It’s no secret that I like the old diatom microscope slides. They can be very well made and provide very photographable diatoms often for much lower prices than modern prepared slides. The one I’m sharing today was by Samuel Henry Meakin and was made in 1945. It is a diatom test slide with examples of a variety of different species. Images were taken on my modified Olympus BHB microscope, and using a range of different objectives (4x Zeiss Planapo, 10x Olympus UVFL, 20x Olympus Splan, and 60x Olympus Splan Apo). Lighting was from below via an Olympus Aplanat Achromat condenser (straight bright field and oblique), and was 450nm LED light. Photoeyepiece was an Olympus 2.5x NFK, and the camera a monochrome converted Nikon d800. No stacking was done for these. Images are shown at reduced resolution where the originals were >1600 pixels across (most of them).

To start with, a low magnification image of the arrangement (4x objective).

The test diatoms are bounded at the left and right by large circular diatoms. Using 2 circular diatoms like this was often done by the slide maker Eduard Thum to help with locating the samples on the slide. They are arranged in a linear fashion, although there has been a little movement of some of them. The slide is shown below.

What is great here is that Meakin has labelled the diatoms present (although not the circular ones on either end). They read as follows, going from left to right on the slide;

1. Triceratium favus
2. Pinnularia nobilis
3. Cymbella gastroides
4. Navicula maculata
5. Navicula lyra coarse
6. Navicula lyra fine
7. Stauroneis phoenicenteron
8. Pleurosigma balticum
9. Pleurosigma attenuatum
10. Pleurosigma angulatum
11. Navicula smithii
12. Cymatopleura solea
13. Navicula lewisiana (also known as Frikea lewisiana)
14. Brebissonia boekii
15. Amphipleura lindheimeri
16. Navicula rhomboides (Cherryfield)
17. Navicula rhomboides fine
18. Nitzschia singalensis

Some of these took some tracking down to get the details right as I struggled with the hand writing at times (especially for the less common ones). Overall the quality of the slide was very good, and the diatoms were intact. This is one reason why these types of test slides can be very nice – lots of intact, well prepared diatoms, great for imaging.

Going in closer, here are 2 images done with the 10x objective, oblique lighting.

Even closer now with the 20x objective (oblique lighting). There is some overlap between the images.

At this magnification, the details were starting to become obvious. So I went in closer to some of the diatoms using a 60x objective (oblique lighting).

One in particular struck me as very interesting, and it was one which I had struggled with for the name – 13. Navicula lewisiana. I’d not seen one of these before, and here is how it looked with the 60x objective (oblique lighting).

At this resolution the fine detail is lost. Below is a crop from the original. shown at original pixel resolution.

Putting this in ImageJ, the spacing between the poroids can measured, and it comes out to just under 400nm (shown as 0.395 in the image below).

As I mentioned, this was one I hadn’t seen before, but is one I shall come back to again to do more imaging on. Here’s some information on it on the Diatoms.org website. Also, it is in the excellent book “The diatoms”, by Round, Crawford and Mann, page 534, where is it mentioned that it is “A rarely recorded monotypic genus.”. Explains why I haven’t seen it before. While I am on the topic of books, I should also note that there is a bit of information about Meakin in “Microscopical mounts and mounters” by Bracegirdle (published by the Quekett Microscopical Club) which is a text I often refer to for getting background on mounters and slide makers.

As always, thanks for reading, and if you’d like to know more about my work, I can be reached here.

EDIT – I did return to the slide and did some more imaging of the Navicula lewisiana. Same objective (60x Splan Apo) but this time with 405nm light and stacking. In order to get a successful stack I reduced the extent to which the light was oblique, so this is essentially a bright field image.

While there is now detail to the edge of the diatom, the image is a bit ‘flat’ – going away from oblique light and stacking makes it look less 3D to me. This one was tidied up more as well. I actually think I prefer the 450nm light image which wasn’t stacked, but this is more of a complete image of the diatom. Horses for courses as they say……

After my recent post about microscopy of a mammal eye, it got me on the lookout (sorry, couldn’t resist) for other eye microscope slides. Within a few days I had a cross section of Dragonfly eye on its way to me. This is a slide by Watson, and I was hoping I’d be able to do some cross polarized imaging on it. Today’s post shows the results of my work.

