UV microscopy – modifying the binocular/trinocular head, part II

Happy New Year everyone. 2021 brings promise of a new start and I hope to bring you plenty of cool science over the next 12 months. I have some pretty special lenses and microscope kit to review, so watch this space. Today though, we start with an update on the UV microscope build. This has been a project I’ve been working on for about the last 6 months, and has required some major modification of my Olympus BHB to make happen (see here for the previous summary of the work to date). Today an update on the modification of the binocular/trinocular head, and the results of some major mechanical surgery.

When I took the Olympus head (I’ll just refer to it as a ‘head’ from now on, rather than repeating myself every time) apart, I realised that the construction was going to be quite difficult to adapt for UV. There was a lot of thick glass in there, along with a prism which acted as beam splitter – see here for the initial assessment. Instead of trying to replicate the prism exactly, I made the executive decision to cut the prism in half, and replace half of it with a simple block of UV fused silica. This would mean that when the prism was in the position for taking a photo, the light would go straight through to the camera, rather than being split between the camera and the eyepieces. This would result in more light for the camera (a good thing for UV imaging), and with most cameras having some form of live view, I don’t need to look down the eyepiece for the final focus. The window in the bottom part of the head also needed replacing, but this would just need a simple disk of UV fused silica. However both parts needed to be custom made and the prism would need cutting in half to make this all work. To supply the fused silica components and cut the prism down, I went to a company I’ve worked with quite a bit over the last year – UQG Optics – who were able to supply the parts I needed and do the work on the prism. When anything needs to be custom made the costs go up dramatically, and given the tolerances involved were tight, it was a case of ‘measure many times’ before the dimensions went to UQG for cutting.

When the two UV fused silica parts arrived, along with the cut down prism beam splitter, I assembled everything and glued the circular window into its metal housing, as you can see below.

New UV fused silica parts for the Olympus BHB head

Transmission of the new parts, compared with the old ones is given below.

Transmission of the new UV fused silica parts compared with the original ones

Note the absolute transmission here is probably slightly higher than in real life (by a couple of percentage points). Even after collimation of the light beam for the transmission test, it will still be slightly divergent. Including such thick pieces of glass / fused silica in the path of the beam means that some more light will reach the spectrometer detector than would happen without the glass / fused silica in place. I have tried to minimise this with the setup of the device, but can’t guarantee that it has corrected it fully. The important thing to note is that the new fused silica components have a flat transmission curve as a function of wavelength, unlike the original glass ones, which block varying parts of the UV. The new UV fused silica parts will allow UVB (and even UVC) imaging down to and below 280nm.

Once the glue had set on these, the head needed re-assembling. Not a simple task, and akin to putting a quart into a pint pot. The picture below give you and idea of the parts that needed to go back into it.

Parts from the Olympus BHB head

Once back together (after a lot of loosening and tightening screws to get everything aligned, and cleaning of any glass after putting fingerprints on it) it looked like this.

Olympus BHB head re-assembled (without the top trinocular tube attached)

The next step was to put the yellow filters in the base of the tubes for the eyepieces – the idea here being to eliminate any UV that could otherwise reach the eyes. This is part of my ‘belt and braces’ approach to safety when using this device. Not only would any UV have to pass through the glass part of the beam splitter, and the glass eyepieces, but now has to go through yellow filters which have a transmission of <0.01% of anything below 400nm. As mentioned before, I’d still use UV protective glasses when using this, the yellow filters just another layer of protection. Once it is up and running, I’ll be measuring the spectral throughput to the eyepieces to see how low it is. If in any doubt, I’ll just use live view on the camera for all the focusing work. Here’s a view down the tube of one of the eyepieces to the yellow filter.

Yellow filter in place in the eyepiece tube

One thing I haven’t got right was the diameter of the filter needed. The eyepiece tubes are 23.2mm, and I assumed 23mm filters would be fine. However at the bottom of the tube the diameter opens up and the filters rattle around a bit unless the eyepiece tubes are screwed all the way down. This can be rectified by using larger diameter filters, or by gluing the ones I have in place, and I’ll see if I can get access to where I need to to be able to do this. For now the tubes will be screwed fully down, locking the filters in place.

There was a lot of glass in the head, all aligned very precisely, and I had real concerns that this would simply not work well enough, leaving me with a very big bill for a new paperweight. Once assembled, for a quick test I did a visible light image of one of the Diatom Lab strewn diatom slides using an Olympus 20x SPLan objective, and the normal tungsten light source on the microscope. Stacked in Zerene Stacker, here is how it looks (I used a Schott S-8612 filter as this was taken with my multispectral camera and I wanted to remove IR from the image).

Diatom image using the modified Olympus head

The image above has been reduced in resolution for sharing on the web, and is a crop from the full area imaged, but demonstrates that this modified head can at least give a usable image in visible light. Note, the fuzzy areas in the largest diatom are more down to my stacking technique than anything else.

In theory this is the final part of the UV microscope build – all the glass parts have now been converted to fused silica, and the next stage is to try imaging in the UV. To start with I’ll use 365nm as this is simpler and safer, and I have a couple of light sources (LED and mercury xenon lamp) to try out. I also have a Diatom 2.0 test slide on order from Diatom Lab, so I can look to see how the resolution of the imaging changes when I go from visible to UV. See, I told you 2021 would be exciting.

The original plan was to use this for sunscreen formulation imaging – to be able to image UVA and UVB absorbing components separately to see where they are in the formula – and I’ll certainly be using it for that first. After that we shall see, as there are plenty of other applications for UV imaging, in biology, forensics and other fields.

Thanks for reading, and if you’d like to know more about this or any other aspect of my work, I can be reached here.

UV Microscopy – Tiyoda UV 1.2-1.4 darkfield condenser

As I write this it is the the last day of what for most has been a ‘character building’ year, and I shall start today’s post with a little question – As scientists, what is it that drives us to do the things we do? See, nothing to serious, and with that slight philosophical diversion, let’s dive into the subject of this post.

With UV imaging, things aren’t always what they seem. Equipment designed for UV work could be only for certain wavelengths for instance – normal glasses can still be used for UVA imaging work, while materials such as quartz and calcium fluoride are needed for shorter wavelength UVB work, as normal glass would just absorb the light. Some microscope objectives are described as ‘UV objectives’ but are not meant for imaging in the UV, rather they have low fluorescence and are used for visible light fluorescence imaging after illuminating the sample with UV. With older equipment, it becomes hard to try and find original source information about it as it often doesn’t exist digitally, so there is little alternative to hands-on testing to try and determine if something is suitable for a specific application.

In my searches for UV imaging kit, I came across a microscope condenser called a ‘Tiyoda UV 1.2-1.4 darkfield condenser’. This piqued my interest as it sounded as though it was a dedicated UV darkfield condenser, and got me wondering whether it was made of quartz or other exotic materials. Problem was, there wasn’t any info out there on the transmission properties of it. I saw a couple for sale, for rather large amounts of money (hundreds of GBP), which was too much to invest just to answer a question. Eventually I found one for sale here in the UK which cost a lot less, but was best described as a ‘fixer-upper’. So I bought it and here it is.

Tiyoda UV 1.2-1.4 condenser

The condenser came in a mount for a Reichert microscope (which I don’t have) but my main interest was to assess its UV transmission properties. Main problem when it arrived, was that the coating around the central part of the condenser was damaged, as shown below.

Damaged coating

The black paint should be present in a ring around the central part of the condenser, and on this copy it was missing on about half of it. The construction of these things is quite amazing and goes back a long way. They are known as ‘cardioid condensers’ and use a specially shaped mirror to project a cone of light onto the subject. Shining a torch through this one from below produces the following.

