The microscope images today come from a Triceratium nitescens slide by the maker R.I. Firth. The slide is marked as ‘Barbados’ and ‘Very rare’, is mounted in Styrax and was made in 1943. There is a single example of the diatom on the slide. The microscope used was my modified Olympus BHB. The condenser was an Olympus Aplanat Achromat, set to slightly oblique and oiled to the underside of the slide. The objective was a 63x Leitz Pl Apo NA 1.40, oiled to the top of the slide. The photoeyepiece was a 2.5x Nikon CF. Lighting was 450nm LED (white LED source with a Thorlabs 450nm, 40nm bandpass filter). 17 images were stacked in Zerene stacker. Camera was a monochrome converted Nikon d850 done by MaxMax. The image has been reduced in resolution for sharing here, although a crop of the main image at original resolution is also shared. Here’s the final image.

And a crop of the image shown at original pixel resolution.

A friend asked me about why I do stacking, and it reminded me that I have written something which briefly covered the reason before (see here). Essentially though, at high magnifications with high NA objectives, the depth of field of a single image is tiny – way less than a micron in the setup used here – making it impossible to get the whole diatom in focus in a single photograph. So I take multiple photos, moving the stage very slightly between each one, and then stack them together using software called Zerene which takes the in focus parts of each image and combines them. Live view on the camera is very helpful here, as I can see what is in focus with each image and make the movements accordingly to capture everything. After stacking, the image still need some cleaning up which I do in Photoshop – making the background smoother, and removing artifacts from dirt on the sensor coverglass. Microscopy will reveal dirt that you never knew was there before in normal photography – the shorter the wavelength of light used, and higher the objective NA, the smaller the features that will be revealed and will need editing out. I tend to capture my images in the camera as Jpeg files, and after stacking output the stack a 16 bit Tiff for subsequent work (outputting the stack as a Jpeg gave me more artifacts especially in the background of the image). Ideally I would do everything as Tiff files, but my computer is old and stacking Jpeg files is a tough enough job for it.

Before I go to cover some UV work I did with this slide, here is the slide itself.

While the slide was on the microscope, I also did some experiments with 365nm UV (the camera is monochrome converted and sensitive to UV, visible and IR light). Keeping the same objective, the condenser was swapped for a Watson Quartz Cassegrain (which I just realised I’ve not written about before so must rectify that), the light source was a 365nm LED torch, and I filtered the light going to the camera by a stack of 2 Edmund Optics 365nm, 10nm bandpass filters. Here’s a photo of the diatom made up from a stack of 4 images with this setup.

The effect wasn’t quite what I was after, but I think it still looks nice and shows the features in a different way. Given the high NA of the objective the effect is produce circular oblique lighting when using the Watson Quartz Cassegrain condenser.

Before I go, one last thing to mention, when dealing with what is effectively extreme macro imaging on the microscope, the settings under which the images are taken can have a huge impact on the quality of the final photograph. I use 3s exposure delay and electronic front curtain shutter, but found out something yesterday which is worth sharing. Initially I tried ‘S’ (Single shot) and the image was quite blurry. I changed to ‘Mup’ (Mirror up) and the image was much sharper. Comparison below using 365nm LED light, crops shown at original pixel resolution.

I tried this a few times, and got the same result each time, so will be using Mup from now on with the d850. I seem to recall Nikon changed how they deal with Mup with the d850 so it can now be used with live view unlike earlier Nikon cameras, so perhaps it is a d850 specific consideration.

Slightly longer post than usual today, but I thought it would be useful to cover off some other things in addition to showing the main image. As always, thanks for reading, and if you’d like to know more about my work I can be reached here.

It’s too warm today to spend much time on the microscope, but I wanted to share the results of a little experiment. Having recently discovered that the Leitz 100x Pl Apo objective had pretty good UV transmission at 365nm, I thought I would give the 63x NA 1.40 a go as well, in combination with a Watson Quartz Cassegrain dark ground condenser, with the idea being to produce a circular oblique illumination setup (due to the high NA of the objective). Here are the results of the initial test. A little less formal than usual, but something to be returned to later.

