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Fluorescence on Optiphot
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ChrisR
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PostPosted: Tue Jan 15, 2019 5:13 am    Post subject: Fluorescence on Optiphot Reply with quote

Inspired by pictures by Jacek and others I watched out for parts for a Nikon Optiphot which is in store at the moment. AFAIK the parts for a Labophot would all be the same.
Long story short, I have two epi illuminators and 4 cubes.

Now I'd like some advice Very Happy

I have one lamphouse, with "burner" whose bulb is broken. I know they get dangerous when old, so it's good that I wasn't temped to try it!

Is it worth getting a bulb and power supply, or would I be better off skipping that and going to LED illumination?

The cubes I have are these:
DM580 (BA590)
DM510 (BA520-560)
UV-2A BA420 DM400
B

Yes the last one just has a B.

I'm having trouble working out the specifications of the Excitation, Dichroic Mirror, and Emission filters are, which are part of these. Does anyone have good reference material on them?
Any comments/information on their usefulness, would be most welcome. Smile

This is the current brochure, my parts are older.
https://m.nikoninstruments.com/content/download/16085/353426/file/Fluorescence_Filter_Cubes_2CE-MRJH-3.pdf

Information on LEDs is quite spread out across the forum.
Pau and Saul have posted, eg
http://www.photomacrography.net/forum/viewtopic.php?p=193198
...and links out from that thread.


As and when I locate things I'll add them here.

UV and near-UV leds have become much easier to find. A few:

https://www.lumitronix.com/en_gb/nichia-nvsu233a-uv-smd-led-witch-pcb-10x10mm-1030mw-365nm-14352.html
(1W, 365nm, 10mm x 10mm, Euros 19.85 + Euros 5-10 delivery)
“(I have the NVSU233A-D1 that has a lens and it's the one that have focusing issues in my system)”
“"3 W" no brand Chinese UV LED and, despite working it's not well powered and produces a large amount of white light.”

“very satisfied with – “
5W UV/Ultra Violet LG3535 Aliexpress, a few $, around 380nm

“COB LEDs are inadequate for microscope illumination at least with the original optics designed for the small arc lamp source.”
Convoy S2 Nichia 3W led torch, 365nm, £16 at Gearbest. Higher elsewhere currently

LED-ENGIN at Mouser and elsewhere shows a good range.

--
Pau's 6 led illumination system: http://www.photomacrography.net/forum/viewtopic.php?t=32736

A pdf to read:
https://www.chroma.com/sites/default/files/HandbookofOpticalFilters.pdf
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Last edited by ChrisR on Tue Jan 22, 2019 8:55 am; edited 3 times in total
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Pau
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PostPosted: Tue Jan 15, 2019 6:17 am    Post subject: Reply with quote

Hi Chris

About LEDs, the linked NVSU233A will be excellent for UV excitation with the UV2A cube, the issue with the NVSU233A-D1 is its narrow angle built in lens, my other LEDs have wider angle lenses. Likely I'll change it in the near future. No lens is a priory the best matching light source with a mercury arc designed illuminator.

For blue the Cree XPE2 is also very good, when buying it be sure to get the 2 version, I first got the older less powerful XPE and the ebay seller specifications were wrong so it burned out after few uses.
If I get it right*, it will work well with the DM510 (BA520-560) cube for blue excitation and green emission and I guess also with the B cube for green to red emission

I suppose that the green Cree XPE2 would also be excellent, I still have a less powered model. It would work with the DM580 (BA590) cube (green EX, red EM) although this combo is less interesting for autofluorescence.

I have no experience with mercury burners, they could be more powerful but given the good results I get with LEDs I wouldn't consider them for myself:
- Heat issues
- Short live
- Contaminant and even dangerous
- Need to have them running to stabilize...
- Easy damage to filters...

The only real advantage I see over my LED setup is the easy to change just the cube for multi excitation images or even to use multiple wavelength cubes.

* lacking the reference, an approximate indication is the filter color, could you tell us the color of each filter?

This is from EPI-FLUORESCENCE ATTACHMENT "TMD-EF" for DIAPHOT - TMD, a microscope of the same era, PM me if you want the pdf


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JohnyM



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PostPosted: Tue Jan 15, 2019 7:13 am    Post subject: Reply with quote

This webpage is quite good:
https://www.microscopyu.com/techniques/fluorescence/nikon-fluorescence-filter-sets

Rule of thumb:
-Amateur would like autofluorescence (many sparkling colors) so gravitate towards left side of the spectrum and wideband (excitation) longpass (emission) cubes. Multiband cubes are also very interesting in this aspect.