Light transmission through damaged condenser

In the image above, the black ring around the central disk should be blocking all the light from below, and it’s pretty obviously damaged. The central disk is actually transparent, and this can be seen more easily if viewed from the side with the torch still on. This is what would sit just below the sample on the microscope stage.

Damaged condenser viewed from the side

The unit consists of two main parts – the main top piece, in and above the Reichert mount, and then a screw-on lens underneath which is marked with the name ‘Super, Wide PAT. PEND.’, as shown below.

Super Wide lens below condenser

The view through this lens is pretty trippy, and it has a dark spot in the centre. It also looks very, very delicate.

View through the Super Wide lens

This lens actually creates a circular ring of light from a point source as can be seen below, as it projects the image of my ceiling lights onto the desk.

Circular ring of light from the Super Wide lens

In use, this ring of light is then projected into the main body of the condenser and reflects off the mirror towards the front of the condenser. A very clever design minimizing the amount of light which would otherwise be lost.

The damage to the top of the condenser was actually a blessing as well as a problem, as it allowed me to measure the transmission straight through, so I can see whether the components were attenuating the UV. Measuring the transmission of the individual parts and complete condenser gave me the following.

Transmission of the Tiyoda UV 1.2-1.4 condenser

Now there is a lot going on in the graph above, so I shall explain. The red line is the transmission through the Super Wide Pat Pend lens by itself. This actually shows good transmission down to about 300nm, which suggests it is a thin layer of conventional glass, rather than something unusual like quartz. The green line is the transmission through the top part of the condenser with the damaged black paint. Measured transmission is very low here, as even with the damage the light still has to bounce off mirrors and through a small hole to get to the detector, which attenuates the total signal. When the two parts of the condenser are recombined, and transmission measured, we get the blue line. Now the incoming light is focused by the Super Wide Pat Pend lens, onto the mirrors and out of the top of the condenser. As can be seen the overall transmission drops from 400nm down to about 320nm, below which it is essentially blocking the light. Note that absolute transmission of the condenser would be higher than this as the diameter of the beam of light I use for my tests is relatively large, and some light is lost by not being able to pass through the small optics of the condenser. The key thing to note is the drop in transmission at the short wavelength end. The Tiyoda UV 1.2-1.4 would therefore be good for the longer wavelength UV down to around 320-330nm, but not good below that as it would prevent the light from transmitting.

OK, so I have a damaged condenser which does not have short wavelength UV capability. What next? First step was to repair the damaged paint, and I did this using Semple Black 3.0 paint which as I have shown in other applications has great light blocking properties. After painting, the condenser looked like this.

Condenser after repainting

Shining the torch through it now, and the light is only coming from the central disk.

Torch through the repainted condenser

If I turn down the exposure on the camera, and capture the torch through the condenser again, I get the following.

Torch through the repainted condenser

Basically the only light now getting through the condenser is coming through the central circular opening, as it should be. However it is pretty obvious now that there are some major scratches in the glass of the circular opening especially close to the edges. This condenser has had a hard life….

Where does this work leave me? I have a condenser in a mount which does not fit any of my microscopes. Even if the mount was modified, the diameter of the condenser means that my microscope stage would need modifying to make it fit. The condenser does not have the great UV transmission I was hoping for (although it will still be good for UVA work). The one I have was damaged, and while I have given it a new coat of paint to cosmetically repair it, I have no doubt that a few uses with immersion oil and that paint would come straight off.

So after all this, what was the point of this exercise? Looking around online I was unable to find anything definitive about the transmission of light through a Tiyoda UV 1.2-1.4 condenser. By buying one myself I was able to test it, and fill a gap in the knowledge base about these old pieces of equipment. It answered a question that was relevant to my research, and has allowed me to definitively say how it works, and where it can and cannot be used. And now I write up and share my experience, so that anyone else who may be wondering this, perhaps as part of their work, has something to refer to.

At the beginning of this post I asked a simple question – As scientists, what is it that drives us to do the things we do? Motivations for the work we can do take many forms. Money – sure, we have to make a living and some questions need answering to push projects forward. To push our own knowledge forward – absolutely. To fill a gap in the knowledge that wasn’t there and share what you know, enabling the work of others in your field to move forward as well as your own. Now there’s a nice thought as we move from what for many has been a difficult year, into one which hopefully will be a better one.

If you’ve made it this far, and would like to know more about this or any other aspect of my work, I can be reached here. Happy New Year everyone.

Leitz UV microscope objectives – historical optics, part II

Working in the area of UV imaging and microscopy gives me 2 options. Firstly, win the lottery (or sell a kidney or two) so I can afford to buy new equipment. Secondly, try and hunt down older equipment which can be re-used. Given that I’m quite attached to my kidneys (literally) and that unfortunately that lottery win still evades me, that leaves the second option as being the preferred route. Even this route presents significant challenges though. Very often these piece of equipment were made in very small numbers, most dealers have never even seen or heard of them, and some are well over 50 years old now so tracking down information on them is extremely difficult. Today, I have a little bit of an update on a post I shared earlier in 2020, and also a new historical Leitz objective that I’ve tracked down.

Firstly, the update. Earlier this year I shared a post which talked about a 300x reflecting objective lens – here. This lens is based on a mirror system rather than refractive optics, and is suitable for UV, visible and IR imaging. I bought it as a bit of a collectors items really, and while I’d love to use it some day, it’s going to take some pretty complex work to make that happen, as the tube length it has is much longer than that microscope I have at the moment. While I did find a mention of what I thought was it, in a catalogue from 1967, I didn’t know any more about it.

Fast forward about 6 months, and I was talking with a dealer in Sweden and he mentioned that he had a couple of interesting items. One was a Leitz microscope condenser which had the markings “Glyc Q” on it, and the other a lens which had “Quarzglas” and “Glyz” on it. Now these make my ears prick up a bit as the use of glycerine (marked as “Glyc” or “Glyz”) often implies they were designed for use with ultraviolet light, as does “Quarzglas” (as quartz is UV transparent). So after a quick discussion they were on their way over to me.

I’ll come back to the objective lens with “Quarzglas” in a minute, and cover the condenser first. The 300x reflective objective I already have was pictured in the catalogue alongside a couple of small condensers (see below).

Leitz reflecting objectives and condensers (1967 catalogue)

Being quartz glass the reflecting condensers these should also be suitable for UV work. The one I have is one of the 0.6 NA ones, mounted in a Leitz holder.

0.6 NA Leitz reflecting objective

While the wording on the condenser is not exactly like the one in the brochure, I think this is the same one as advertised. The difference is probably down to being different batch or production run, as I’ve seen small differences like this before.

How does the transmission of this condenser look. I measured transmission between 280nm and 420nm and got the following.

Leitz 0.6 Q Glyc condenser

At first glance the transmission of the condenser doesn’t look great. However it should be kept in mind that the light gathering part of the condenser is quite small, smaller than the diameter of the light beam in the my transmission measurement setup, therefore some of the incoming light is not hitting the open part of the condenser. This of course limits the max transmission possible. The key thing here is that there is no significant drop between 420nm and 280nm, indicating that it has good transmission in the short wavelength UV region, and as such would be suitable for UVA and UVB imaging. Hurrah.