First a large field of view image showing the whole sample, using a 2x Olympus Splan Fl objective. Here are the images – cross polarized and plane polarized.

In the images above, the eye itself is towards the bottom of the slide, and the rest of the head towards the top. The eye shows some amazing structure from its compound arrangement. It looks like the very surface of it is most optically active. Within the head there are more regions of optical activity. I’ll come on to those later.

Something to note, the cross polarized images aren’t completely black in the background. I did not use Pol objectives, and some of them had fluorite in which may well be messing with the polarization a bit. Essentially I set the polarizer angles to give the nicest images and show the regions of optical activity. Photos were taken using a Canon Eos R7 camera just using auto white balance. Light source was a white LED light, and the microscope my modified Olympus BHB. Plane polarized imaged are essentially like non-polarized ones in appearance. It was easier to do that than keep removing the polarizer from the photoeyepiece.

Going in a bit closer now. First on the surface of the eye, with a 4x Zeiss Planapo objective, and a 10x Olympus UVFL objective, and again, cross polarized and plane polarized.

Cross polarization shows the nice optical activity in the surface of the compound eye.

Now going into the head itself let’s a have a look there, again with the 4x and 10x objectives.

Moving in closer to the bundle of fibers in the middle of the image we get these.

I’m guessing these are a bundle of muscle fibers used to move the eye around. They are very optically active under cross polarized imaging.

Before I wrap this up, here’s the slide itself (a Watson one).

I was hoping that this slide would allow me to image it in cross polarized lighting (unlike the mammal eye one which had an optically active mount) and it did not disappoint. What we capture in an image depends strongly on how we image the subject, and changing things like polarization of the light can have a huge impact on what becomes emphasized in the final photograph. These old slides continue to amaze me, and I look forward to sharing more of them in the future. As always, thanks for reading, and if you’d like to know more about my work I can be reached here.

Continuing with my microscopy journey, I thought I would try imaging an eye cross section to get a better understanding of the visual organ. After having missed out on a couple of old slides of eye sections, I ended up getting one from a seller on eBay (here described as Eye Entire VS). I’m fairly sure this a ‘mammal’ eye, not specifically a ‘human’ eye, but it’s interesting nevertheless. Here’s what it looked like.

First an image of the whole specimen with a 1x objective.

The eye itself is towards the bottom of the image (if that wasn’t obvious). The lens is the oval feature which looks purple/green/yellow. The eyeball itself is squashed here so is no longer spherical. I had hoped to look at the sample with cross polarized light to look at the construction of the lens itself, but the slide mount is crystalline and so this wasn’t possible. Note to self, keep looking for an older sample in case that doesn’t have a crystalline mount.

How about if I look at the sample with a 40x Olympus Dplan Apo UV objective, what can be seen? Images using this higher magnification are given below.

There are some beautiful features in the sample of the slide. What are we looking at though? In the first of the three images above it is showing the lens of the eye and you can see the cross section through the lens fibers. I had hoped to look at this using cross polarised light, but as mentioned this wasn’t possible due to the mount used to make it.

The second and third images are the tissue at the rear of the eye – the retina region. I found a nice paper which shows the different layers present (The Role of Photodynamic Therapy in Non-malignant and Malignant Eye Disorders, by Nowak-Sliwinska et al.). I took one of my images, flipped it horizontally and put it below their schematic.

Although slightly different thicknesses, the schematic lines up quite well with my image. It’s interesting to see the rods and cones in the retina, and it makes me realise how complex it is back there.

I did return later to the slide and looked at it with my 60x Olympus Splan Apo NA 1.4 objective, and my monochrome Nikon d800. This was done with white LED light and is a composite of 2 images stitched together. It has been hugely reduced in resolution for sharing here – this one is 1600×517 pixels, while the original was 14540×4701 pixels.

