features – poroids – in their structure using 365nm UV light.
First an unstacked image of a Pinnularia from the slide, taken using my modified Olympus BHB microscope and a 365nm LED light source (image is shown at original pixel resolution).
Going in close to the top right of the image starts to show a hint of of the poroids as dots in the structure.
Using ImageJ, I measured 929nm between 6 of them, giving a spacing of 186nm centre to centre. Poroid imaging in Pinnularia is seen as a huge challenge, given how tiny these features are, and it is normally recommended to use circular oblique lighting and cross polarization. This image was done using bright field with slightly (linear) oblique lighting using a Lomo OI-14 Aplanat condenser, and no polarization. The objective was a 63x Leitz Pl Apo NA 1.40, with oil immersion. The condenser also used oil immersion. The benefit here comes from using 365nm UV light, with the shorter wavelength offering improved resolution.
Even in my optimistic frame of mind would accept that the image above barely has enough resolution to see the poroids, however I did image are few other examples of Pinnularia on the slide, one example of which is shown below.
Going in close and doing some measurements with ImageJ gave the following.
With this one, the spacing worked out as 190nm centre to centre, and they are a bit clearer here. Other examples gave poroid distances of 235nm and 251nm so there was a bit of variation between samples.
As always, an image of the slide made in 1998.
The slide uses Sirax as the mountant and quotes a refractive index of 1.8(!). Sirax refractive index is quoted between 1.65 and 1.81 depending on sources, and I’ll be getting more into this when I get round to writing a full article on this slide. High refractive index is great for imaging diatoms due to the improved contrast it offers. There are lots and lots of different diatoms on this slide, and I think it warrants a proper paper showing more images from it. Another article for the list of ones to write…..
As always, thanks for reading, and if you’d like to know more about my work I can be reached here.
On the 12th August 2023 I attended Microscopium which is an annual event held by the Quekett Microscopical Club where members and visitors meet for the buying and selling of surplus microscopy related items. There were some fascinating items for sale, including some absolutely amazing microscopes, and I came away with a couple of boxes of microscope slides of diatoms (and other things) which I am now gradually working through. Today I’d like to share some of the initial images of the slides.
First is a slide by Frederick Marshall of three Coscinodiscus oblongus diatoms. This slide is beautifully decorated with an engraved pattern all over its surface.
The diatoms from the slide are shown below.
Unfortunately at some point in its life, the coverslip has been damaged, as shown in the composite image below.
It turns out that these engraved Frederick Marshall slides are quite rare and desirable, and I got lucky with this one as it was in with a box of 100 diatom slides. For more about Frederick Marshall see here.
Next is a Brightwellia coronata diatom from a strew slide from Jackson’s Paddock, Oamaru, New Zealand.
This is a strew slide with a lot of material present, as can be seen from a wider field of view below (B. coronata in the middle of the image).
I’ve yet to find an intact one of these on a slide, and this is about the most complete one I have come across so far. Apparently the ‘LI’ on the right hand side stands for ‘Little Imp’. An image of the slide is given below.
The next one is an interesting curiosity, and also from a Jackson’s Paddock strew slide, although this one was made by Steve Edgar as one of his Meakin Collection slides.
I’m not entirely sure what this is, and I initially thought this was something artificial and a contaminant, although having spoken with a diatom expert it could be part of a sponge spicule. It looks really cool in dark ground imaging.
It reminded me of a punched card and I went back and did a high resolution image using a 63x Leitz Pl Apo NA 1.4 objective and a stack in Zerene.
It still amazes me as to how artificial this looks. Very curious and I shall look out for similar examples in other slides. Speaking of which here’s the slide it came from.
Next is a locality slide from Moulton, California made by Northern Biological Supplies (NBS).
And the slide itself.
The image I took of this was not stacked, and I’ll return to it when I have more time to do a better one.
Finally for now, back to Oamaru, and a 4 form slide (unknown maker).
Here’s the slide (if you know the maker drop me a message please).