As a slight aside I also found out a little more about the 300x objective I have. In a paper from from 1964 (Klaus Weber, “New developments in microscopes at Leitz”, Applied Optics, 1964, 3(9), 1045-1048), it describes the mirror objectives as follows; ” The mirror objectives expand the application of microscopes into an additional spectral range; the objective Q 170/0.50 and the glycerin immersion Q Glyc 300/0.85 are suitable for a spectral range of from 2200A [220nm] to 7000A [700nm]; and they are fully achromatic. Suitable condensers are also provided. Mirror objectives consist of spherical mirrors on quartz bodies. The main characteristics of the Leitz mirror objectives are the very small central zone (only 5% of the pupil size), thus guaranteeing very good contrast even for extremely fine object structures. The special type Q Glyc 300/0.85 s, which has a somewhat reduced light transmitting capacity, produces maximum contrast even for finest structure elements over the entire spectral range. It is particularly suitable for microphotometry.”. My 300x one is marked with the ‘S’ on the end, indicating is is one of the slightly higher contrast ones.

Now for the second item that arrived. This is an objective lens with a 50x magnification and is shown below.

Leitz Pv 50x Quarzgl objective
Leitz Pv 50x Quarzgl objective

There’s a lot going on on the label, so what does it all mean. ‘170/0.18’ means it is designed for a 170mm tube length, and a 0.18mm thick coverslip. So far, this is fairly standard. ‘Pv Glyz. 50:1’ means it is for phase contrast (Pv), meant to be used with glycerine as an immersion fluid, is 50x magnification. It has an NA of 1.00 (‘A 1,00’). The phase ring can be seen in the photo below.

Phase ring in the Leitz Pv 50x Quarzgl objective

The next line on the lens is really interesting – ‘Quarzgl.’ as this should indicate it is quartz so should in theory have good UV transmission. After that, the ‘A 1,00’ has already been discussed. Next we have ‘Muster-Fasserei’ in red. This is an odd one, and not something I have seen before. Talking with a couple of optics expert the overall consensus is that this was a prototype or manufacturing pattern for this lens design, making it a rather interesting piece. While Leitz did make a few refractive optics for UV work (which I have), the ones I have seen so far were not phase contrast ones, again potentially adding to the uniqueness of this one.

What about the history of this 50x lens, what was it designed for? This proved to be a little harder to figure out. I eventually found reference to it in a Leitz document “Image-forming and illuminating systems of the microscope”, where it showed the following (in the line for Pv Glyc 50);

Leitz 50x Pv Glyz objective document

This seems to suggest that it actually wasn’t for use on a normal microscope, but rather a microspectrograph. So far I have found a couple of references to Leitz microspectrographs and microspectrometers from approximately the era of this lens, but nothing that specifically mentions this lens yet, so for now it remains a bit of an enigma. In the document above, the coverglass is given as being Quartz rather than glass, which explains the ‘Quarzgl’ on the barrel of the objective.

As with the 0.6 condenser above, I measured the transmission spectrum through this objective and got the following.

Leitz Pv 50x Quarzgl objective transmission

Hhhmmm, this is where things stopped going to plan. As with the 0.6 condenser, this objective is relatively small diameter, so the light source is probably being clipped slightly by the objective, which limits the maximum signal I am measuring. However unlike the 0.6 condenser, the objective is blocking the light below 320nm. Not good, and means it wont be any use for UVB imaging. This is a bit odd, as it has ‘Quarzgl’ on the objective lens barrel, and it should have better UV transmission based on that. Does it have a glass element in there in addition to some quartz ones? Does it have coatings on the lenses or adhesives between them? If it is a mock up, pre-production lens, have they just used glass instead of quartz? I shall probably never know the answer to the last question, unless I ever manage to find another one to compare it with. I shall get some images using it once my glycerine arrives, so for now it is just a technical assessment.

Tracking down old optics has been a fascinating part of my UV imaging research. Often there is very little information on these items, as when they were made they were done so in very small numbers. As with all research the road to knowledge is not always a smooth one, as is the case with the objective lens described above. Sometimes what we thought was the case proves not to be so. This is the very nature of research though, and we must take the rough with the smooth. We should not see it just as a negative – if everything we did was right, how can we learn anything? Very often I have seen data which didn’t fit the hypothesis discounted, with every effort made to discredit it, rather than have someone ask the awkward question “Was the original hypothesis right?”. As scientists we should never be afraid of that question, and always be ready to ask it of our own work as well as that of others. Thank you for reading, and if you’d like to know more about this or any other aspect of my work, you can reach me here.

UV Microscopy – Summary and new UV fused silica optics

For a few months now I have been working on converting an Olympus BHB microscope to create something that I can use for UV microscopy down to below 300nm. This will be of use with my work on skin and sunscreen development as well as other areas. As can be seen here, the existing optics of the BHB very effectively block the UV below about 340nm which means the microscope needs modifying for it to be able to do this. To make it UV capable below 340nm means getting rid of all the glass optics and replacing them with UV transparent items such as UV fused silica, quartz and calcium fluoride. I’ve been providing updates as I’ve been doing the work on different aspects of it, but these are spread across a number of posts. I think it is time now to give a bit of summary about where I have got to, and also discuss the latest part of the work – replacing some of the optics with UV fused silica lenses.

Summary first, and I’ll give links to other pages which go over the topics in more details. In the Olympus BH series brochure (from Alan Wood’s excellent Olympus site) there is a nice cross section of a BHB microscope, and this is shown below.

Olympus BHB cross section diagram

With the cross section above I’ve added some labels to help explain the areas I’ve been working on with the UV conversion. Colours are purely there to help identify each section.

Starting at the light source (lower right). The conventional light source is Tungsten filament bulb which gives no UV. The aim will be to use either xenon or mercury xenon light sources for the UV imaging, as shown here. I may also use LED’s especially for the 365nm imaging, but not sure yet on that.

After the light, there is a set of lenses used for high magnification work. These can be moved in and out the beam as needed, depending on the objectives being used. These are glass, and I wont be replacing them, basically because I don’t need to. Some of the mercury xenon lamps I have are focusable, which means I can focus the light without needing these additional lenses, so I can just leave them out of the beam when they are not needed. This cuts down on cost and complexity for me.

After the high magnification lenses there is a mirror which turns the light through 90 degrees. That mirror is actually a good UV reflector so I am leaving that as it is. Then we come to the field iris lens, the auxiliary lens and the condenser, as discussed here and here. All of those are being replaced with UV Fused silica versions, and this will covered in more detail in the second half of this post. The condenser I am building is a simple one with just 2 lenses (simpler than the one shown in the image above).

Above the condenser, but not shown on the diagram is where the slide sites. Even that and its coverslip need to be made of UV fused silica or quartz, as discussed here.

Above the slide is the objective. I’ve written a few pages on these, as I have a range of different ones which can be used for imaging below 300nm. These include the Leitz 16x UV, Leitz 40x UV and 100x UV, Lomo 10x UV, and some others such as Beck reflecting objectives and a 32x Zeiss Ultrafluar which I have not written about yet on here.

Above the objective sits the binocular/trinocular head. I’m going to be swapping out the window and part of the prism for UV fused silica as mentioned here and parts have been ordered for that from UQG Optics. In the top part of the head is the photo eyepiece. Even this needs to be changed from the normal Olympus ones, and I’ve got a range of Lomo ones which are made from quartz as shown here.

After that comes the filters for selecting the UV wavelengths to be imaged and the camera, but that is a whole different issue and will be discussed another day. Phew, we got there in the end, but I hope that gives you some idea of the complexity of converting a microscope to be able to image UV down below 300nm.

Now the the latest update. As mentioned before, part of the conversion requires replacing the field iris and auxiliary lenses, and also the condenser assembly (which in its simplest form on the BHB has 2 lenses which I refer to as ‘Abbe top’ and ‘ Abbe lower’). UV fused silica parts for these came from a couple of optical suppliers in the UK – Laser2000 and Knight Optical – based on what was available from the ranges they had without things needing to be custom made (to help keep costs down). Let’s start by looking at the transmission spectra of the 4 lenses, as measured using my Ocean FX spectrometer and the lens transmission setup I designed.