The image above shows the tissue from the rear of the eye (the retina) on the left, the remains of the vitreous humor in the middle of the image, and actually part of the eyeball lens itself on the right (the features you can see in there are the lens fibers). I had hoped to get more detail on the rods and cones, but wasn’t able to do that here.

Overall it seems to be a nice little slide, and one I will try and come back to with higher magnification to look more at the rods and cones. As always, thanks for reading, and if you’d like to know more about my work I can be reached here.

A bit of a change from my normal microscopy work today. For one thing it’s colour images. Another is the subject – a thin section of coal. Also the technique used – cross polarized microscopy. Let’s get in to it.

The sample is a slide by Charles Morgan Topping and likely dates to around 1860. I’ll show a picture of the slide at the end of the post. It has a thin section of coal on it and is described as “Trans sect[ion] of coal. Warwickshire”. The section of coal is about 1cm across and mounted under a coverslip. First a normal bright field image (using a 1x Olympus Splan FL NA 0.04 objective).

And now the same sample imaged using cross polarized light.

Note that to get the whole sample in the field of view, I had to remove some of the extension tubes from between the photoeyepiece and the camera. While the increases the field of view it does mean that the edges of the image (which wouldn’t normally be seen in the standard setup) are included. These parts of the images above have some pretty severe aberrations present which is why the edges are nowhere near as sharp as the middle of the image.

The cross polarized imaging sends the background very dark, and now crystalline material which is present becomes very colourful as it rotates the polarized light passing through it. At this scale, this is especially visible in the white band towards the left of the coal sample. This is where my knowledge of coal is getting a bit stretched. I think the dark bands running almost vertically in the images are growth rings from the original plant material – think tree rings.

Going in closer with a different objective now (a 2x Olympus Splan FL NA 0.08). All the images from now on will be cross polarized.

For the observant amongst you, you will have noticed that these images look more than 2x magnified compared to the images with the 1x objective. The extension tubes were put back in between the photoeyepiece and camera, having the effect of increasing the magnification (and getting rid of the aberrations at the end of the field of view). The dark bands running from top to bottom are now more clearly visible, and there are small ‘dots’ present as well in between the bands, which are coloured. The strongly coloured area towards the left of the image is the crystalline region shown towards to the left of the 1x images. This is very crystalline as can be seen from the colours present in the image.

Going in even further with a 10x Olympus UVFL NA 0.4 objective, the ‘dots’ become more visible.

At this scale the ‘dots’ become little stained glass windows, taking on a kaleidoscope of colours. As I said before I think these areas were originally cells in the plant matter when it was alive. These have then become filled with crystalline mineral during the fossilization process. The slide says ‘Warwickshire’ on it, and if so the coal fields of Warwickshire date to 300-350 million years ago. If these features were originally cells in the plant, then they are 300-350 million year old cells.

Some additional information. Here’s the slide.

I’ve been told this was made by Charles Morgan Topping and dates to around 1860. There’s more on Topping here.

Also, the two low magnification objectives – Olympus 1x and 2x SPlan Fl.

Olympus 1x and 2x SPlan Fl objectives

As always, thanks for reading, and if you’d like to know more about this or other aspects of my work I can be reached here.

I’ve had a bit of a catch up this weekend and looked at some slides that have arrived recently. They are both pretty and interesting (for different reasons), so I thought I would post them here. All have been reduced in size for sharing.

First is a Pinnularia slide by Arthur Cole.

There was a single example of the diatom on this slide. The mount was amazingly clear and needed very little cleaning up. Imaging was simple bright field, using 450nm light. Date for this is likely the late 1800s.

Next we have a Triceratium scitulum slide by Thomas Russell of London.

A nice arrangement of 4 diatoms. Again the mount was very clear and needed very little cleaning up. Imaging was brightfield with 450nm light. Likely date for this one, late 1800s.

I returned to this slide the next day and took a picture of one of the diatoms using the Watson Holoscopic condenser to give a dark ground image.

