Is anyone doing UV transmission microscopy?

[kds315*]

Klaus, thanks, I know the Ultrafluar is good, but very expensive. Are the other ones you mentioned really good? For both visible and UV light, or just UV? I would like to use through-the-objective UV lighting to excite fluorescence.

And why do so many UV objectives require a coverslip? Doesn't a coverslip reduce transmission? Or is it so thin that it doesn't matter?

Lou, the Ultrafluar is fully collor corrected from about 200nm to 1100nm (see link). The UV Mikrotar seems to be just a quartz lens. The reflective lenses mentioned (Edmund Optics) are also fully color corrected.

I forgot to mention the various LOMO UV lenses, quite cheap to find.

If you want to do high quality UVIVF then the Zeiss Fluar lenses should work well, as they were designed for that once and allow enough UV light to pass, see here:
https://www.micro-shop.zeiss.com/en/us/ ... ar-5x-0.25

Older, finite low power (low magnification) Fluar objective can be had used for just around $150. Modern infinity ones are $$$$

[Lou Jost]

Thanks Klaus. I had seen those UV Lomo objectives. On this forum we've seen a very wide variance in the quality of different Lomo objectives, from "best in class" (3.7x) to unusable. Is there a Lomo UV that is really good?

[kds315*]

Thanks Klaus. I had seen those UV Lomo objectives. On this forum we've seen a very wide variance in the quality of different Lomo objectives, from "best in class" (3.7x) to unusable. Is there a Lomo UV that is really good?

Can really say, I have a few but packed away deeply and I haven't really made use of them. They do need compensating UV eyepieces! Without them I would not be surprised about crappy results...

[kds315*]

Peter Höbel http://www.mikroskopie-ph.de/ occasionally shows UV images of diatoms for increased resolution in the German forum. He uses a 365 nm LED, oblique illumination, a selection of finite objectives and a monochrome C-mount camera.

High intensity UV and long aquisition times are quite incompatible with live objects. The trend in live imaging is to use at least violet illumination instead (405 nm). But for prepared diatoms it's great!

Examples:
https://www.mikroskopie-forum.de/index. ... ic=14712.0
https://www.mikroskopie-forum.de/index. ... #msg138602
https://www.mikroskopie-forum.de/index.php?topic=1096.0

I know Peter's work from teh time when I started using the early NICHIA 365nm UV LEDs. He was an early adopter, too. He used it for Microspcopy, me for reflected UV photography.

Lous, this answers your question about light sources for UV induced visible fluorescence: I would use a Nichia UV LED for that with a LINEAR current controller (flickerfree!)

[Lou Jost]

Thanks for that advice. I have a Nightcore UV flashlight which I think uses a Nichia LED...

[jmc]

So Lou, when you say "I would like to use through-the-objective UV lighting to excite fluorescence." this gives you much more choice in terms of objective lenses, and you wouldn't need to ultra expensive ones designed for imaging in the UV.

As mentioned above the Olympus UV Fl ones are worth looking at in addition to the Zeiss Fluars, as they pass loads of UV and have inherently low fluorescence.

When I think UV microscopy, I'm thinking more about imaging the UV, rather than using the UV to induce fluorescence and imaging that fluorescence. This puts a different set of requirements on the objective and the imaging setup.

[kds315*]

Thanks for that advice. I have a Nightcore UV flashlight which I think uses a Nichia LED...

Their drivers often are switching, so I'm not sure those are flicker free.

[kds315*]

So Lou, when you say "I would like to use through-the-objective UV lighting to excite fluorescence." this gives you much more choice in terms of objective lenses, and you wouldn't need to ultra expensive ones designed for imaging in the UV.

As mentioned above the Olympus UV Fl ones are worth looking at in addition to the Zeiss Fluars, as they pass loads of UV and have inherently low fluorescence.

When I think UV microscopy, I'm thinking more about imaging the UV, rather than using the UV to induce fluorescence and imaging that fluorescence. This puts a different set of requirements on the objective and the imaging setup.

Indeed Jonathan, the Olympus ones are an alternative to consider, as well as the ones from Nikon, too. Both rather affordable compared to Zeiss, Leica and Mitutoyo. Not to forget companies Reichert and Wild.

Reflected UV seems to be not really done often, as I noticed that only few objectives were made for that, guess since it cannot be directly viewed and needs image conversion per camera or sensor system into the visible.

[Lou Jost]

Jonathan, I'd like to do both direct UV imaging (specifically for higher-res stacks of the diatoms I am finding on my orchid leaves) and epi fourescence with UV excitation. Those do put different demands on the objectives. For the epi lfluorescence application, transmission is important but there is no need for the objective to be color-corrected into the UV range; good visible-light color correctoin is important. In direct imaging for high-res monochrome subjects like diatoms, there will be no visible light and only a single wavelength oif UV light, so color correction practially does not matter, but transmission matters a lot.

