ISO optiphot fluorescence info

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TomFid
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ISO optiphot fluorescence info

#1 Post by TomFid » Thu Mar 31, 2022 7:04 pm

I have an Optiphot with BD objectives and the BF/DF epi illuminator. We use it primarily for low-power examination of botany and rock specimens, mainly in reflected light but we do have the condenser/stage for transmitted. An upcoming project will involve identification of symbiotic fungi on wild orchid seeds.

I just spotted a fairly cheap epi-flourescence illuminator (pics below). I bought it, mainly out of curiosity, but we're hoping it'll contribute to the botany fun. Everything's in good shape, except that the lamphouse is missing the mercury bulb and carrier, and there's no power supply, which may be OK because I was hoping to avoid UV anyway.

I have several Optiphot manuals, but it turns out that none I can find covers the epi-fluor illuminator in any detail. So, I have a few (probably naive) questions:

- Is there a manual for these around? I searched the forum, but a couple links I found had gone dead unfortunately.

- An unexpected bonus was that it included 2 cubes. How do you get those little guys out? I assumed they just slid in/out on a dovetail, but these seem pretty stuck. I understand that caution is in order with the optical surfaces, so I thought I'd ask before just pulling on things.

- My plan, such as it was, was to use blue excitation with longpass, something like a Nikon B-2A or B-3A. The included cubes are DAPI which I understand to be UV, and an Omega XF18, which looks like it might be close to the Nikons, with a dichroic cutoff around 500nm. Should I hang onto these, or look for something different to start with?

- I hadn't thought about tube length. It looks like the epi-fl illuminator includes some compensating optics. Will it work alone with the 210 BD objectives, or do I need to have additional space in the train?

- Is the empty slot (red arrow, 2nd image) for a polarizer, as it is on some of the other illuminators?

Any thoughts, rants, excuses for getting more objectives, etc. would be much appreciated!
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Hobbyst46
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Re: ISO optiphot fluorescence info

#2 Post by Hobbyst46 » Thu Mar 31, 2022 8:47 pm

UV is emitted by high-pressure mercury lamps that are used for fluorescence. Staring directly into the lamp would be hazardous.
However, the epi-illuminators are designed to protect the user from intense excitation light.
If the lamp housing misses important parts like the reflector mirror, and in addition there is no power supply (these are not "general purpose" power supplies, rather they are dedicated PSs for the illuminator) I would opt for a LED illuminator instead. There are 15W or more LED sources for fluorescence that do not emit UV. They are mocnochromatic so an excitation filter can be omitted.
The first question to ask yourself however, is what is the fluorophore. When you know its excitation spectrum and emission spectrum, you know which wavelengths of excitation, which dichroic mirror and long pass filter to use. I doubt if DAPI is relevant for botany but not an expert on it.
Commercial LED lamps for fluorescence microscopy are fairly costly, but DIY solutions have been posted.

I might be able to provide some input about the usability of the BD objectives, within a few days.

PeteM
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Re: ISO optiphot fluorescence info

#3 Post by PeteM » Thu Mar 31, 2022 10:03 pm

The cubes some out by unscrewing a set screw at the front upper side. There's a sort of cam action holding the dovetail and they will slide right out and forward (where the cover is). You might use tweezers carefully on one side to get them out.

Unlike some of the other Nikon series, far as I know, the epi (210mm) and fluorescent (160mm with a 1.25x compensating lens) illumination heads don't cross over. You might be able to cobble together an epi cube for your fluorescent head and add a regular lamp (halogen, LED) to the back - but I've not seen a brightfield cube for it. Perhaps someone else has?

As Hobbyist says, be careful with UV. Get it wrong (say a burned-through barrier filter) and you can do real damage to your eyes.

The slot above the cubes might be for a secondary barrier filter, though you could certainly put a polarizer there and use it for normal diascopic viewing.

I haven't see a manual either. Hope someone has a link?

TomFid
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Re: ISO optiphot fluorescence info

#4 Post by TomFid » Thu Mar 31, 2022 11:05 pm

Hobbyst46 wrote:
Thu Mar 31, 2022 8:47 pm
UV is emitted by high-pressure mercury lamps that are used for fluorescence. Staring directly into the lamp would be hazardous.
Right - I have a couple UV mineral lights that freak me out, even though I'm careful about eyewear and handling. Also, the mercury lamphouse, bulb, and power supply are gigantic and expensive - possibly incompatible with family harmony. I built myself a nice white LED illuminator, and was thinking of doing the same thing in blue for this unit.

I'm not sure about fluorophores (not my department for the orchid investigation) but I know that one choice mentioned was Acridine Orange, for some mix of cheapness and usefulness. That was the original motivation for a blue/longpass filter cube.