Using the Holoscopic condenser has brought out the dot features present within the polygonal structures which were barely visible in the bright field image. The ‘dots’ look to be about 500nm across.

Now an arrangement of Actinocyclus by Eduard Thum, Leipzig.

A typical Thum arrangement using two large circular diatoms to mark where the arrangement is. Imaging was oblique brightfield with 450nm light. Likely date for this one, again late 1800s.

Last but not least is a Bacteriastum furcatum slide by Watson.

Imaged using 450nm light and a Watson Holoscopic condenser (discussed here) to give the dark ground image. Single images, no stacking.

There are always lots of these interesting old diatom slides available on places like Ebay and in my experience they offer great value for money for those wanting to image slides, so don’t discount them because they are old. As always, thanks for reading and if you’d like to know more about this or other aspects of my work, I can be reached here.

A special slide to share today. I’ve shared a few images from slides made my Horace Dall before, and this is another one. Dall was an optics genius, and experimented with using titanium dioxide and aluminium layers to improve the visibility of diatoms. This slide is from 1960 and is marked up as being ‘Pleurosigma Balticum‘. Here are some images from it taken on my modified Olympus BHB microscope using 450nm light. All have been reduced in resolution for sharing here.

The aluminium coating has the effect of improving the contrast for the diatom, making the features more visible. These were imaged with simple bright field illumination from below. I’m not happy with my stack with the 40x objective, as there are artefacts in there, so I shall redo that at some point.

Here’s the slide itself.

The slide says it is ‘best seen with vertical illumination and with oil imm[ersion].’. However my system is not really setup for illumination from above, which is why I lit it from below. This slide appears in the Quekett Microscopical Club book, “Microscopical Mounts and Mounters” by Brian Bracegirdle (plate 13, D). It’s an early example for one of Dall’s aluminium coated slides, and looks different to the others I have which are shiny like mirrors. I suspect it is an early experiment using the technique.

EDIT – after a chat with an expert, while the slide is marked as Pleurosigma balticum, (new name, Gyrosigma balticum) it is not that, and is likely to be one of very big Pleuorsigmas like Pleurosigma formosum. This highlights an issue I have had with a few different diatom slides – that of naming. I’ve had a few slides where the name on it did not match the subjects.

As always, thanks for reading, and if you’d like to know more about my work I can be reached here.

A while ago I shared an image from a diatom which had been sputter coated with gold to help provide increased contrast (see here). Since then I have been working on a paper about metal and metal oxide coated diatoms for microscopy which I am aiming to publish later this year. In the mean time I’ve been talking with a nice gentleman who coats diatoms with gold using sputter coating and he has sent me a few slides to examine. I’d like to share some of the images from those slides today.

For background, my modified Olympus BHB microscope was used for these, and the objective was a 60x Olympus Splan Apo NA 1.4 oil immersion one. 450nm light was used to illuminate the samples from below. Stacking was done using Zerene. Other details are shared for each image and the image sizes have been reduced for sharing here.

Please forgive me if the names of some of these are wrong, I am still learning the complexities of diatom nomenclature. First one, a Stephanopyxis diadema Ehr. from a fossil diatom strew from Dunkirk, Maryland.

This has an amazing 3d structure, and looks to be hemispherical. There were 13 images in the stack. The tiny depth of field from the objective can be seen if you look at a video where the stage is moving.

Next is a Stauroneis (perhaps a phoenicentron) from Mud lake, Ottawa, Ontario.

This was mounted in Pleurax and was a lovely slide to image as there was virtually no debris around it, and the diatom was almost flat. Only 3 images were used in this stack.

The small features don’t show up too well in the file which has been resized for sharing, but this is what they look like at original pixel resolution.

Final one is a Heliopelta of some description, again from a fossil diatom strew from Dunkirk, Maryland.

Why do this process? The gold sputter coating of the diatoms makes the silica of the structure darker and so improves their contrast, so that even simple bright field imaging (as used here) can be used successfully.