Transmission of original Olympus lenses (individually and combined)

As can be seen above the 4 lenses cut the UV between about 280nm and 310nm, and when looked at together are effectively blocking everything below about 320nm. Obviously no good for UV imaging below 300nm. This transmission is similar to the modeled data shown here, and given that there were a lot of assumptions in that model, it is not surprising that it isn’t a perfect match. So what do the UV fused silica replacement lenses look like?

Transmission of UV fused silica lenses (individually and combined)

The UV fused silica lenses have essentially flat transmission spectra down to 280nm which is far as I can currently measure the lens transmission. However based on the specs for the fused silica, they would be good for use down to 250nm and even below.

Success. I now have 4 replacement lenses which will transmit the UV below 300nm. But the big question, do they work – can I get an image using them if I mount them in place of the existing Olympus lenses? For this test I just did a brightfield visible light image (this is purely a test of whether they can control light in a manner by which I can generate images of a subject, so at this stage of testing UV is not needed). For this I used a strewn slide of Simbirsk, Russia diatoms from Diatom Shop (very high quality mounted slides) and an Olympus 20x Splan objective. Images were stacked in Zerene (Pmax method). No additional sharpening or image modification has been done to enable comparisons to be made more easily.

Firstly, with all the original lenses still in place (these are low res versions of the original images).

Diatom image using the original Olympus lenses

Now, the same slide imaged with all 4 lenses replaced with UV fused silica ones.

Diatom image using the 4 replacement UV fused silica lenses

And then finally an image taken with the UV fused silica field iris lens, and the other lenses kept as original Olympus ones (as I want to be able to use this microscope for normal visible light work too and didn’t want to compromise its functionality).

Diatom image using the UV fused silica field iris lens but normal Olympus Auxiliary lens and condenser

The good news is that the replacement UV fused silica lenses still allow me to capture images. On the low resolution images above it is difficult to draw comparisons between the different setups, although the UV fused silica lens image looks to be a little lower in contrast. However I also created a set of cropped images from each of the original ones, and they are shown below at actual pixel resolution. Firstly, all 4 original Olympus lenses.

Cropped diatom image using the original Olympus lenses

Next, with all 4 UV fused silica lenses.

Diatom image using the 4 replacement UV fused silica lenses

And finally, with the UV fused silica field iris lens, and the auxiliary lens and condenser as standard Olympus components.

Cropped diatom image using the UV fused silica field iris lens but normal Olympus Auxiliary lens and condenser

What is going on here? Firstly replacing the field iris lens with a UV fused silica one (but keeping the Auxiliary lens and Condenser standard), I get a very similar image to when the field iris was left standard. Good, I can leave the UV fused silica field iris lens in all the time and still use the microscope for normal visible light work. Hurrah. Next, with all 4 UV fused silica lenses, I get a good image, although perhaps not as high contrast as with the original Olympus components. That’s ok, I can live with that as after all I am modifying a device designed and manufactured by a major microscope builder and I wasn’t expecting final image quality to be just as good. Interestingly, with the original Olympus Abbe condenser, there is evidence of strong chromatic aberration (red and green fringes) on the diatoms, but I do not see that with my UV fused silica lenses, and I’m not sure why at the moment. I somehow doubt I have created an APO lens setup, so more work needed to look into that.

It’s been a bit of a large update today, but I thought it was necessary to try and bring everything together as there is so much work that has gone in to getting this far. The UV fused silica lens replacements have enabled me to create an image, and of course their transmission will allow me to work down below 300nm once the final few components for modifying the binocular/trinocular head arrive towards the end of the year.

I’ve been told I am a bit mad (in a light hearted way I hope – anyway, I prefer the term ‘eccentric’) for trying to modify a microscope to image down below 300nm, and that starting with an Olympus BHB was not the right way to go about it. However by using this microscope it has been possible to modify and dismantle and reassemble it easily, something which wouldn’t have been possible with a more recent ‘plastic’ microscope. It certainly hasn’t been straightforward so far but if I look back over the last 8 or so months, it’s made me learn loads about a whole new area of research. It’s that drive to learn new things that keeps us as scientists going, and while yes I’ll use this for research in future, the learning of new things is in itself a worthy goal.

Thanks for reading, and if you want to know more about this or any other aspect my work, please contact me here.

UV Microscopy – modifying the binocular/trinocular head

Earlier this year I started my journey into the world of microscopy, and after a few weeks in thought ‘wouldn’t it be such fun to build a UV microscope’. This idea came along with all the optimism and naivety of someone who really didn’t know what he was letting himself in for. After all, I’ve been researching UV photography for a while and everything can just be reapplied, right. It’ll be easy, right. Well as Arnie said in Commando, that cinematic classic from the 80s, “Wrong”. Even though it has been challenging, it has been great fun and scientifically rewarding. You don’t really know how something works until it is in bits on your work bench.

A few weeks ago in an earlier post, I discussed about the glass elements in the binocular head which contains the eyepieces (covered here). The issue here was that the 2 elements – a circular window and a prism – which were present, blocked the short wavelength UV and would be effectively opaque below 300nm. Now this was a bit of a tricky situation. While I could potentially just get rid of the binocular head and have a tube which the camera would connect to, I wanted to keep the eyepieces and make it look like a normal microscope. To do this, there was only one thing to do, and that would be the replace the window and the prism with UV fused silica or quartz (or calcium fluoride) versions. The problem with this is that both parts would need to be custom made, and anything custom made out of these types of materials is expensive, especially something as complex as a prism made of multiple components.

For help with this I approached UQG Optics, who I have bought filters from before and who have always been good to work with. After drawing up a set of plans they are able to make components in the sizes that I need. Firstly the window. Being a circular this was relatively easy, however the size was unusual so is a custom build. The prism presented more of a challenge. As shown in the previous post, the prism is held within a metal slider. A few screws loosened, and a bit of gentle persuasion with a thin bladed screwdriver, and the prism was released from its cradle to reveal the following.

Binocular head prism

The prism is split into 2 halves – left and right in the image above. When the right hand side is slid into place, the light coming from underneath is bounced into the eyepieces, and none of it can pass directly upwards towards where the camera would be (when the vertical tube is added to the binocular head to make it a trinocular). When the prism is slid across so the left side of it is in the optical path, the light is split with some of it going to the eyepieces, and the remainder going vertically upwards to the camera. To replicate all of this in UV fused silica would be a stretch. It’s a big block of glass, and with some complex angles and shapes. Rather than trying to replicate the whole thing, I decided on a different approach – to cut the prism down the middle, and keep one half while replacing the other half with a simple block of UV fused silica. By keeping the right hand side of the prism, I can keep the side which directs light to the eyepieces and allows focusing. By replacing the left side with a block of UV fused silica, the light would go straight up to the camera. In this configuration, all the light would go to the camera, helping to improve optical efficiency and final focusing for the camera can be done using the live view function. The block of UV fused silica would simply be glued to the remaining part of the prism, and the whole thing mounted back into the the slider. Being a block with parallel sides that is smaller than the overall prism it is also much easier (and cheaper) to make.

Both the UV fused silica window and block have been ordered, and the prism will be sent off to be cut down and prepared. These should all be back with me in December, and are the last UV fused silica components required to convert the microscope.