These were some of the nicest slides I have found so far from the Microscopium event. Most of the ones I got were strews, and they aren’t all like these, but even so I’m more than happy with what I managed to find there. I also came away with a box of slides of different Bacteria which it will be interesting to look through as they are completely different to my usual subjects.
The Microscopium event was great and although you don’t need to be a Quekett member to attend I can well recommend membership to the club. There is an incredible depth of knowledge in the club and the USB copy of the old Quekett Journals is well worth getting. For a summary of this years Microscopium event see here. One thing I would say about Microscopium it is not for people to go along and pick up bargains to then subsequently sell, this is for those passionate about microscopy.
As always, thanks for reading and I hope you enjoyed the images. If you’d like to know more about my work I can be reached here.
It’s well known in diatom microscopy that the refractive index (RI) of the mountant has a huge impact on successful imaging. But why is this the case? Today I’ll show some examples of Amphipleura pelludica – a particularly challenging diatom to image – mounted different ways, and how the final images look.
I’ve chosen three different mountants with different RIs. First a dry mount by Watson. Second another Watson slide with Hyrax used as the mountant. Third is something a bit more unusual, a Horace Dall slide which uses titanium dioxide (TiO2) as the mountant. All images were done on my modified Olympus BHB microscope, using 365nm light from a Zeiss 50W HBO mercury xenon lamp. A 63x Leitz Pl Apo NA 1.4 objective was used with oil immersion. A simple Olympus Abbe condenser was used, again with oil immersion, to give brightfield images. A 2.5x Nikon CF PL photoeyepiece was used to project the image to a monochrome converted Nikon d850 camera. I’ll share the images and then an explanation of what is going on. Images have been reduced in resolution for sharing here.
The dry mount is where we begin, and the name itself is a little bit confusing. Dry mounts are typically thought of as there being nothing between the coverslip and the slide, but that is not always the case. With diatoms there is typically a thin adhesive layer on the coverslip holding the diatoms in place, although they can also be ‘burned on’ by using high temperatures. After a chat with a few people on the Diatom Images Facebook page, it seems as though these ones are likely attached with a thin adhesive layer rather than being burned on. Here’s an image of one of the diatoms on the slide.
Here’s the slide (by Watson).
The outline of the diatom is clearly visible, although overall contrast on the original image was a little low. The structures within the diatom cannot be resolved here.
Next is a Hyrax mounted slide, again by Watson.
And the slide.
The diatoms on this slide are larger than the ones on the dry mount, and at this resolution it is s little hard to see the details, so a crop of the main image is given below.
In the crop the striae (lines) in the diatom can easily be seen. Measurement of the spacing between them using ImageJ gave a distance of 2.603 microns for 10 lines, or 260 nm each which is in keeping with the expected values. Overall, while the lines are visible, the contrast between the diatom and its surroundings was still relatively low but better than the dry mount.
Finally is the TiO2 mount.
The TiO2 forms a layer over the coverslip and diatoms present, and a small hole in the layer can be seen as a white area in the image above. The diatoms were again smaller in this slide, and the striae are very clearly visible. Other images from the slide are below.
And the wonderful slide (I am a fan of Horace Dall’s work).
Mounted in TiO2, the diatoms have much better contrast with the surroundings. The final diatom image above shows some interesting features where it looks like the diatoms have only been partially coated.
Measurement of the striae distances from this slide gave spacings of between 260 nm and 264 nm, so again in keeping with what would be expected for A. pellucida.
What is going on here? There seems to be a clear difference in the appearance of the diatoms between the different mounts (keep in mind all the other settings were kept the same).
The key thing here is the difference between the refractive indices (RI) of the diatom and what it is mounted in. The bigger the difference between the diatom and its surroundings, the more visible it becomes. This is known as the ‘visibility index’ and is discussed further in “Special Methods in Light Microscopy” by Robert McLaughlin.