As always, I remain fascinated by these amazing structures and the worlds that can be viewed with a relatively simple light microscope. Thanks for reading, and if you’d like to know more about my work I can be reached here.

As it was Easter recently, I had a bit of spare time to do some microscopy. This time I returned to one of the diatom slides I had made using a quartz slide and coverslip so it could be used in short wavelength UV light. I thought this would be a good time to try out a trio of Leitz UV objectives (16x NA 0.25, 40x NA 0.65 and 100x NA 1.20) for imaging at 313nm. However as we shall see, things didn’t quit go to plan.

For the imaging I used my UV modified Olympus BHB microscope, and a monochrome converted Nikon d800 camera. Condenser was a Zeiss quartz one (simple brightfield illumination). Light source was a Zeiss mercury Xenon lamp. Edmund Optics 313nm, 10nm bandpass filters (2 stacked together) were used to filter the light going to the camera. The subject was a diatom slide which had a Gyrosigma (balticum, maybe) along with 2 other diatoms. Images have been reduced in overall size for sharing here.

First, a stacked image using the 40x objective, showing part of the Gyrosigma (stacked using Zerene).

Plenty of detail to be seen with the 40x objective. Even being NA 0.65, the short wavelength light results in improved resolution compared with visible light imaging under the same conditions. As I had the system setup, I thought next I would try the 100x NA 1.20 objective (again as a stack). Unfortunately the depth of focus this objective has meant that I could not move the stage enough to get all of the diatom in focus, and the image only shows part of the structure in focus.

This highlights the issues with these high magnification, high NA objectives, i.e. very shallow focus depth. This make imaging thick samples a problem. Perhaps a thinner coverslip would work here, although likely with a bit of loss of image quality (more on that later). Final image, using the 16x objective, not stacked this time.

The 16x image shows the 3 diatoms on the slide. Speaking of the slide, this is it.

Here are the three objectives.

These objectives are interesting and I have been able to find out very little about them. They seem to be the Leitz equivalent of the Zeiss Ultrafluars and are very well made. The 40x and 100x are designed for glycerine immersion, and as I am writing this I have just had a thought. I use a 50:50 mix of glycerine and water for my immersion as this was what I got from a microscope supplier when I started out. This will have a different refractive index compared with pure glycerine. I wonder if this reduced the depth of focus for the 100x objective? I will park that idea and check later. The only mention I have found of ‘Leitz UV objectives’ is in a brochure from 1985 which I have shared before (here). Thing is in the brochure the coverslip thickness is quoted as 0.17mm, not the 0.35mm ones written on the objectives themselves. All very odd. Perhaps a thinner coverslip (0.20 or 0.25mm) might be an option and get me to image slightly thicker samples, although whether this would drop the resolution with the 100x objective would need to be checked.

A good friend asked me recently what was the actual benefit of these quartz based optics and UV light over conventional methods for improving resolution. Funnily enough I have written a bit about that before (here) and there isn’t much between say UV imaging at 313nm and imaging with blue light and a high NA objective and oblique illumination. I do it because I enjoy it and it was a scientific challenge to make the microscope. However where it will get more interesting is below 300nm, as the shorter the wavelength the greater the resolution. It is proving challenging that far into the UV though, and more work needs to be done. The microscopists of the early 1900s managed it though, so I know it can be done.

As always, thanks for reading, and if you’d like to know more about my work I can be reached here.

With microscopy it is important to know how big the object is that you are imaging. The simplest way to do this is to use a stage micrometer (also called a graticle or graticule). These are small rulers and designed to be imaged using the same optics setup as being used to image the subject. The ‘pixels per micron’ can then be calculated and used to make a scale bar on the image of the subject. Today I’ll show you a couple of the ones I use. But first a nice image from an old Leitz one showing their name as well as 250µm of the micrometer.

The ones I tend to use on a daily basis were made by Graticules Ltd in the UK (now know as Graticules Optics Ltd). I have a 2mm one with 10µm divisions) and a smaller one which has 2µm intervals which I use for for higher magnification objectives. Both of these were bought second hand from ebay for about £30 to £40. Here’s what the 2mm one with 10µm divisions looks like.