In all of this the safety of the user (i.e. me) needs to be considered. UV light sources for microscopy tend to be high power, and when focused to tiny pinpoints are extremely dangerous. Couple this with the light then being passed through eyepieces, and the risk of eye damage becomes a very real threat. Retaining parts of the original glass prism for the light going to the eyepiece is actually an advantage here, as the glass itself will block some of the UV, especially the UVB. However there would still be a lot of UVA passing through. How to address this? I wear glasses anyway which have UV blocking layers on them, and I would also wear UV safety glasses when using the device, but it would also be good to have some UV blocking filters in the eyepieces themselves to block anything below 400nm. The eyepieces are 23.2mm diameter so rather than having some long pass filters custom made, I wondered whether there were any 23mm yellow camera filters out there. A quick on eBay search led me to Vintage Cameras Ltd, who had some yellow and orange filters in 23mm diameter for substantially less than they would have been to have custom made. There were two light yellow, two dark yellow and two orange filters as shown below.

23mm filters

Measuring the transmission spectra of the filters using my Ocean FX spectrometer gave the following.

Transmission spectra for the yellow and orange filters

In the scan above, Yellow 1 and 2 are the light yellow filters, Yellow 3 and 4 the dark yellow ones and Orange 1 and 2 the orange ones (if that wasn’t obvious). Yellow 3 and 4 were almost identical, hence the lines overlap. All showed well defined long pass behaviour, although the light yellow and orange filters had a bump in the transmission below their cutoff. Zooming in to the short wavelength end gives the following.

Short wavelength filter transmission

The light yellow filters (Yellow 1 and 2) start letting some UV in at around 300nm and then transmission gradually rises from there. However the darker yellow and orange filters are effectively blocking all the UV light from 400nm to 250nm (less than 0.05% transmission). As the goal is to block the UV while still retaining usability the darker yellow filters are the best trade off between UV blocking and visible transmission, and these will be the ones I use in the binocular head for the UV microscope. It should be emphasised though, that these filters do not obviate the need for UV protective eyewear, they just reduce the risk of damage when using the device while enabling it still be to be used like a normal microscope. I shall still determine the light coming through the eyepieces spectroscopically in the final build.

This UV microscope build has been a really interesting scientific challenge, teaching me more about optics (and microscopes) than I thought there was to know. All the replacement UV fused silica components are now ordered so hopefully in early 2021 I’ll be able to give an update on how it is working. Thanks for reading, I hope you enjoyed it and if you want to know more about this or my other work, you can reach me here.

UV imaging – Yooperlite

Come on, I bet you were not expecting to see a title saying ‘Yooperlite’. What even is Yooperlite? Well, Yooperlite is a very unassuming looking grey rock, in which is hidden a heart of burning fire. However in order to see its flaming heart requires a mystical light that you can’t see. Ok, enough of that and back to the science. Yooperlite is a a Syenite rock which is rich in fluorescent sodalite, and is found in Michigan. While it looks grey in visible light, when illuminated with long wavelength UV parts of it shines yellow and orange, as shown below.

Yooperlite UV induced visible light fluorescence

Just on case you were wondering, the rock doesn’t look quite as interesting when imaged using normal visible light.

Yooperlite imaged under normal visible light

Indeed, it is equally uninspiring when viewed using UV reflectance imaging.

Yooperlite viewed using UV reflectance imaging at 365nm

It really does shine with a fire when illuminated with a UV source. This type of UV induced visible light fluorescence is not uncommon in minerals, with many of them glowing with different visible wavelengths under UV light, however Yooperlite is extremely bright and pretty to look at.

The images above were taken with a macro lens on a normal camera. However, once you realise what needs to be done to carry out this type of imaging, it can also be applied to microscopy. The image below was taken using an Olympus BHM reflectance microscope using a 5x objective, as a stack of about 20 images.

5x magnification image of Yooperlite

Under the microscope, the Yooperlite material looks truly 3d, looking as if there is a fire within underneath a layer of ice.

UV induced visible light fluorescence photography is an interesting and powerful photographic technique, used in a wide range of research fields, from dermatology and forensics, to art conservation, biology and microscopy. As it uses a standard visible light camera and lenses which transmit visible light, it does not need some of the specialist equipment required for UV reflectance photography. However there are still a few things which need to be done to make it work. The light source needs to be filtered to let only UV light through to the subject (I used a Baader U filter on a 365nm torch), and the camera lens needs a filter which blocks UV from reaching the sensor. ‘Ah’, I hear you say, ‘my camera has a filter on the sensor which blocks UV, so I don’t need an additional filter’. Well, yes and no. Normal visible light cameras do have a UV blocking filter, but not all of them block all the UV, so additional UV blocking filtration is also needed on the lens in that case. it is also good practise to use one to eliminate the chance of elements inside the lens fluorescing on UV exposure. I used a 420nm long pass filter which had low inherent fluorescence, so did not give an additional colour cast from UV exposure. Also, you need to image in the dark to get a true fluorescence photo. Really, really dark. All extraneous visible light needs removing for a true fluorescence image. The Yooperlite fluorescence is so strong though, that even in daylight, if you shine a 365nm UV torch on to it you can see the yellow.

The sample of Yooperlite I used was bought from the Caledonian Rock Shop and is about 2cm across. I used a 365nm torch, with additional filtration from a Baader U filter for filtering the light. The camera was a Canon EOS 5DSR and the lens a Rayfact 105mm f4.5 macro lens, with a 420nm long pass filter on the front. The Yooperlite was placed in a box painted with Semple Black 3.0 paint to effectively removed UV reflection and fluorescence around the rock.

Thanks for reading and if you want to more about this or any other aspect of my work, you can reach me here.

UV Microscopy – designing a UV condenser

I’m a bit of dinosaur when it comes to the idea of ‘modelling’. As an experimentalist, I like build and test and then refine my idea, rather than rely on models. Also I’ve been involved in far too many projects where the term ‘model’ and ‘hypothesis’ get used interchangeably, resulting in huge issues which could have been resolved by just doing a few experiments. However there is no doubt that at times and when used properly, modelling can produce useful insights for projects.

With my UV microscope build I am converting an old Olympus BHB microscope into something I can use for doing transmission microscopy down to and below 300nm. At these wavelengths glass becomes opaque and lenses need to be made from materials such as quartz, UV fused silica, or calcium fluoride. And in a microscope there are a lot of lenses which need to be swapped out (as discussed here).

The condenser lens is positioned just before the sample and focuses the light onto the sample – a rather important role. The standard Abbe condenser on my BHB actually did a good job of letting UVA though (see here), but was blocking the UVB. Not unexpected really, as it is just glass after all. In fact, after being reflected through 90 degrees by the mirror below the stage the light then goes through 4 lenses before reaching the sample – the field iris lens, the auxiliary lens, and then 2 lenses in the Abbe condenser – none of which are suitable for UVB. To try and understand a bit more about the light movement and behaviour in these lenses, it was time to delve into the murky waters of optical lens design software. So it was time to grab a fresh cup of extra strong coffee and jump in…..

As a novice in this area, I thought I’d try a free piece of software which had some online tutorials – WinLens3D. The first thing to try was to produce a model of the 4 lenses below the stage. Unfortunately one of the lenses in the condenser is an odd shape, so I had to make an assumption for that one that I’d treat it like a half ball lens (a hemisphere). Creating a model with 4 lenses, gave the following.

4 lens stack using BK7 glass as the lens material

Moving from left to right, the 4 lenses in the stack are the field iris lens, the auxiliary lens, and then the 2 lenses from the condenser. This is a rough model, based on some of my measurements, so will not be 100% accurate. The good thing about this type of modelling software, is that in addition to looking at how the light moves through the lenses, the optical properties of the materials can be used to calculate a theoretical transmission spectra for the system, and this is shown in the graph in the image above. Assuming they are made from BK7 (a common glass for lenses) transmission starts to drop at 400nm, before reaching essentially zero at 300nm and below. With this you can see why normal glass is no good for UVB imaging. However with the model, you can change the materials the lenses are made from and see what effect that has. Changing from BK7 glass to UV fused silica gives the following.