On the Thum slides, he used large disc shaped diatoms at either side of the main subject to help with locating for viewing. It is one of these disc shaped diatoms that is the subject today. Here’s a stack of 7x images taken using 365nm light on my Olympus BHB with the 63x Leitz Pl Apo NA 1.40 objective (resolution reduced for sharing here).

The lighting has produced a nice 3D effect (even though the condenser is a dark ground one, the high NA of the objective means the images isn’t dark ground). Here’s a crop at original pixel resolution.

There is certainly lots of nice detail in the image. Refocusing on the edge of the diatom gave the following (single image, no stacking this time).

And again a crop of the image at original resolution.

I save my images as jpegs in the camera, and then if I am stacking use them to create a 16 bit TIFF file for further work. Interestingly the single image of the edge of the diatom shows a lot more artifacts (and was just processed as the jpeg) vs the stack even though everything was just saved a jpeg in the camera. I may try saving as BMP files in camera in future, especially when dealing with these UV images which show up all the small features and noise. The slide itself came from the Antiques Microscope ebay shop (see here), and here’s the full arrangement.

And the slide itself.

I shall return to the slide to look at the main subjects when it is a bit cooler here. In the mean time thanks as always for reading, and if you’d like to know more about my work I can be reached here.

Some of the microscope slides I get are quite expensive, either rare examples, unusual mounts, or sought after makers. Others however are not. Today’s post is one with slides of the latter. I got two slides by Smith, Beck and Beck, and for the pair they cost me the princely sum of £5 (plus £3.50 postage). As it turned out, one of them was pretty much useless – degradation over time – but the other still had some good diatoms on it.

The images as per usual were done on my modified Olympus BHB, this time using 450nm oblique illumination. The 40x images used a Nikon UV-F NA 1.3 objective with glycerine immersion. Photoeyepiece was a Nikon 5x CF. Camera was a monochrome converted Nikon d800. Image resolution reduced to 1600 pixels across for sharing here.

Here is a stack of 10 images from the slide described as ‘Pleurosigma balticum’.

As well as there being plenty of P. balticum, there are various other diatoms on the slide including quite a few Surirella gemma. Quite a few of the examples have degraded somewhat, but there are still some usable ones on there. Most of the detail in the image above will be lost because of reducing the size for sharing, but the crop below is at original resolution.

As can be seen from the crop, there is plenty of detail in the P. balticum diatom.

A wide field of view using a 10x objective shows how more of the slide looked showing the variation and density of the slide.

Unfortunately the other slide, marked P. quadratum, was pretty much unusable (10x objective image below).

And of course, here are the slides themselves.

At first glance the slides look similar, but the printing and importantly the addresses are different. They were at 6 Coleman Street from 1847-1856, while at 31 Cornhill from 1865-1880 (according to Bracegirdle’s ‘Microscopical Mounts and Mounters’) which makes these some of the older slides that I have, especially the P. quadratum one. Last week I spent £5.40 at a service station for a sandwich while visiting the Archives of the Science Museum near Swindon to go through documents by Horace Dall (more on that soon after I have gone through my notes), so for less than my sandwich I got a nice slide to examine.

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

A few days ago I took a look at a Klaus Kemp slide of diatoms called Cymatopleura elliptica with 450nm light and a Leitz 63x Pl Apo NA 1.4 objective and, well, the image was not as good as I had hoped. Today I decided to revisit the slide with a different approach, and this post shows the results of that work. The image was done on my modified Olympus BHB microscope, light source was a Zeiss Mercury Xenon lamp. Objective was a Leitz 100x Pl Apo NA 1.32-0.60 (oil immersion). Condenser was a Watson Holoscopic one (again with oil). Objective iris was closed down slightly with the aim of trying to produce circular oblique lighting (COL). Photoeyepiece was a Nikon 5x CF. Filters – 365nm (10nm bandpass, Edmund Optics, 2 stacked together), 405nm (10nm bandpass) and 450nm (40nm bandpass) from Thorlabs. Camera – monochrome converted Nikon d800 from MaxMax. At 365nm, a stack of 18 images processed in Zerene stacker. Image cropped slightly and reduced in resolution for sharing. The camera produces slight colour casts and I have kept those here as I like the effect. And after all that, here it is.