UV, V, BV, B cubes will give most colors in most subjects. Those wavelengths are weak in halogen source, and just slightly better with led.

-Specialist is interested in special type of fluorescence, so he want to get rid of all the others, so he would gravitate toward right side of the spectrum and narrowband (excitation and emission) cubes.

G, R , Y, IR specialist cubes, easily found on all lightsources.



Now, you'll get all of those excited with almost any lightsource. Led's are just NOT worth the hassle IMO, unless you can grab system like "Colibri".
Still HBO is vastly superior to Colibri in terms of signal strength until we hit ~550nm (green).

If you want my advice: go with HBO. If you're afraid, just get halogen lamphouse - it will work with UV. You're just get very faint signal (noisy image).
LED will be a slight improvement in terms of signal strength, at cost of all the DIY hassle and changing emitter for every cube. It doesn't suit me, might suit you.
Halogen is best of both worlds, middle ground i would say.

In my scope, i use 3 lightsources for fluorescence:
- Halogen for quick checks (will it show autofluorescence?)
- HBO for "serious" fluorescence work
- XBO (Flash) for quick action / high photobleaching fluorescence.





I just want to address that with some first hand experience:
Quote:
I have no experience with mercury burners, they could be more powerful but given the good results I get with LEDs I wouldn't consider them for myself:
- Heat issues
- Short live
- Contaminant and even dangerous
- Need to have them running to stabilize...
- Easy damage to filters...

-Heat is real, but it's not something i really notice anyhow. It's just as annoying and as noticeable like heat from any of 100W 12V lamps im using everyday for DIC work.
-200/300 hours of guaranteed lifespan. 600-900 hours of actual workspan.
Still much more expensive with comparison to LED / Halogen.
This is not something you'll hear from salesman of find in a brochure but:
If lamp is old it will be very difficult/impossible to ignite, also it will begin to flicker heavily - that means it's time to replace it... after that ~600-900 hours.
-In case of burst (never happened to me) room need to be evacuated for few hours, that's about it. I don't see it being a problem, but i can see how it can be an obstacle for others.
- Running to stabilize... it's not really true. You will get some EV differences in the image, yes. It's all gone after 2-3 minutes, not after 15+ like they say. It become more obvious and longer to stabilize as the lamp grow older. But... LED lamps flicker NON STOP, although it's very subtle. I would say, if you haven't heard about it, you wouldn't notice it.
- In my workspace we have Olympus AH and Zeiss Axio. I personally own Microphot. After all those years, all filters are still operational, including especially "vulnerable" multibands.

Additionally:
-Power, it's no competition. Imagine using F5.6 zoom in a church. Then switch it to F1.4 prime. It's something like that.


Like i said, you can get the image with halogen / led no problems nowadays. I take photos of fluorescence that's invisible in eyepieces. Only camera with high iso and long exposure time can see it.
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ChrisR
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PostPosted: Tue Jan 15, 2019 7:23 am    Post subject: Reply with quote

Thanks Pau.
I'm glad I have two B cubes then - they came with the second illuminator Smile.

So to be clear, looking at the table you kindly supplied
Eg for "UV"
The intended illumination is 365nm -
So, "ideally"
The the EX filter is a Bandpass filter 330 - 380
The mirror reflects below 400nm (onto the specimen)
and any fluorescence emitted above 400nm goes through the mirror to the eyepiece, and
the Absorption filter also stops anything below 420nm getting to the eyepiece.

I notice the B cube doesn't have at least one filter. That makes sense if they're using the mirror to cut all the short wavelengths.
I'll get the cubes together and see what comes out of their colours..

Is it typical to have to add an(other) excitation filter after the light source??

The "XPE-2" I've found is red, so I was thrown completely,Rolling Eyes but I see that designation covers the whole range:

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JohnyM



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PostPosted: Tue Jan 15, 2019 7:25 am    Post subject: Reply with quote

Cube Excitation Emmision
DM580 (BA590) G-2A / G-1A Green Red only
DM510 (BA520-560) B-1E / B-2E Blue Green only
UV-2A BA420 DM400 UV-2A UV 365nm All the colors
B Most common B cube is B-2A

You can unscrew excitation filters, you'll be able to read the marking then and can tell for sure which cube you have.
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ChrisR
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PostPosted: Tue Jan 15, 2019 7:50 am    Post subject: Reply with quote

JohnnyM.
What a fantastic answer!! Thank you very much.

