Wandering into fluoroverse

A forum to ask questions, post setups, and generally discuss anything having to do with photomacrography and photomicroscopy.

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ModelZ
Posts: 38
Joined: Sun Nov 01, 2020 4:34 am
Location: Northern Europe

Wandering into fluoroverse

Post by ModelZ »

A regularly appearing impression in this forum and elsewhere seems to be that getting started with epi-fluorescence microscopy is a very complicated and expensive proposal. This does not need to be the case - let me just present a small case study on this matter.

First clarification: fluorescence with a DF-condenser and a UV-LED illumination is very easy and cheap to do. While the results can be good that way, too, to keep things finite, I won't discuss that technique here. There are articles on that in this forum and elsewhere.

A while back I got lucky and found a fine but neglected piece of hardware, Reichert Universal Mikroskop, Model "Z". Superb engineering, still tight and smooth like at its birth some 85 years ago. Presumably the world's first modular microscope. But humidity had gotten the better of some of its optics. The binohead prisms were covered with fungus and screws around them rusted. As a matter of principle I don't drill, file or do other brutality to this kind of masterpieces - only gentle methods are acceptable. No-go so far with the bino. So what to do? Well, it is modular scope after all so perhaps Herr C. Reichert allows me to use it with a different head, ja?

DSCF7452-1.JPG

This is about an auto-fluorescence set-up, no fluorochrome talk here. When investigating these ideas and digging up various net and paper resources I found about the Lomo OI-17. It's a epi-head building block, an optical lego if you like, a little black metal cylinder. Zeiss male&female bayonets at ends and inside a 440nm dichro and about 480-490nm barrier together with a couple of lenses (1.63x mag total). Sells for about $30-40 if you keep looking at ebay.

If you search around you will find e.g. Laurent Delvoye's or David Walker's descriptions how to use this lego piece. So primary credits should go to these gentlemen.

Below is a sketch how to I constructed the epi-head around OI-17 (A). No lathe or other serious equipment needed, basic garage gear will do: metal saw, files, hand drill. The body (C) is made from a leftover piece of aluminum profile and another piece of alu that slides snuggly on it (D). You could use plastic tubes, but I hate plastic and besides aluminium looks way better and conducts heat away, too. Around the aluminium core there is a thin steel cover to keep any stray UV inside.

OI-17epi-v2.jpg

The illumination is by UV LED. Nichia 3W 365nm attached to a processor cooling fin (F). Quite narrow bandwidth, intense enough and works well once the upper tail is killed with a suitable filter (E). A 3W generic Chinese deep violet is also usable. These are 20mm diameter cobs attached with two screws to the fin, which itself is y-z-plane adjustable. Condenser is Lomo 10x eyepiece (O). Zeiss KPL 10x works, too, but is larger and less handy. A filter slot is essential (for UG1/BG3 resp.) but diaphragm isn't. I was first worried about this part of the construction but once the relative positions of the condenser and LED are (experimented) right and the LED centered, the light is very uniform on the sample.

With LEDs there is no need to control the intensity with an aperture since the power source does it. Any old adjustable, stable power will do or e.g. DIY unit using BuckBlock.

At the top of the OI-17 unit there is a depression where you can put additional barrier filters (B) in a rather flexible manner, diameters 15-35mm work. Above it the upper department is vanilla Lomo photo tube, at it't top a 7x photo eyepiece, Lomo MFN-beam splitter and an appropriate camera adapter. When doing fluoro I mostly observe via the camera screen. I don't mind the monocular since I only use it when setting-up the specimen under visible light (from a DF condenser). The whole thing rests on a hollow wooden block that has in it the visible light source (not shown, e.g. Lomo OI-35 works fine).

DSCF7455-6-1.jpg

Sans hours, the total cost of this project was around $120. This included the parts above (OI-17, LEDs, eyepiece, LP430 interference filter, excitation filters, MFN+tube and a polycarbonate visor in front, not shown in the pics). Excluded are the power unit, cam adapter and safety goggles. The Reichert achros perform well under UV and initially I didn't need any new objectives. Later got some Lomo Lumams for their higher NA - this enables somewhat shorter exposure times.