4 lens stack using UV fused silica as the lens material

Changing to UV fused silica alters the light movement through the lenses slightly – as expected as the actual focal lengths of a specific lens design will change with refractive index of the material it is made from. However the biggest difference is in the transmission spectra. UV fused silica has great UV transmission, and as you can see the predicted transmission plot now shows a pretty much flat transmission curve down to 250nm, well into the UVB region. The software then does something a bit odd though – the transmission plot drops straight to zero at 240nm. I actually think this is a quirk in the software, as the fused silica should be good down to around 220nm. I suspect the data for the silica just stops at 250nm. Overall, moving to UV fused silica looks good – the model seems to predict good transmission, and the change from glass while slightly altering the light path should not make it unusable.

What to do about the condenser? I have a vintage Zeiss quartz microscope condenser currently waiting for me in the US to be collected the next time I visit, but Covid has put all those plans on hold for now. In the mean time, is it possible to modify an existing condenser by replacing the glass elements with UV fused silica ones? Using an existing condenser is the preferred option, otherwise it’d need custom machining work in addition to new lens, and the costs would rise very quickly. Taking a spare condenser apart presented the first challenge. One of the lenses was secured by a ring that had been glued in place in the factory. Having tried polar solvents, non polar solvents, heat, cold and various other methods to free it off nothing worked, and I resorted to the tried and tested approach of ‘hit it with something hard’. I got the lens out but not in any form which was useful. It did however leave me with the metal condenser housing which could be reused. The lens itself was 35mm diameter, which is an odd size for my supplier to source a replacement. To fix this I bought a 30mm filter ring and glued it in place where the old lens was. This allows the use of 30mm diameter lenses, which are much more common. I currently have one on order, and will update when it arrives.

The top part of the condenser has a very odd shaped lens in it. It looks to be based on a ball lens, which then has a section sliced from it, and a chamfer ground into it as can be seen below.

Olympus Abbe condenser lens

This is a not a common shape, and after trying multiple suppliers have currently given up on trying to find someone who can make this in UV fused silica. If I win the lottery, I’ll come back to getting one custom made, but it’ll have to wait for now. Looking at the shape though, it got me wondering whether a half ball lens would work. In fact it was a half ball lens that I used in the WinLens3D modelling above, and it looked like it should do the job. Half ball lenses are a bit more common, although I was looking for a large diameter one, which is much rarer. In the end I found an 8mm diameter UV fused silica one at Knight Optical here in the UK. This was much smaller than lens it would replace, but was worth getting to test the idea. The two lenses are shown below, highlighting the differences in shape.

Comparison of the half ball lens with the original one

The smaller lens left me with a bit of a problem – how to mount it in the condenser? The original lens was held in place with a screw ring, but the new lens was much too small for that. I resorted to my old friend JB Weld, which is a 2 part epoxy adhesive used for fixing anything as far as I can tell. The good thing is that it is opaque as well, and I just glued the lens in place using the JB weld to fill the gap around the lens. The photo below shows the original (left) and the modified condenser (right), and you can see how small the UV fused silica half ball lens looks compared to the original one.

Original and modified condensers

Making one of these is all well and good, but if it cannot illuminate a sample it really is not going to be a good condenser. As a quick test I imaged a sample slide of Gorgonian sea fan spicules (from Diatom Lab). These guys make high quality microscope slides great for microscopy testing and imaging, and I’d recommend checking them out if you want slides for imaging. The objective was an Olympus 20x SPlan, and the lighting a tungsten bulb in the microscope. This is a visible light image taken with a normal camera, just to test to see if the condenser would work. The warm cast to the image comes from the tungsten bulb. Images were collected as a stack of about 15 shots and processed in Zerene stacker. UV testing will come later. Firstly one of the spicules taken using the original unmodified Abbe condenser.

Image with the standard Abbe condenser

Now an image where the standard Abbe condenser was replaced with just a single 8mm UV fused silica half ball lens.

Image with the 8mm UV fused silica half ball lens

While slightly lower contrast than the standard Abbe condenser the 8mm UV fused silica half ball lens was still able to provide good lighting for an image of the spicule. Success. Also the condenser with the 8mm half ball lens is currently missing the second lens which is present in the standard one, so hopefully image quality should improve further with both lenses in place.

There are some drawbacks to using such a small lens. Sample size is limited, along with the choice of objectives. I need to use 10x or higher magnification objectives with it, but as my UVB capable objectives are 10x and above this is not a problem. The light leaving the half ball lens is highly convergent, so condenser position is crucial to getting good lighting. How this will work in UV remains to be seen as focal length will be different to that in visible light and may leave me with slightly less working distance to play with. The image with the half ball lens was slightly darker than with the standard condenser leading to longer image acquisition times (approximately one stop difference).

Where does this leave me? The conversion of a microscope to being able to do UVB imaging is a complex job, certainly more complex than I originally thought it was going to be. As such it takes time and testing. Modelling of the optical properties of the lenses has proved useful, and testing so far is looking good. I’ve been able to modify an existing condenser to install a lens which has great UV transmission characteristics and use it to create good quality microscope images. More work to be done for sure, but the UV BHB is coming along, and I am now a step closer to completion. If you want to know more about this or any other aspect of my work, you can reach me here.

Addendum – after writing this article, I’ve been able to source some larger diameter half ball UV lenses (12mm as opposed to 8mm) and they have been sourced for future testing.

UV Microscopy – All aboard the optical train

When doing normal visible light photography, the question of whether the wavelengths you’re interested in capturing can even reach the camera, tends not to come up. Lenses are transparent to visible light, and our eyes and camera sensors are sensitive to it, so we can see just what we are going to be imaging. Simple. However with UV photography that is not always the case. Normal camera lenses block varying amounts of UV and if you want to image the UVB region, you need to delve into the realm of dedicated UV optics without normal glass. Even with those considerations, with UV photography once you have a suitable lens and filter, and a modified camera you are good to go. The concept of doing UV microscopy though, takes the things you need to consider for photography and amps everything up to 11. Today I’ll bring some of the work that I’ve been doing to build a UV microscope together and see what I have still got to do.

The optical train for a microscope is quite complex. In my microscope, starting with the light source, the light then goes through between 1 and 5 lenses, and bounces off a mirror, all within the body of the scope before going through the condenser. Then we have the slide and coverslip with the sample. After that we have the objective lens. Above the objective lens we have the head with the eyepieces, which also contains the photoeyepiece. And it is only all of this that we have the camera. Some of these I have discussed before, and are linked in the description above. I’ll now go through some more recent work looking at whether the internal lenses, mirrors and windows within the microscope are suitable for UV, and if not how to address that.

Within the body of my BHB microscope there are a set of lenses and a mirror that the incoming light has to traverse before it even reaches the condenser. These are to focus and direct the light so it can reach the sample with enough intensity. 3 of the lenses aren’t absolutely necessary for the UV imaging, as the xenon and mercury xenon lamps I have have some degree of internal focusing. However this still leaves 2 lenses, and mirror which are necessary. The two lenses can be seen in the image below.

Two lenses for focusing the light

The top lens above is the Auxiliary lens, and this isn’t always needed depending on the magnification you are using. Below that with the numbered scale is the Field Iris lens. Below the Field Iris is a mirror which bounces the light from horizontal to vertical. With the Field Iris lens removed, you can just about see the mirror underneath.