Moving to 365nm light produced a noticeable improvement in resolution, although that is probably lost a little on the image above which is 1600 pixels across compared with the original (5826 pixels across). As an example of the resolution of the original, the image below is a crop before it was reduced in size.

There is plenty of detail in the image, much more than my initial attempt with 450nm light. Going in closer and checking in ImageJ, some of those features are about 280nm apart.

While the slide was on the microscope I took a series of 3 images of it at 3 different wavelengths (365nm, 405nm and 450nm). These are shown below.

The images above are unprocessed and not sharpened, but the microscope was refocused each time. It demonstrates nicely how the shorter wavelength light brings out the features in more detail (as expected by Abbe).

The slide actually has 4 examples of the diatom on it. The larger one was the one that was imaged.

It looks as though there may something unusual with the fixative, as shown by the droplets in the image which were very visible on this slide. These were not on the outside of the slide or coverslip. Here’s the slide.

The mountant is Hyrax which was one reason I decided to try 365nm light (it has pretty good UV transmission at 365nm). What was more surprising was the objective – I expected the Leitz 100x Pl Apo to block a lot of the UV and it actually performed really well. I must remember this for future work. Diatom naming is still well beyond me, but this one is also sometimes referred to as Surirella undulata (see here).

UV microscopy presents its fair share of challenges, but imaging at 365nm is the more straightforward end of the spectrum to try and provides some quite visible improvement in resolution compared with visible light. Be safe though if you plan on trying this – I use UV safety specs whenever dealing with UV light sources. As always, thanks for reading, and if you’d like to know more about my work I can be reached here.

A couple of weeks ago I had a box of assorted slides come through which have turned out to have some real gems in it (I shared some initial images here). Today’s post looks at another slide from the box – an Actinoptychus heliopelta by Eric Impey from 1970. Images were done on my modified Olympus BHB microscope using 450nm light (slightly oblique). The objective for the main image was a Leitz 63x Pl Apo NA 1.4 which recently arrived from JB Microscopes. A Nikon CF 5x photoeyepiece was used. 27 images were stacked in Zerene. Images have been reduced in resolution for sharing.

Here’s the main image.

The sample is beautiful and there is plenty of detail on there, some of which is lost on here as I had to reduce the resolution for sharing. Below is part of the image shown at the original resolution.

The slide itself has 3 examples of the diatom on it, shown below (single image, not stacked, using a Nikon 20x Plan Apo objective). The one in the main image is actually the one on the right hand side of the image below.

And the slide itself. The diatoms came from Dunkrik, Maryland. The mount is Naphrax, and it is a very well made slide, which even has a slightly decorative ringing around the coverslip.

As an aside, I think I have spent too much time looking at diatoms this weekend. This was what I saw when I looked down inside a plastic coke bottle…..

Before I go, being an equipment geek, I’ll also share the objective as I was very impressed by the quality of the image it produced. It’s a Leitz 63x Pl Apo NA 1.4, oil immersion. Here it is alongside it’s big(ger) brother, the 100x with an adjustable iris, which is also an excellent performer.

At the risk of sounding like a broken record, I continue to be amazed by the quality of some of these older slides, and would well recommend them for anyone interested in looking for samples to image. This slide cost me all of £5.50 plus postage. If you don’t want to buy slides but relish new slides to look at, the Postal Microscopical Society offers members the chance to borrow slides from their collection and is well worth checking out. Thanks for reading and if you’d like to know more about this or other aspects of my work I can be reached here.

Today’s post has come about as the result of a chat with a microscope shop who I use to check over my objectives when they need fixing (JB Microscopes Ltd in Newcastle). A few weeks ago I bought an 20x Olympus Splan Apo that had some optical issues (the price was right and I had hoped it was worth the gamble). To see if it could be saved I sent it to JB Microscopes to be looked over and hopefully fixed. Unfortunately it could not, but while I was chatting with them I asked what objectives they had on their shelves. It turned out that they had a couple that I had been looking for for a while – a Nikon 40x UV-F glycerine immersion objective, and a Leitz 63x Pl Apo oil immersion objective. This post will cover some initial results with the Nikon 40x UV-F.