On the flash - is that a Nikon fitting? Is the UV filter removed from the flash, or is there just so much light anyway that it's not necessary, or using Wood's glass, or......?
Which model 'scope is it - maybe one day? Smile


I just dismantled an old Sony 3MP camera, only keeping the screws. But I did wonder if the bare flash tube could be used - it's nice and small.

I doubt I'd ever get to use multi-passband filters, with the exotic dyes and specimen prep skills I don't have, but I bought these on ebay for not much:

1 x 5g Rhodamine B500
1 x 5g Rhodamine 6G
1 x 10g Fluorescein
1 x 10g Bromofluorescein
1 x 10g Acridine Orange
1 x 10g Green Fluorescent Dye
1 x 25ml Optical Brightener
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ChrisR
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PostPosted: Tue Jan 15, 2019 8:06 am    Post subject: Reply with quote

For Chlorophyll then

A Royal Blue source would work with both
DM580 (BA590) G-2A / G-1A Green Red only
DM510 (BA520-560) B-1E / B-2E Blue Green only ( <--- or not ? )
as well as B?
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JohnyM



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PostPosted: Tue Jan 15, 2019 8:38 am    Post subject: Reply with quote

ChrisR wrote:

1)On the flash - is that a Nikon fitting? Is the UV filter removed from the flash, or is there just so much light anyway that it's not necessary, or using Wood's glass, or......?
2)Which model 'scope is it - maybe one day? Smile


3)I just dismantled an old Sony 3MP camera, only keeping the screws. But I did wonder if the bare flash tube could be used - it's nice and small.

4)I doubt I'd ever get to use multi-passband filters, with the exotic dyes and specimen prep skills I don't have


1) It's not nikon, because i needed 3 light sources, and nikon provide only 2. It's called "dual lamphouse adapter". Asking price is ~2k USD, sold few on ebay IIRC ~800USD range. Zeiss made a lot of those for standard and now for axio and are much cheaper.
Flashgun DOES have filter removed. I didnt tested it at all, just removed it straight ahead as it was a fresnel lens and my adapter is koehler ready. So it MIGHT be unnecessary to remove it.

2) This is Nikon "Microphot"... with everything and more Smile
Only bit of exipment i miss is "mirror box". Seen it only once, now i regret not even saving auction pics.

3) In case of flasguns... i like things big Very Happy

4)Multiband cubes are outstanding for autofluorescence! Especially the old ones! They were hard to make and first models (Optiphot scopes time) were considered weak... because they provided much of autofluorescence obscuring scientific signal. That's outstanding from amateur POV, many radiant colors.

Take a look at those images:
https://get.google.com/albumarchive/110216219979417982302/album/AF1QipMJIfpAFa9yRJ3cxZof03ci2nP15g_FuRFJOiWs

Those are spiderwort leafes with order: G, G, V, UV, DIC, B, G, MULTIBAND.

Note how it's EXTRAORDINARY as it's producing WHITE fluorescence. extremely rare and hard to obtain. Not saying it's especially beatufull, but it's something that you can only do with multiband cube.
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JohnyM



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PostPosted: Tue Jan 15, 2019 8:45 am    Post subject: Reply with quote

I do not know what "royal blue" means.
B-1E and B-2E have peak at ~ 470-480nm.
G-2A and G-1A have peak at ~420-430nm.

Both of those cubes would excite chlorophyll.
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PostPosted: Tue Jan 15, 2019 8:48 am    Post subject: Reply with quote

Quote:
I notice the B cube doesn't have at least one filter. That makes sense if they're using the mirror to cut all the short wavelengths.
I'll get the cubes together and see what comes out of their colours..


No, that's very bad.
No mirror - obviously wont work.
No barrier - there will be bleeding.
No exciter - also bleeding UNLESS you use monochromatic source, like LED.

Even tiny amount of bleed through can ruin weaker signals, and will significantly imair stronger ones.

Nikon marks their "darker" cubes as "B" which means darker background.
Cubes marked "A" are stronger signal, but might show somewhat gray backround. Now imagine removing one of those carefully selected filters, what will happen to the image.
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PostPosted: Tue Jan 15, 2019 3:05 pm    Post subject: Reply with quote

Thanks again. Learning fast!
Royal Blue is he name they give the XP -E2 LED above, 460-465nm.
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Pau
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PostPosted: Wed Jan 16, 2019 3:55 am    Post subject: Reply with quote

Johny, thank you for sharing your experience and thoughts, very interesting.
JohnyM wrote:

Now, you'll get all of those excited with almost any lightsource. Led's are just NOT worth the hassle IMO, unless you can grab system like "Colibri".
Still HBO is vastly superior to Colibri in terms of signal strength until we hit ~550nm (green).