The RI of silica in diatoms is quoted as 1.434. The adhesive layer on the dry mount slide is likely something gelatin based with a relatively low RI of around 1.5. With relatively little difference between the the diatom and it’s surrounds, the visibility will be low, resulting in low contrast and making it hard to discern details such as the striae.
Next is Hyrax and I have seen this quoted as having an RI of between 1.65 and 1.70. This bigger difference to the silica of the diatom, increases this visibility index, and a higher contrast is seen making it easier to see the fine striae.
The final on is TiO2. It’s RI is very high, and is written on the slide as 2.90. This is hugely different to the diatom silica, and the result is a much higher contrast image, making the diatom and its structures easier to see.
As you can see, going to a higher refractive index is beneficial for imaging diatoms as it improves contrast and helps with visibility. It should be remembered that what is being enhanced is contrast and not resolution (which is defined by wavelength of light and NA of objective and condenser). The resolution of the setup is the same for all these slides, but changing the mountant varies the contrast and therefore the visibility of the features. Interestingly, although perhaps not visible on the images at the resolution on here, the lines on the TiO2 coated slide were sometimes a little less well defined than the Hyrax slide. I think what is happening here is that the TiO2 is forming a layer over the diatom which is ‘a few’ nm thick and with minor variations in thickness. I think it is these variations in thickness which are being imaged and resulting in a slight waviness of the lines.
If you’ve made it this far, well done, and I hope you found it interesting. As always, if you’d like to know more about my work I can be reached here.
Today’s post contains the images from four diatom slides by the slide maker William Allott Firth (W.A. Firth) who is widely renowned as one of the finest slide makers there has been. For more information about W.A. Firth, take a look at the excellent writeup here. These were taken using my modified Olympus BHB microscope and 450nm LED light and have been stacked using Zerene. All have been reduced in resolution for sharing as the original file sizes are too big to upload efficiently.
The first one is Opephora schwartzii. This was imaged using dark ground, using a 100x Leitz Pl Apo NA 1.32-0.60 objective with oil immersion, and a Reichert Neo 1.42/1.18 dark ground condenser. The iris on the objective was closed to make the dark ground image. There are 2 examples of the diatom on the slide.
These were small diatoms, but even so the original has been reduced in size for sharing, and some of the details are lost in the image above. Below is a crop of the image shown at the original pixel resolution.
The dark ground image has done a good job of bringing out some of the sub-micron structures in the diatom. Got to love pushing the limits of optical microscopy. Before I move to the next one here’s the slide.
Next is Aulacodiscus orientalis. This was imaged using brightfield with an Olympus Aplanat Achromat condenser (oil immersion), using a 40x Leitz Pl Apo NA 1.00 objective with oil immersion.
Here’s the slide.
This one confused me a bit at the beginning as it looked like it said ‘Hulacodiscus’ for the name.
The next slide is Surirella macraeana. Back to a smaller diatom, and a 100x Leitz Pl Apo NA 1.32-0.60 objective was used (with oil immersion), and the iris closed down slightly to make circular oblique lighting. A Leitz Heine condenser was used along with oil immersion.
There were 6 examples of this diatom on the slide.
And of course the slide.
The final one today is Stephanopyxis corona. This was imaged with a 40x Leitz Pl Apo NA 1.00 objective, oil immersion, and an Olympus Aplanat Achromat condenser, slightly oblique, again with oil immersion.
There was actually 3 examples of the diatom on the slide, but one of them had gone walkabout almost to the inner edge of the ring, as can be seen with this lower magnification image.
I had a bit of trouble selecting an objective to images this slide. They were quite small, and my original choice of 100x and 63x Leitz ones did not have sufficient working distance to reach the diatoms, and a 40x Nikon UV-F NA 1.30 couldn’t didn’t get the full depth of the diatom. Hence I ended up using the 40x Leitz, which had a slightly longer working distance. The diatoms must be quite thick (dome shaped) and this can be more easily seen if you look at a video of me moving the microscope stage.
It also proved difficult to capture the two rings of spikes in the structure in the flat photo, and they can be more easily seen in the video.
Here’s the slide.