For the image I used an aluminium coated slide by Horace Dall. The slide is marked PA indicating it as Pleurosigma angulatum diatoms. Imaging was done on my modified Olympus BHB using 365nm light from an LED torch, an Olympus Abbe condenser (glycerine immersion), the Nikon 40x UV-F NA 1.30 objective with glycerine immersion, and a Nikon 5x CF photoeyepiece. Light was filtered with an Edmund Optics 365nm, 10nm bandpass filter. Camera was a monochrome converted Nikon d800 by MaxMax. 8 images were stacked in Zerene. The main image has been reduced in resolution for sharing.

The final image was about 6200 pixels across and has been reduced to 1600 here. The stack produced plenty of detail, and to try and show this better, there are a couple of crops below which are shown at original pixel resolution.

And of course, here’s a photo of the amazing Horace Dall slide.

What about the objective? Here it is.

Why is this a special objective? Firstly, it’s a 40x objective with high NA. It is also quite rare, dating to around the mid to late 1980s. Originally developed for fluorescence imaging work, so it has good UV transmission (for longer wavelength 365nm UV anyway). There’s not much info about these objectives out there, but Charles Kreb’s site shows a Nikon CF brochure which has the 100x version of the objective, here. These aren’t Plan objectives nor are they Apo, so expect some field curvature and chromatic aberration if not using monochromatic light. What they are designed for is the let a lot of light through, and they certainly do that.

I have a 100x version of the objective, which has also been used for imaging at 365nm. The 100x is nice as it has an adjustable iris making it great for darkfield. Here are the 40x and 100x together.

If anyone has the 10x and 20x from this series that you are looking to sell, please let me know as I am on the lookout for those two for my UV work.

We often do not thank our suppliers, but they make the work we do possible. As such, thanks again to JB Microscopes – great customer service and worth contacting if you are in need of something specific (they do not list their stock on their site). As always, thanks for reading, and if you’d like to know more about my work, I can be reached here.

Occasionally I’ll buy a little bit of a ‘lucky dip’ selection of slides to image on the microscope, especially if the same seller has a number of them for sale at the same time. At the weekend a box of 8 slides came through, and I wanted to share some of the images from a few of the slides. Images were done on my Olympus BHB microscope and have been reduced in resolution for sharing here.

First is a diatom slide, an Actinoptychus hexagonus by W.A. Firth, imaged using a 60x Olympus Splan Apo NA 1.4 objective with oblique 450nm illumination.

Lots of lovely detail in the 60x image (this was a stack of 24 images). There are actually 2 diatoms on the slide, and here’s what they look like together, using a 10x objective and oblique light.

The main 60x image was of the one of the right. Here’s the slide itself.

Next is another diatom slide, this time a strew from Oamaru. I only have a very preliminary image of one of the diatoms I found on this, as I then went on the Diatom Images facebook page to get an ID for it (I’d not seen one before). It is apparently Brightwellia coronata. Single image with a 20x objective.

Finally for now, something a bit different – Salicine crystals imaged using cross polarized light and 4x Zeiss Planapo objective.

And the slide itself.

There’s a few more in the box to look at….

This box of slides cost me about £8 per slide including delivery, and will certainly give me plenty of enjoyment with trying to image them. I’m a huge advocate of second hand and older slides, and they are well worth checking out.

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

For those who frequent my blog, you’ll have heard of the slide maker Horace Dall before. I’ve been a keen collector of his metal and metal oxide coated diatom slides for a while (and am currently writing an article on them which I hope to publish later this year). I recently came across a slide of his for sale which was a bit different though – not a diatom slide but a whole Soldier Beetle. Today I’ll share some images from this slide using both visible light and Infrared (IR).