A comparison to arc lights. I very strongly recommend the LED route to fluorescence over old mercury lamp units sold at ebay etc. While being outdated technology they are nevertheless still offered at hideous prices that I certainly cannot justify. The arc bulbs are expensive, short lived, often hard to find, clumsy to use, fry filters, dangerous to the environment etc. Their shortcomings are really numerous. Sometimes they even explode spreading mercury vapor in the air endangering everyone in the room. Absolutely a dead-end tech that I feel nobody should support.

Below a couple of moss pics for you to get some view through this Frankenscope.

mozz220-DSS-1-1.jpg
mozz315-DSS-1-1.jpg

While I've since renovated and converted to LEDs another fluoro rig that I now mostly use, I'm quite fond of this set-up. Its partly nostalgia, partly pride in rescuing fine metal. But while it has definite limitations, it also has further potential. Indeed I made another epi-unit like this just "for reserve". But then on an odd lazy moment an idea struck me: could it also be used upside down, under the condenser stage? Well, crazy you, no, no,... yes. Yes!

The idea is to make the illumination/excitation richer. Firstly epi-lightning is rather flat, but with DF (and oblique) you can remedy this in various ways. Illuminating surface structures from the sides and by bringing in multiple narrow wavelengths, pinpoint the excitation. You can do this on the fly without any change of parts (which would be detrimental since you easily loose alignment of sample structures if you fiddle with the light train). With the OI-17 upside down and carefully centered under the DF condenser you can add another UV-wavelength on top of simultaneous visible light. You need multiple power sources, but you can play them all independently. Note that you can also use the rotating oblique light above the stage with UV or visible. It of course needs focusing optics & careful aiming to work.

DSCF7460-1.JPG

This latter part of the project is only pretested, still work in progress and obstacles may show up. But I like to think this set-up with it's four channels of illumination as sort of a "poor man's Colibri" ;-)

If anyone has related tinkerings on the way I'd be happy to discuss these things.

Cheers, Karl

Pau
Site Admin
Posts: 5412
Joined: Wed Jan 20, 2010 8:57 am
Location: Valencia, Spain

Re: Wandering into fluoroverse

Post by Pau »

Very nice project! and results, BTW

Time ago I made a similar but more complex and much less brave device, now a bit improved with more and better UV LEDs and a big Leica light shield for the LEDs
viewtopic.php?p=201937#p201937
Pau

Scarodactyl
Posts: 685
Joined: Sat Apr 14, 2018 10:26 am

Re: Wandering into fluoroverse

Post by Scarodactyl »

What a beautifully assembled frankenscope! I'll admit based on your caveats I wasn't sure what to expect but your example images are superb.

ModelZ
Posts: 38
Joined: Sun Nov 01, 2020 4:34 am
Location: Northern Europe

Re: Wandering into fluoroverse

Post by ModelZ »

Thanks Pau and Scarodactyl for nice comments!

Pau, I read your article a way back with great interest. That's a very through and clean conversion you made. I also seem to recall that before entering to that project you asked here about the usefulness of OI-17 but didn't get much response. Later on working with it I was a bit surprised by that. Unknown piece of optics, hmm...
Before doing this project I considered modding the Zeiss fluorohead to this scope but then rather went this way. It turned out to be the best choice for me. Some time after completing this I found a rare, top quality original epi-fluoro head for my workhorse, Zetopan (these things are funny, the seller didn't even know what he was selling...). I Modded the thing for multilight LED, got filter stage upgraded etc. These pieces are just so sweet when you get them working.

Pau
Site Admin
Posts: 5412
Joined: Wed Jan 20, 2010 8:57 am
Location: Valencia, Spain

Re: Wandering into fluoroverse

Post by Pau »

Thanks for your comments, I love this kind of DIY frankenscopes and yours is really notable :D
Some time after completing this I found a rare, top quality original epi-fluoro head for my workhorse, Zetopan...
Would be nice to see it!
Pau

ModelZ
Posts: 38
Joined: Sun Nov 01, 2020 4:34 am
Location: Northern Europe

Re: Wandering into fluoroverse

Post by ModelZ »

Zeto later, it's now embedded in a jumble of wires, lights, power units etc. I'll shoot it after its next reincarnation.

seta666
Posts: 1026
Joined: Fri Mar 19, 2010 8:50 am
Location: Castellon, Spain

Re: Wandering into fluoroverse

Post by seta666 »

This is a very nice project with beautiful results; I got a BH-2 fluorescence attachment but is missing the G B dichroic mirrors so is kind of useless, maybe I will find them one day..

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