Do you know the emitting power of Colibri LEDs?
When designing my multi LED illuminator I have browsed all the info I was able to find and didn't find it, without a reference I have the impression that they are low powered
Quote:
If you want my advice: go with HBO. If you're afraid, just get halogen lamphouse - it will work with UV. You're just get very faint signal (noisy image).
LED will be a slight improvement in terms of signal strength, at cost of all the DIY hassle and changing emitter for every cube. It doesn't suit me, might suit you.
Halogen is best of both worlds, middle ground i would say.

At least for UV I don't think that halogen could compete at all with modern LEDs (if so it would be extremely dangerous!)
Quote:
In my scope, i use 3 lightsources for fluorescence:
- Halogen for quick checks (will it show autofluorescence?)
- HBO for "serious" fluorescence work
- XBO (Flash) for quick action / high photobleaching fluorescence.

Very nice equipment you have! Love the triple source setup. Would be nice to see a detailed post about it.
How well does work the electronic flash as excitation source? Does it provide enough UV? (exposure data of an example taken with it will be very interesting)
Quote:
I just want to address that with some first hand experience:
...

Very useful info
Quote:
- In my workspace we have Olympus AH and Zeiss Axio. I personally own Microphot. After all those years, all filters are still operational, including especially "vulnerable" multibands.

I've seen lots of filters heavily damaged, mainly excitation ones, although not knowing their history, likely they came from labs and had been in not careful continuous daily use for many years, a very different situation of amateur use.
Quote:
Additionally:
-Power, it's no competition. Imagine using F5.6 zoom in a church. Then switch it to F1.4 prime. It's something like that.

Again, could you kindly provide exposure data of an example?
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PostPosted: Wed Jan 16, 2019 4:31 am    Post subject: Reply with quote

ChrisR wrote:
So to be clear, looking at the table you kindly supplied
Eg for "UV"
The intended illumination is 365nm -
So, "ideally"
The the EX filter is a Bandpass filter 330 - 380
The mirror reflects below 400nm (onto the specimen)
and any fluorescence emitted above 400nm goes through the mirror to the eyepiece, and
the Absorption filter also stops anything below 420nm getting to the eyepiece.

Yes. Actual combos can vary a bit from these data, but this is the idea.
Quote:
I notice the B cube doesn't have at least one filter. That makes sense if they're using the mirror to cut all the short wavelengths.

As Johny has explained you need all the filters, and well matched.
At my illuminator thread after some questions asked by another member I did some tests removing filters and the results were bad:
Be aware of:
- Filters are not perfect, so double filtering highly improves the results. DM filtering is not enough.
- Fluorescence emission in most cases is much weaker than excitation, so if the system lets to pass a small amount of excitation light the image will lack contrast of even will be completely ruined
- Single color LEDs are not really monochromatic (ones less than others). With HBO or halogen lamps the need is even higher
Quote:
Is it typical to have to add an(other) excitation filter after the light source??

Sometimes filter cubes have two excitation filters and illuminators often provide place to put additional filters. If you're referring to the filters at the lamp side of the illuminator, they are likely heat filters, needed with HBO or halogen lamps but not with LEDs
Quote:
The "XPE-2" I've found is red, so I was thrown completely,Rolling Eyes but I see that designation covers the whole range

At https://www.ledsupply.com/leds/cree-xlamp-xp-e2-color-high-power-led-star you can find different colors, at ebay it changes a lot
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PostPosted: Wed Jan 16, 2019 5:04 am    Post subject: Reply with quote

Prompt for query:


Query: how important is Köhler illumination ?
I understand that not using it wastes light and may affect contrast but is there a problem other than that as long as there's no image of the led/filament appearing anywhere?

---
To all:
By the way, if you find useful references such as lists of old cubes/filters/handy fluorophores or equipment documents, I'll put a link in the original post. Send a pm if you prefer.
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pbraub



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PostPosted: Wed Jan 16, 2019 5:11 am    Post subject: Reply with quote

Hi,

there is a paper out detailing a LED illumination axis made with simple off the shelf components (Arduino, Thorlabs, etc.). They also have a comparision of critical and Koehler illumination:

https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0143547

Edit:
I think besides having less light the problems come mostly from projecting the LED pattern onto the sample if you use critical illumination. This may be a problem depending on the LED type.

kind regards
Peter
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