Mirror beneath the Field Iris lens

In order for the microscope to be usable for UV imaging, the lenses need to be able to transmit the UV, and mirror needs to be able to reflect it. I removed both lenses and measured their UV transmission using my lens transmission testing rig, and got the following.

Auxiliary and Field Iris lens transmission

Both lenses looks like they’d be OK for 365nm imaging, but at the shorter UV wavelengths the transmission drops quickly. This makes sense as they are glass lenses, and normal glass absorbs UVB. So they’d be no good for UVB imaging. Determining whether the mirror would be suitable for UVB was a bit more problematic. I do not have a setup for accurately measuring specular reflection from shiny surfaces, and I didn’t want to remove the mirror unless absolutely necessary, as they can be annoying to realign. This meant trying to test it in-situ. To do this I used a Xe lamp. I measured the spectrum of the lamp by itself. Then shone the lamp off the mirror and measured the spectra again. I then normalised these for max irradiance (which occurred at about 400nm) and calculated how much light was reflected from the mirror vs the light output from the lamp alone for between 250nm and 410nm, and got the following.

Mirror reflection from 250nm to 410nm

The mirror reflection shows no major dips down to 250nm indicating good reflection in the UVB. This is not a perfect test, as I am assuming that 100% of the light is reflected at 400nm. However it is good enough for seeing whether there are any big dips in the reflection spectra. The next job was determining focal lengths for the lenses. The Auxillary lens was 40mm diameter, planoconvex and about 7cm focal length. The Field Iris lens was 40mm diameter, biconvex and about 13.5cm focal length. For replacement UV suitable lenses, I’ve gone through Laser2000, who have good range of lenses in different sizes and focal lengths and went with a 7cm planoconvex, and 15cm biconvex. For the biconvex the available focal lengths were 12cm and 15cm, however as the wavelength decreases the focal length does too for these simple lenses, so the 15cm is nearer to the original in the UV. These lenses should enable good transmission of UVB to the condenser. Will this prove to be the right choice – I shall see when then lenses arrive. Watch this space…

The next thing to consider what happens to the light between the objective lens and the camera. On the Olympus BHB there is a binocular head which fits on top of the microscope which allows the user to view the sample. This can also be fitted with a third vertical tube (making it a trinocular) which enables a camera to be fitted. The head however also has some optics in it – a thick window at the base, and a beamsplitting prism to enable the light to be split between the eyepieces and the photoeyepiece for the camera. Of course, every piece of the glass in the optical train has to be assessed for suitability when thinking about UV imaging. How do these components impact UV transmission? Thankfully the BHB head is straight forward to take apart (I love old mechanical designs). Doing so reveals a thick window at the base of the head and the prism beamsplitter assembly, as shown below.

Thick window from BHB head
Beamsplitter assembly from BHB head

The window is thick, very thick. The size is 20.9mm diameter and 9.9mm thick, but it does look to just be a window and not a lens. It also looks to be a simple single element. The beamsplitter is more complicated. In the position shown in the image above the light is sent to both the viewing eyepieces, and the photoeyepiece. While not obvious in the photo, this requires two prisms – one on top of the each other – and there is a glued interface as well, making it quite complicated. There is no path to the photoeyepiece without going through this prism arrangement. The transmission spectra for these two components are shown below.

Transmission spectra through the BHB head optics

Both the window and the beamsplitter show good transmission at 365nm, but the transmission does drop quickly as the wavelength gets smaller, and is essentially zero by 300nm. For imaging at and below 300nm both of these will block all the UV and be opaque. This could be a tricky one to fix – if I want to use the head for UVB, so I can view the sample before taking the photo, I’ll need to figure out a way of getting the UV through these components. I can just use a vertical monocular tube for the photos, as that has no optics in it, so there is another approach, but I’d like to keep the optical eyepieces if possible. More thought needed here, especially with regards to making the use of eyepieces with UV light sources safe…… After more thought (and more coffee) I shall take some measurements and source suitable components to test.

Just over 6 months ago now I started my journey into microscopy, and it has so far proved fascinating and challenging in equal measures. Setting myself the challenge of making a UV microscope able to be used down below 300nm have really made my life difficult. But then science is often difficult, and a challenge is not something that should be shied away from. Being able to image in UVB and UVA will be useful for my sunscreen research and for imaging sunscreen formulation structure. If I were to try and have a UVB suitable microscope built, I doubt I’d get much if any change from 100,000USD so until I win the lottery is a bit outside my R&D budget. In fact a couple of the objective lenses alone if bought new would cost more than I have spent on my entire project to date, so a self build is the only option. I’m not there yet, but am gradually working through the issues and will update as and when I have more results. If you’re interested in this or any other aspect of my work, I can be reached here.

Microscopy – reflectance imaging of a modified camera sensor

It’s always the same when I attempt some DIY – I start out with a plan, and a pile of bits, and I invariably end up with parts left over at the end, and a nagging question about where they should have gone. It’s been a little like that with Project Beater, my microscope build. As I’ve gone along buying spare parts for it and bits of donor equipment, I’ve ended up with some stuff which I wasn’t really sure what to do with. A couple of weeks ago it dawned on me that I almost had enough parts to make a reflection microscope, to go along with the transmission setup I already had. So back on eBay (for a few more missing parts) and that is precisely what I did….

Reflection microscopy relies on top illumination and is used for non-transparent samples. It’s often used for looking at gems, metals, feathers, fabrics, etc etc. Before I show you a picture of the setup though, here’s one of the sample pictures I took to try it out once it was assembled. I’ll leave you to figure it out what it is for now, and come back to it later…..

Can you tell what it is yet?

My setup was based around an Olympus BHM chassis, a reflected light setup placed on top of the microscope, and then a normal or trinocular head on top of that (depending on whether or not I want to take photos). The objectives are old Olympus Neo ones, designed specifically for this type of ‘top down’ imaging as they have a channel around the lens to allow the light to be shone down there rather than through the objective itself. Here’s what it looks like from above.

Olympus BHM reflection microscope setup

The original light source is a 15W 6V Tungsten filament bulb, but these are both expensive and difficult to source now, and give a very warm colour cast to the image, so instead I just used a 10W cool white LED source I built previously. This gives out plenty of light, and with very little colour cast. Plus it has the added benefit of being virtually immortal.

So, back to the test object. Did you figure out what it is? Here it is in its entirety.

44 Remington Magnum inert round

The image was one of the ‘4’s from an inert 44 Remington Magnum round, taken with a 10x Neo objective, imaged using a camera phone through the eyepiece (overall magnification 100x).

While looking at old brass is one use for these types of microscopes, I did have a different use in mind when I built it. I have some monochrome converted cameras for my UV work, and for a while I’ve been wanting some nice pictures of a camera sensor to show what it looks like as a result of the conversion. So I got a damaged sensor which had been partially converted to monochrome, and as a result had regions which were left untouched, regions which had the microlenses removed, and other parts which had had the microlenses and Bayer filter removed. Here it is (with the area where the Bayer filter has been removed to the left of centre);

What does a camera sensor look like in its normal form? Here we have a reflection image of the sensor from part of it which hadn’t had anything removed, taken with a 40x Neo objective.

Camera sensor including microlenses and Bayer filter

You can see the very neat arrangement of red, green and blue filtered pixels (2 green to 1 red and 1 blue). On top of each coloured region is a microlens. This focuses the incoming light through the coloured filter and into the sensor to be collected. In a conversion to monochrome, these microlenses and the Bayer filter are removed, leaving the bare sensor behind. Here you can see what it looks like as the microlenses start to be removed.

Microlenses partly removed.

And then, with the microlenses fully removed.