With diatoms, I normally have to go to high magnification to see them. However with this slide the problem I had was going to a low enough magnification, as the whole sample is about 20mm across. To image the whole beetle, I took 4 images using a 1x Olympus Splan Fl objective and stitched them together in Photoshop. Here’s what the sample looks like (resolution has been dropped from the original 9390×10687 to 1600×1821 for sharing).

A higher magnification image of the head of the beetle using a 4x Zeiss Planapo objective.

And to finish with a couple of images using a 10x Olympus UVFL objective.

While chatting with a friend on a microscopy forum he mentioned to me that IR light can be useful for imaging insect microscope slides as it makes some parts become more transparent. To try this out I swapped the LED light source for a Tungsten bulb, and placed a Heliopan 780nm long pass filter over the field lens, and then imaged the slide again.

Here’s the composite of 4 images using IR light (the IR images have a slight pink tinge in the JPEG for straight from the camera).

The IR light does indeed make parts of the insect appear more transparent, and the overall effect is to produce a lower contrast image. Next up the head using the 4x Zeiss Planapo objective.

Again, the increased transparency if obvious. Finally some images using the 10x Olympus UVFL objective.

I did an additional image here of the eye with the 10x objective. The IR cuts through the darkly pigmented area and shows more of the structure of the eye. I think I need to come back to this with a higher magnification and try a stack. The 10x objective has a bit of a hotspot in the middle of the image in the IR photos. This behavior is often seen in IR photography when the lenses aren’t suitable for IR, and I presume that is the case here.

As a final set of images, I thought I would try imaging the eye region in IR with a 20x objective (a 20x Nikon Plan Apo NA 0.65). 3 different images are shown here; a single image at a given focus point (bright field), a stack of 4 images (bright field), and a single image using oblique bright field. These images were desaturated to remove the pink tinge from the IR images.

The single image using the 20x objective shows a surprising amount of detail especially in the surface of the eye. The stack of 4 images shows even more detail and some of the tubular structures of the eye lens elements. The oblique image is more of a cross section through part of the eye but does nicely highlight the structure of the individual lens elements. Interestingly this 20x Nikon Plan Apo seems to have very little hotspot in the IR so could be a good one to use again in future.

As per usual here’s an image of the slide as well.

Every now and then it is nice to have a break from imaging diatoms and look at a different sample. By moving from visible light to IR it was possible to seem through some of the dark areas of the beetle’s structure. Thanks for reading, and if you’d like to know more about my work I can be reached here.

While most of my imaging outside of the visible spectrum is in the Ultraviolet, I do also like to take photographs at the long wavelength end in the Infrared (IR), especially for landscapes. Many lenses suffer from hotspots when used in the IR adding complexity when processing images. A few years ago Zeiss released their ZF-IR lenses (25mm, 50mm and 85mm) which had coatings on them to produce better IR transmission and reduce hotspots. These lenses weren’t available for long before Zeiss stopped selling them and they have now reached something of a cult status making them hard to find (and expensive) second hand. I recently managed to source examples of the 25mm f2.8 and 50mm f1.4 ZF-IR lenses from the photography dealer Jo Geier at Mint and Rare in Austria, and today I wanted to share some initial thoughts on them.

I’ll come to the transmission through the lenses in a minute, but to start with a visual comparison of the front elements of the 25mm ZF-IR and a normal version of the lens the 25mm ZF.2. This shows the reflection of sunlight through my kitchen window.

The coating on the front elements is very different between the two lenses as can be seen by their colour. Couple of things to note here. Ideally I would compare the ZF-IR with the ZF version, but I happened to have a ZF.2 copy of the lens. The ZF.2 came after the ZF but I would think the coatings would be very similar as the main difference was the addition of electrical contacts for providing aperture data. Note I have blurred the last 2 digits of the serial numbers on the lenses.

I measured the transmission through the 3 lenses I have (Zeiss 25mm f2.8 and 50mm f1.4 ZF-IR and 25mm f2.8 ZF.2) using my Ocean Insight spectrometers and a couple of light sources – xenon and halogen lamp. This allowed me to measure transmission from 300nm to 1000nm, although as I will explain with a couple of caveats.