Using a 10x Olypus UVFL objective this is what the scale bar from the Graticules Ltd slide looks like.

In the image above, each larger division is 0.1mm (100µm) with 10µm smaller divisions between the larger ones. I also picked up an antique Leitz one, which is also 2mm long and has 10µm intervals. Here’s what that slide looks like.

Interestingly the Leitz one has a coverslip attached which the Graticules Ltd one does not. Here is an image of the micrometer scale on the Leitz slide using the same setup as for the Graticules Ltd slide above.

As can be seen from the images of the micrometer, both slides are giving the same scale – about 830µm from the top of the image to the bottom. The Leitz one also has the makes name on it below the scale as shown in the first image in this post.

I also bought another Leitz one which I thought was a micrometer, see below.

Turns out this wasn’t a stage micrometer at all, it was actually a haemocytometer, used for counting cells (you can read more about them here). Here’s an image from the slide.

I should also point out that there are plenty of new, cheap, stage micrometers available from places like ebay, however I have no idea how accurately made they are, which is why I tend to look out for older, second hand ones. In addition to these types of glass ones, they are also available on metal or opaque substrates for reflection microscopy.

The measurement side of things tends to often go unnoticed when compared with some of the beautiful subjects which can be imaged, but it is an important aspect of microscopy. As always, thanks for reading, and if you’d like to know more about my work I can be reached here.

A short update today. I returned a diatom slide I have imaged before – the Amphipleura linheimerii slide originally imaged here. I wanted to try imaging it at 365nm, but using a Watson Quartz cassegrain condenser that I have on loan (a very special condenser, which I will try and write more about in the future). In summary, the condenser is a high NA dark field condenser, and being made from quartz has great UV transmission. This post shares an image from the slide using 365nm dark field illumination.

First though a quick note about the setup. Microscope was my UV modified Olympus BHB. Light source was a 365nm torch slid into the light port of the microscope. Condenser was a Watson quartz cassegrain one, which was used with glycerine/water as the immersion fluid. The objective was an Olympus 40x NA 1.3 silicone immersion lens, which I used with glycerine/water as the immersion fluid. Photoeyepiece was an Olympus 2.5 NFK. Then I used 2 stacked Edmund Optics 365nm, 10nm FWHM bandpass filters. Finally a monochrome converted Nikon d800 which is UV sensitive.

Ok, the image – this has been cropped slightly, and has been reduced in resolution for sharing.

There are two examples of the diatom on the slide, and part of the other one can be seen in the lower left of the image above. There is plenty of detail in the image, but perhaps that does not show up as well with the lower resolution image being shared here. Here’s a crop, at original pixel resolution.

With the cropped image some of the smaller features of the diatom now become visible. The distance between the small white dots was measured on using ImageJ was was found to be about 380nm (length 0.379 in the image below, as it was calibrated in microns).

A few things to note with this setup. Glycerine/water was used as the immersion fluid, while the objective was designed for silicone. Glycerine/water was used for a few reasons – firstly, I had some, secondly, it is a similar refractive index to the silicone, and finally it is much, much easier to clean. However I have to acknowledge this may impact performance of the objective. Being quite a low magnification objective, the field of view was large. As such the resolution achievable by the objective is getting close to the actual pixel resolution of the camera. I did try taking RAW files and then converting them to monochrome in Monochrome2DNG to get more resolution, and I even tried frame averaging in the camera, but neither of these improved things noticeably and I just stuck with the original single frame jpg captured in the camera in the end. The slide itself was not very UV transparent. I suspect if I measure the transmission spectrum of the slide, it will not be great at 365nm. As such this was not the ideal subject. I am also not sure I got the iris on the objective fully optimized, so perhaps a little more resolution can be squeezed from this setup.

Diatom imaging in the UV continues to be a fascinating journey, and one i will continue to pursue. As always, thanks for reading, and if you’d like to know more about my work, I can be reached here.