Microlenses removed

With the microlenses removed, you can now see the coloured squares of the Bayer filter. Oddly enough the green ones look smaller than the red and blue ones, and I’m not quite sure why that would be the case. With the monochrome conversion, this coloured filter layer is also removed, and the bare sensor is revealed.

Monochrome converted region

With the monochrome conversion, all trace of the coloured Bayer filter is now gone. It also reveals why the microlenses play an important role in the sensor design. The darker squares are, I believe, the sensitive parts of the pixels. The microlenses gather light from a larger area than the pixel itself, and focus it down into the sensor to be collected. Sensor architecture also plays a significant role in their effectiveness at this task, but that is a story for another day…

The sensor I had for imaging was damaged, and quite fascinating to look at under the microscope. I found a region where something sharp had been dragged across it, scraping away the microlenses and Bayer filter, revealing the sensor underneath. It enables the different regions to be captured in one image – raw, untouched, sensor with microlenses, microlenses partly removed, microlenses completely removed from the Bayer filter, and bare sensor with the Bayer filter removed – as shown in the image below taken with a 20x Neo objective.

Scratched surface of the camera sensor

As a quick note for the microscopists amongst you, I used bright field imaging for these images. With this the light is directed down the objective optics, before being reflected back up along the same path. This reduces the image contrast somewhat. The Neo objectives have a path around the edge of the optics for the light to travel, ending up with an image which I suppose is a bit like a dark field transmission image, but I did not use there here as the bright field illumination photos looked nicer.

When you start on a new adventure, it isn’t always clear where you are going to end up. When started with microscopy about 6 months ago, I’d assumed I would be doing transmission imaging. By learning as I go along, I’ve now ended up with a reflection microscope, which has allowed me to visualise what I had theorised about when writing about monochrome camera conversions and UV imaging. As scientists we should always be open to letting the work guide us, in addition to just ‘running the experiment’.

Thank you for reading, and if you’d like to know more about this or any of my other areas of work, I can be reached here.

UV leakage through stock camera sensor filters

When it comes to UV fluorescence photography, setting up of the lighting, subject and imaging system are key to getting good results. An important part of this is knowing how your equipment works, along with its limitations. Today I’d like to share the results of testing which show how UV is leaking through the internal filter stacks of some of my cameras, and how that effect can be rectified.

This all came about when I first measured the transmission spectra of the filters which were removed from a camera I was having converted to multispectral imaging (a Canon EOS 5DSR). The filter stack consisted of a green IR blocking filter, and a dichroic ICF filter which is supposed to block UV and IR. However when I measured the transmission through the two filters I got the following spectra.

Transmission through Canon EOS 5DSR internal filters

What was worrying was the ICF filter had a significant UV transmission peak at around 365nm, which means that UV light can actually reach the sensor in a stock camera (the red line is the combined transmission through both filters). This got me wondering what the end result of this type of leakage would be in a fluorescence image.

Fast forward about a year, and I was doing some fluorescence imaging for an article, which was using UV light to look at the fluorescence of a glass vase, and seeing what the effects of different blocking filters on the camera lens were. A quick guide to the setup. Light source was a Hamamatsu LC8 with a 200W Xe lamp. A Baader U was used on the output of the light source, to make it UV only and remove the small amount of visible and IR light being emitted. Subject was a painted glass vase, placed in a box painted with Semple Black 2.0 paint which I’ve found to be both highly light absorbing and very low fluorescence making it ideal for UV imaging work. Camera was a stock, unmodified Canon EOS 6D with a Rayfact 105mm UV lens. Imaging was done in the dark apart from the UV light source (obviously). All exposures where for the same length of time.

Firstly, the vase imaged with no filtration on the camera lens.

Vase fluorescence under UV light, with no lens filter

The vase shows the expected blue fluorescence, but there seem to be red areas in the image and they look to be reflection rather than fluorescence based on the parts of the glass they are showing up on. It is also red on some of the black painted cardboard. The ever present dust also shows up with blue fluorescence (damn you, optical brighteners).

The next stage was to add a UV blocking filter to the front of the camera lens. First one was the Schott KV418 which is well known in the UV fluorescence imaging world as having good UV blocking and low inherent fluorescence.

Vase fluorescence under UV light, imaged with a KV418 blocking filter

Adding the Schott KV418 to the front of the camera lens removed the areas which appeared red in the unfiltered image, confirming my suspicions that those areas where to do with UV reflection which was making its way through the stock internal camera filters and hitting the sensor.

As a final confirmation that reflected UV was indeed the cause of the red highlights in the unfiltered image, I removed the Schott KV418 filter and replaced it with a LaLa U filter from UVIROptics. This filter lets UV through, but very effectively blocks visible and IR light, and is a great choice for UV photographers. Doing so, gave this image.

UV image of the vase, using a LaLa U filter

Using the LaLa U filter, the blue fluorescence is now gone (as expected as the filter is blocking the visible light), but the red still remains in the areas shown in the original unfiltered image. It is a little darker as the LaLa U filter is absorbing around half of the UV light, due to its transmission curve. Remember, this is a stock camera with internal filters which are supposed to be blocking UV.

Ok, so it is fairly obvious that the stock Canon EOS 6D filtration is not enough by itself to block reflected UV light with the fluorescence images. Schott KV418 is good at blocking that reflected UV, but, Schott KV418 is no longer widely available (and do you homework on anyone claiming to be selling it – the only legitimate source I know of was ITOS in Germany, and I’m not even sure they have any anymore). However I found that Zeiss T* UV filters also did a good job of blocking the UV and were themselves low fluorescence. This is what the vase looked like when imaged with using a Zeiss T* filter on the camera lens.

Vase fluorescence under UV light, imaged with a Zeiss T* UV blocking filter

As a final image, and why not, here is the vase imaged using a Tiffen 12 yellow filter, as the blocking filter.

Vase fluorescence under UV light, imaged with a Tiffen 12 filter

The Tiffen 12 image doesn’t really add to the story, I just think it looks funky.

So, some comments on the images and the experiment above. The transmission spectra for the filters I have was for a Canon EOS 5DSR, and the photos were taken using a Canon EOS 6D. The cameras were different, and I do not have the filter stack from a 6D to check. I therefore cannot be sure that it would be exactly the same as the 5DSR one. However the images back up the hypothesis that the stock filters are leaking some UV. I used a Rayfact 105mm UV lens for the images. That is a nice sharp macro lens, but has high UV transmission, which emphasizes any UV leakage. I have done some other work with different (normal) camera lenses, and the effect is still visibly, although reduced in intensity. I used a Zeiss T* UV filter for one of the images. Not all UV filters block UV – some are just plain glass. Unfortunately this is an example of where marketing trumps science, and claims are made about products which do not stand up to testing. In the absence of owning a spectrometer, if you shine a UV torch (which has the black looking filter on it to remove visible light) through a UV filter onto white paper, if the UV filter is not blocking the UV light then the paper will fluoresce. If the paper does not fluoresce then the UV filter is blocking the UV light, giving you a rough indication of how good a blocking it has. This of course needs to be done in the dark.

I should note that I have tested some Nikon and Sony sensor filters stacks for transmission, and the ones I tested at least showed much less UV leakage than the Canon ones, so not all cameras behave the same.

My message to you here, is that it is good practice to ALWAYS use an external UV blocking filter in front of the camera lens when doing UV fluorescence imaging. While it is easy to say that the internal filters on digital cameras are designed to block UV light, that internal filtration setup is not always sufficient. Knowing how your kit works is vital to generating good reliable scientific images for your work, as is understanding its limitations. When you understand what it can and can’t do, you are in a much better place to explain any unusual or unexpected results. I hope you enjoyed reading this, and if you’d like to know more about this or other aspects of my work, you can reach me here.