I think flicker rate doesn't matter at all because the exposures are going to be fairly long.

I've just rigged up mu ordinary visible-light epi illuminator (not a proper fluorescence illuminator) so I can attach my Nitecore flashlight and a visible-light-blocking filter (Schott UG1) and I have seen my first biological fluorescence in leaves! Yippee! But exposures are 60 seconds per shot.

[Ichthyophthirius]

Klaus, thanks, I know the Ultrafluar is good, but very expensive. Are the other ones you mentioned really good? For both visible and UV light, or just UV? I would like to use through-the-objective UV lighting to excite fluorescence.

And why do so many UV objectives require a coverslip? Doesn't a coverslip reduce transmission? Or is it so thin that it doesn't matter?

Hi Luo,

The Ultrafluars were good for microscopy in UV (as there were no alternatives) when we are talking about Zeiss finite objectives.

They were corrected achromatically from 230 - 700 nm with "practically no focus shift when changing from one wavelength to another". However, there are very few images around taken with these in visible light. The image quality doesn't seem to be great. The main application seems to have been microphotometry. See pages 28f, 58 http://www.science-info.net/docs/zeiss/ ... ystems.pdf

For typical fluorescence microscopy (near UV-excitation + visible light emission) Zeiss made Neofluar objectives which give excellent, high contrast and bright images (they have high NA). Unless you want to do UV-excitation + UV-emission or UVB excitation, you have a lot more options with the Neofluars.

The coverslip: that's probably from the microphotometry applications. It protected the sample from the objective and the glycerol immersion. The lowest magnification dry objectives (Ultrafluar 10/0.20) can probably be used without a coverslip (not from my first hand experience but from looking at the usual coverslip thickness tolerance at such a low NA). At NA 0.20, the coverslip is probably just needed for parfocality.

The higher NA ones need the coverslip. It's quite thick (0.35 mm) because Quartz glass coverslips had to be ground and polished to that thickness (normal glass coverslips are "simply" cast from molten glass).

Regards, Ichty

[Lou Jost]

Ichthyophthirius, that's very good info about the Ultrafluars. Neofluors are much more affordable and based on your information, they are also probably better for fluoresence applications. Thanks, you've saved me lots of money!

The higher NA ones need the coverslip. It's quite thick (0.35 mm) because Quartz glass coverslips had to be ground and polished to that thickness (normal glass coverslips are "simply" cast from molten glass).

Most of the UV objectives I have seen on eBay, including high-NA ones, ask for regular 0.17mm coverslips. I've seen a few objectives, especially Leitz, that ask for quartz 0.35mm slips, but those are definitely in the minority, at least on eBay...Maybe the more modern and professional objectives want the quartz coverslips and those don't appear as often on eBay?

[Ichthyophthirius]

Hi Luo,

The early specialised UV objectives (Zeiss Ultrafluar; Leitz UV (still on list from 1985)) used 0.35 mm quartz windows.

Maybe more recently they switched to 0.17 mm glass because the users didn't want to pay $5 for a cover slip any more?

A typical coverslip glass is Schott borosilicate glass D 263 M https://www.schott.com/advanced_optics/ ... index.html When you look into the "Technical Details" you can see that this glass is zero transmission below about 290 nm. So glass was not an option for the very short wavelengths (below 300 nm) that the Ultrafluars were designed for - only quartz worked.

Maybe modern UV objectives use normal cover glasses because they were not designed for these very short wavelengths. I think some Olympus UIS objecives, like UAPON-340, refer to 50 % transmission at 340 nm but they have no transmission below 300 nm, so they don't need quartz windows: https://www.olympus-lifescience.com/en/ ... /uapon340/

[Lou Jost]

Maybe modern UV objectives use normal cover glasses because they were not designed for these very short wavelengths.

That seems like a good explanation for the difference. And it makes sense for many applications, since most camera sensors aren't able to register shorter wavelengths than that.

The graph in your link shows that normal coverslips are completely transparent at 365nm.

[jmc]

The early specialised UV objectives (Zeiss Ultrafluar; Leitz UV (still on list from 1985)) used 0.35 mm quartz windows.

Hi Ichty. do you have any documentation which mentioned the Leitz UV objectives. I have one (a 16x), but can't find anything about it anywhere. It'd be great to know a bit more about it.

[Lou Jost]

Jonathan, yesterday I found this, which covers many old Leitz objectives. I don't know if it includes yours:
http://microscope.database.free.fr/500_ ... rriers.pdf