Thanks!

Scarodactyl
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Re: ISO optiphot fluorescence info

#5 Post by Scarodactyl » Fri Apr 01, 2022 8:27 am

These are designed for 160mm objectives, so adding some empty space would be advisable. It would probably be pretty easy to 3d print an empty spacer.

TomFid
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Re: ISO optiphot fluorescence info

#6 Post by TomFid » Fri Apr 01, 2022 4:04 pm

I thought about using the other illuminator as a spacer, but it has a protruding tube on the bottom that would probably conflict with the top element of the fluor illuminator. That means the spacer would have to go on the bottom, leaving the arm of the fluor illuminator hanging, which seems less than ideal.

That's a long way of saying that printing a spacer seems like a good idea. I'll put the part on Thingiverse when I get around to it (if there isn't already something out there).

TomFid
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Re: ISO optiphot fluorescence info

#7 Post by TomFid » Fri Apr 01, 2022 4:37 pm

PeteM wrote:
Thu Mar 31, 2022 10:03 pm
The cubes some out by unscrewing a set screw at the front upper side. There's a sort of cam action holding the dovetail and they will slide right out and forward (where the cover is). You might use tweezers carefully on one side to get them out.
You're right. I had assumed that the hole at the top-right corner was empty, but there is indeed a tiny set screw way down in that hole. It took a 1/16" Allen key, which seems odd, but after loosening the cubes came right out.
cube.jpg
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As Hobbyist says, be careful with UV. Get it wrong (say a burned-through barrier filter) and you can do real damage to your eyes.
I discovered that the cube labeled DAPI has no excitation or emission filters, and the mirror looks neutral (though maybe I just can't see the UV reflection band). Maybe it was just used for brightfield? Good thing I didn't try it with UV!

The XF18 cube does seem to work though. With white light into the illuminator, I get a nice blue spot on the sample, and illuminating the sample directly, I get a nice yellow image at the eyepiece. So far so good, but I think I'll need a LOT more blue light.

Hobbyst46
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Re: ISO optiphot fluorescence info

#8 Post by Hobbyst46 » Fri Apr 01, 2022 9:22 pm

The XF-18 cube can yield red autofluorescence from plant, alga and moss green parts. A common cool-white LED (6000-6500K) has a very strong blue component, that might be enough for that fluorescence - just guessing. Although not at the optimum wavelength that is 400-425nm.
Nevertheless, I would try it with a 3W cool-white LED. On the Photomacrography forum there was a post that demonstrated successful fluorescence microscopy based on a DIY multi-3W LEDs for excitation. A couple of years back.

Excellent that you did not try an empty cube with UV... yes !

TomFid
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Re: ISO optiphot fluorescence info

#9 Post by TomFid » Sat Apr 02, 2022 12:28 am

Hobbyst46 wrote:
Fri Apr 01, 2022 9:22 pm
The XF-18 cube can yield red autofluorescence from plant, alga and moss green parts. A common cool-white LED (6000-6500K) has a very strong blue component, that might be enough for that fluorescence - just guessing. Although not at the optimum wavelength that is 400-425nm.
Nevertheless, I would try it with a 3W cool-white LED. On the Photomacrography forum there was a post that demonstrated successful fluorescence microscopy based on a DIY multi-3W LEDs for excitation. A couple of years back.
This is great to know. I have a fairly high power Cree LED on an Indus star, I've forgotten the details but I think I can run 6W at least. My initial test was kind of a flop, partly because I didn't have a fluorescent target, but I suspect mostly because I was shining the LED straight down the pipe. Without a collector lens, most of the energy was probably wasted. I'll have to retrofit something into the lamphouse, or harvest the collector, and try again.

I see that the Cree XP-E2s now come in several colors, with a Royal Blue peaking at 450nm and Blue at 465, which I think is closer to the sweet spot for Acridine O. The cool whites seem to share the Royal Blue peak. I'll experiment and report back.

TomFid
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Re: ISO optiphot fluorescence info

#10 Post by TomFid » Sun Apr 24, 2022 7:26 pm

I've done a little more experimenting. The 210 BD objectives will definitely need a 50mm spacer if I want to use them with the fluorescence illuminator. They seem sharp enough at 160, but the various magnifications are no longer parfocal, which is a pain. I recently scored an RMS turret on ebay, so it might be better in the long run to find some 160 objectives.

I also experimented with my existing white LED lighting, which should have a strong blue peak at 450nm, which is right in the window for the XF18 cube. I do get a pretty good blue spot from it, but it's a long way from filling the field of view of the objective. This is a little puzzling, because the LED die should be about the size of a filament or arc, and I'm using the stock collector lens with about the same spacing. I'm wondering if this is another tube length issue, resulting in the BD objective projecting an image of the source?