I really enjoyed looking at these slides as their quality was so good. Amazingly, they were relatively cheap for such good quality slides – I paid about £15 for them. As always, I hope you enjoyed the images, and thanks for reading. If you’d like to know more about my work I can be reached here.
Diatoms make great subjects for microscopy given the fine details in their structure. In addition to slides with a strew of material and single examples of given species some slide makers have produced amazing arrangements either to show what species are present in a given area, or to create fantastic works of art. While more common in the Victorian era, the production of these artistic arrangements has continued into the 20th and 21st century with a few very skilled practitioners. Today I’d like to share a few examples from the amazing Klaus Kemp (now sadly passed away) which I feel very privileged to have in my collection. All images were taken on my modified Olympus BHB microscope, using 450nm light, and with a Leitz Heine condenser. They have been reduced in resolution for sharing here, so some of the fine detail present will have been lost.
First up an exhibition rosette with 136 diatoms.
This slide has unfortunately had an accident at some point in its past. However it will be well looked after and at some point I may try repairing it.
Next, another exhibition rosette, this time a 145 form one.
And the slide.
A smaller example now, a 25 diatom exhibition star.
And the slide.
Next is a circular arrangement, again with 25 diatoms.
And the slide for the circle.
For the final couple of examples, we’re moving away from the more artistic arrangements to ‘Type Slides’. Both of these are with 50 forms, and both images are much smaller than the original file sizes to allow me to upload and share (for example Type Slide 2 was made from a composite of 4 stacks of images and the original file size was 24750×5504 pixels!!).
These slides really are works of art, crafted by a hugely skilled diatomist. Thank you Klaus for the beauty you brought to the world.
As always, I hope you enjoyed the post, and if you’d like to know more about my work I can be reached here.
This post finishes off something I started yesterday (see here) where I tried pushing the resolution I managed to get on a couple of diatom slides while only using 450nm light. The N.B.S. slides I imaged had diatoms with small features on them, but one really difficult diatom to photograph using visible light is Amphipleura pellucida, as this has features down around 250nm in size. While I have done some work with this using UV light (for example here), my challenge was to try using 450nm blue light in combination with circular oblique illumination and even cross polarization.
Equipment wise I used my Olympus BHB microscope with the Reichert Neo condenser, in combination with a 100x Leitz Pl Apo NA 0.60-1.32 objective. Kept wide open, this objective has such a high NA, the result is that instead of dark ground, I get a circular oblique illumination. 450nm LED light. No stacking was done, but for the non-polarized image I did try averaging 10 images in camera. Cross polarization was done by adding one linear polarizer just above the field lens, and another on top of the photoeyepiece, rotating the bottom one until the image was at its darkest. The images showing the full diatom have been reduced in size for sharing here, but I’ll also include crops at original resolution to better show the features.
First the non-polarized image, so just using circular oblique illumination.
The striae are nice and obvious with this setup, and can be more easily seen by looking at a closeup of the top right of the diatom.
Putting this into ImageJ and doing some measurements, we have 10 striae in 2.659µm (or 37.6 striae per 10µm). This matches pretty well with the slide which says 40 lines per 10µm. Think of it another way, those lines in the image above are 266nm apart.
Cross polarizing in combination with oblique lighting is well known as a way of showing the punctae on this diatom, so I tried that approach. I placed a linear polarizer on the field lens (below the condenser), and then another on top of the photoeyepiece. I rotated the one on the field lens until the image was at its darkest which would be when the polarizers are aligned at 90 degrees to each other, and then took an image of the diatom.
Even looking at the whole diatom, the lines appear a bit more broken up, but this can more easily be seen on closeups of the top right and bottom left of it.
With cross polarization, I’m just starting to see a hint of punctae of the diatom (dotting), where the lines are breaking up into individual features, so has been beneficial for the imaging here. For using 450nm light I am happy with this result, especially at the first attempt.
Before I go, here’s the slide.
As always, thanks for reading, and if you’d like to know more about my work, I can be reached here.