First the comparison between the 25mm ZF-IR and ZF.2 lenses.

The ZF-IR transmission is lower at the blue end of the spectrum than the ZF.2 but while the ZF.2 transmission drops sharply above 700nm the ZF-IR does not. This matches what I’d expect to see from the Zeiss product data sheets. My system sensitivity drops sharply above about 920nm, which is why the data gets very noisy up there, and I don’t think the drops and peaks at around 950nm are real features. The curves are shown in 3 sections as I used 3 setups to measure them. Overall though the ZF-IR certainly offers better IR transmission above 700nm.

Now the Zeiss 50mm f1.5 ZF-IR and 25mm f2.8 ZF-IR.

The Zeiss 25mm and 50mm ZF-IR have very similar shaped curves, which is what would be expected assuming they have the same coatings.

I did some quick landscape test shots with the 25mm f2.8 ZF-IR using my monochrome converted Nikon d800 camera from MaxMax and a Heliopan 780 IR filter, and these are show below.

The lens certainly seems to live up to its reputation for not producing hotspots in the IR. I have heard that the 50mm ZF-IR is more prone to hotspots, although I have not been able to replicate that yet. To finish with, a not often seen photo of the 25mm and 50mm ZF-IR lenses together.

Overall, I’m very happy with the two Zeiss ZF-IR lenses and they will be a great addition to my non-visible light imaging capability. As always, thanks for reading and if you’d like to know more about my work, I can be reached here.

I have an unusual (and rather rare) diatom slide to share with you today – an example of Hydrosilicon mitra, prepared by S.H. Meakin. This was made in 1933, and is mounted in Hyrax (which will become important later). The images shared were taken on my modified Olympus BHB microscope, with a monochrome converted Nikon d800 camera. The images have been reduced in resolution for sharing.

The first is a simple bright field image. This is a stack of 7 images and was done using 450nm filtered LED light. The objective was a 40x Olympus Dplan Apo UV (NA 0.85). Condenser was an Olympus Aplanat Achromat.

The sample is not flat, and dips towards one side. This means that stacking was necessary with the 40x objective (in fact I missed the focus on the top left of the diatom – ” ‘B-‘ must do better next time”). As can be seen there is some damage to the fine structure of the diatom, but it is still a very pretty specimen.

I wanted to see what this looked like with dark ground illumination, so I got out my Watson Holoscopic condenser (some info about that here) and used glycerine as the immersion fluid to attach it to the underside of the slide. The objective was a 20x Olympus Splan NA 0.46, and again 450nm light was used. No stacking this time, just a single image.

I love the dark ground effect, but being a single image part of the diatom is out of focus due to it not being flat. I’ve not got my head around stacking dark ground images yet.

As a final image, I wanted to try something different. As mentioned above the mount is Hyrax, and this has pretty good UV transmission at 365nm. I wanted to try imaging with 365nm light (to potentially help improve resolution), and also use a different method of illumination – circular oblique light (COL), also known as annular illumination. To do this I used a 40x Olympus UVFL PL NA 1.3 silicone immersion objective in combination with the Watson Holoscopic condenser. This objective has an adjustable iris – wide open and the NA is bigger than that of the condenser and the image is bright field. Iris closed all the way down and the image becomes dark ground as the objective NA is now smaller than the condenser. In the middle ground COL can be produced, when the NA of the objective is close to that of the condenser. This gives a nice 3D effect to the image. Both the condenser and the objective were used with glycerine as the immersion fluid. The image below was a stack of 13 images, using 365nm LED light, and with a COL setup.

COL illumination gives a more 3D effect to some of the features. But, and this is a big ‘but’, the resulting image was very noisy, and was a bit of a nightmare to stack. Not sure why it was so noisy, as I used ISO200 on the camera. I really like the end result though and will definitely try this technique again.

As always, here’s the slide itself as well.

Not too bad for a slide made 90 years ago. The slide was bought on ebay for about £30 and there is a single example of the diatom on it. As always, thanks for reading, and if you’d like to know more about my work, I can be reached here.