Along the way, I discovered that an $8 violet laser pointer makes tons of autofluorescence on moss samples. The filters block all the 405nm output, but I'm still not crazy about the idea of looking at a laser through a microscope, so I also have a short lens as a beam expander. It seems odd that a cheap 5mw diode puts out a ton more usable light than the vertical illuminator (so far) - makes me wonder if direct LED illumination might be the way to go, using the cube only as an emission filter.

Onmregnin
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Re: ISO optiphot fluorescence info

#11 Post by Onmregnin » Sun Jun 12, 2022 1:29 am

These are intended for 160mm objectives, so some extra space is recommended. An empty spacer would most likely be fairly simple to 3D printdrift boss

TomFid
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Re: ISO optiphot fluorescence info

#12 Post by TomFid » Mon Jun 13, 2022 8:13 pm

I'll put the spacer design on Thingiverse if I end up doing it. In the meantime, I picked up a couple 160 phase contrast objectives that seem to work well (in addition to satisfying another interest). Interestingly, the 5x BD objective works extremely well on pond life slides with the Ph3 condenser setting - a really nice contrasty semi-darkfield effect.

Hobbyst46
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Re: ISO optiphot fluorescence info

#13 Post by Hobbyst46 » Mon Jun 13, 2022 8:37 pm

TomFid wrote:
Sun Apr 24, 2022 7:26 pm
I also experimented with my existing white LED lighting, which should have a strong blue peak at 450nm, which is right in the window for the XF18 cube. I do get a pretty good blue spot from it, but it's a long way from filling the field of view of the objective. This is a little puzzling, because the LED die should be about the size of a filament or arc, and I'm using the stock collector lens with about the same spacing. I'm wondering if this is another tube length issue, resulting in the BD objective projecting an image of the source?

Along the way, I discovered that an $8 violet laser pointer makes tons of autofluorescence on moss samples. The filters block all the 405nm output, but I'm still not crazy about the idea of looking at a laser through a microscope, so I also have a short lens as a beam expander. It seems odd that a cheap 5mw diode puts out a ton more usable light than the vertical illuminator (so far) - makes me wonder if direct LED illumination might be the way to go, using the cube only as an emission filter.
The 405nm is exactly the peak for excitation of chlorophylls, much better than 450nm (although 425nm would be fair excitation).
A laser beam through the microscope would IMHO be a dangerous game. On a confocal microscope for example, viewing is done with an ordinary epi- arc lamp, the lasers serve for sensor acquisition, not direct vision.
We note that the bandwidth of a LED is wider than that of a laser.
Still, direct LED excitation is possible, but AFAIK, a powerful LED it should be - say, 10-15W. Say, a 10W violet LED.

TomFid
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Re: ISO optiphot fluorescence info

#14 Post by TomFid » Wed Jun 15, 2022 3:54 pm

Hobbyst46 wrote:
Mon Jun 13, 2022 8:37 pm
The 405nm is exactly the peak for excitation of chlorophylls, much better than 450nm (although 425nm would be fair excitation).
I couldn't resist fooling around with direct illumination with a cheap 405nm laser pointer a while ago, and it yielded really good autofluorescence on moss and little creatures living in it.
A laser beam through the microscope would IMHO be a dangerous game. On a confocal microscope for example, viewing is done with an ordinary epi- arc lamp, the lasers serve for sensor acquisition, not direct vision.
However, as you say, it seems like a poor habit. Probably negligible 405nm light will get through the filter cube, but it would be easy to catch a stray reflection off a slide. Also, after researching a little, I was dismayed to discover that the shortwave diode lasers pass a LOT of infrared - I guess they must be IR diodes with frequency-doubling crystals? I tested mine with a full-spectrum camera and it had a strong NIR spot through a Hoya R72. Even more alarming, after shining it on a wall, there was a hot spot discernible with a thermal camera several seconds after switching it off. And that's from a $9 pointer that can't be more than a couple mw. Since IR might go through my longpass cube, my enthusiasm for that approach is diminished though some mods could probably cut the IR.
We note that the bandwidth of a LED is wider than that of a laser.
Still, direct LED excitation is possible, but AFAIK, a powerful LED it should be - say, 10-15W. Say, a 10W violet LED.
I worked out the issues between LEDs and the fluorescence illuminator - it was partly the 160/210 mismatch and partly finicky spacing and centering. I now get a pretty good 450 blue spot from my white LED. There are quite a few cheap 405nm LED options on ebay, but I don't think my XF18 will pass that, so I'll have to find a violet excitation cube I guess, or solve the direct laser problems.

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