Amateur uses for fluorescence microscopy?

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Beatsy
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Amateur uses for fluorescence microscopy?

Post by Beatsy »

I recently purchased an Olympus BX61 with a couple of very nice high NA PlanAPOs included. It's ideal for dedicating to high-resolution diatom imaging which was my sole reason for getting it.

It came configured as a fluorescence scope with all six positions occupied in the filter turret. I had to remove one cube to get "full-spectrum" light for transmitted illumination (to do BF, DF and COI imaging). The dichroic mirrors degrade sharpness a bit too, but you have to pixel-peep at 200% to reliably detect that - so it's not a significant issue (and no issue at all with a cube removed for bright field work).

I intended to ignore the fluorescence stuff entirely but the set of cubes piqued my curiosity. I did some Googling to find out what was fitted and hopefully get some idea of the kind of work the scope was originally configured and used for. I found most of the filter specs but still haven't a clue what they'd apply to. It all looks very, very specialist and complicated (and expensive).

Help!

The filters installed - posn. "name" (excitation nm)-(emission nm) [usage notes]

1. "U-MNUAZ (DAPI)" (360-370)-(420-460) [narrow band U excitation for colour separation]
2. "30-150232 GREEN" (484-504)-(517-537) [???]
3. "30-150132 ORANGE" (534-544)-(576-596) [???]
4. "U-NS1004 v2-F/R" (??????)-(??????) [ B/G?]
5. "U-N44001- HQ FITC" (467-498)-(513-556) [FITC]
6. "U-MW1Y2" (545-580)-(600-???) [???]

The labels on the turret might offer relevant hints, but didn't help me much. I understand the principles and techniques used in fluorescence, but my knowledge of potential applications is very, very sparse, and the possibilities far too diverse.
20221213_162519--pmn.jpg
With a minor mod, the first filter does look immediately useful to me. Swap the emission filter with a UV-blocker (allowing visible through) and it looks ideal for auto-fluorescence work. I verified that a Convoy S2+ (365nm UV torch) shining into the EPI illumination port has enough power to induce *very* strong fluorescence - so I can avoid using a mercury lamp too. Winning!

The last filter surprised me. I thought fluorescence used UV for excitation but this is green light causing red emission. I later discovered the other filters use long excitation wavelengths too (as in visible light, not UV). Something learned. Again.

My question is - could any of this be useful or interesting to the amateur tinkerer? Mostly in an imaging context for me. If so, then what for? I expect the answer to be "not much" and will just revert to ignoring the capability if that's the case. No big deal. But you never know...

Thanks in advance for any and all info or pointers.

Pau
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Re: Amateur uses for fluorescence microscopy?

Post by Pau »

You're right, they seem specialized cubes with both excitation and emission narrow spectral bands. They will be good for matched fluophors and for sequential imaging of multiple stained samples, likely with confocal LSM microscopes

For amateur use (mainly autofluorescence and not much specific stains) most of us use old style wide band filters that produce more intense fluorescence and in some cases like UV and blue EX more emitted colors although also less clean images.

Having a band pass excitation seems very desirable. If after testing you find that you want more colors likely changing only the emission filters of the UV (as you said) and Blue cubes would do.
Except #1 they are labelled (or described) with the emission color (in most cases excepted LOMO is more usual the excitation color naming)

1. "U-MNUAZ (DAPI)" (360-370)-(420-460) [narrow band U excitation for colour separation]
The excitation band is very narrow but very good for Mercury lamps and good UV365 LEDs like the Nichia. Likely it will limit the emission to Violet-blue, does it?
2. "30-150232 GREEN" (484-504)-(517-537) [???] *
blue/cyan-green
3. "30-150132 ORANGE" (534-544)-(576-596) [???] *
green/orange
4. "U-NS1004 v2-F/R" (??????)-(??????) [ B/G?] **
no idea, of course. Test it
5. "U-N44001- HQ FITC" (467-498)-(513-556) [FITC] **
blue - green
6. "U-MW1Y2" (545-580)-(600-???) [???] **
green- red???
Found only one reference: file:///C:/Users/USuario/Downloads/Phylogenetic_Metabolic_and_Taxonomic_Diversities_S.pdf. Seems adequate for the Cy5 dye, if so, it would be mislabeled

* Spectrum Green and Spectrum Orange are names of (now discontinued) filter cubes from Chroma:
https://www.chroma.com/products/sets/mf ... lot-left-1 The EX does not full coincide
https://www.chroma.com/products/sets/mf ... lot-left-3 The data fully agree

DAPI, FITC and Texas Red are names of usual fluorescent dyes, you will easily find their spectra online.

** The "U___" labels seem to refer to Olympus cubes, maybe Olympus papers or tech service could provide more info

***If as you said you don't want to use Mercury burners or other expensive and/or dangerous light sources I would try putting your halogen lamp house in the upper position as excitation source for all cubes excepted the "DAPI" Ultraviolet one, and an UV LED for it like you have tested with the flashlight. Thee are more powerful models like the Nichia NVSU233B UV 365 I use in my (now improved) Multi LED setup https://www.photomacrography.net/forum/ ... 37#p201937

Good subjects for testing are fresh plants like ferns with spores, pollen but also white and colored plastics (many of them are fluorescent) and even white paper and fabrics. There are some vendors selling online stained fluorescent slides. Also there are proper test slides with fluorescent microscopic spheres or other calibrated subjects.

I have quite some documents and links I could share upon your request.
Pau

pbraub
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Re: Amateur uses for fluorescence microscopy?

Post by pbraub »

Hi,

this looks like a standard setup for in situ hybridisation (e.g. for genetics or pathology).

DAPI is used as a counterstain in many fixed pathological/biological samples.

Spectrum green and spectrum orange are proprietary fluorophores used by abbott. These filter sets are very "narrow" and give good contrast with these probes.
(spectrum green captures some of FITC/Cy2 and orange some of TRITC/Cy3)

The B/G Filter set is most likely a dual-band filter for observation of the green and orange channels combined (e.g. to evaluate co-localisation of the orange and green signal, or to count ratios between orange and green...)

FITC is similar to spectrum green but should be wider. this gives you more light (for both excitation and emission) at the cost of less contrast for special purpose probes but covers more fluorophores (like FITC, Alexa488 or Cy2)

Texas Red is a fluorophore with green ex and red emission. Should be well above spectrum orange (wavelength wise).

For hobbyist purpose one of the things to try would be to exchange the emission filter of the DAPI cube to a longpass (LP 400 or higher).
The green and dual band filters also give some interesting autofluorescence in some subjects. But just try around.

All in all I like the BX61. How are you controlling yours? I'm not totally happy with mine (controlled by micro-manager).

Peter

Beatsy
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Re: Amateur uses for fluorescence microscopy?

Post by Beatsy »

Thanks for all that info Pau. I had gained further glimmers of understanding with subsequent searches and your post adds more. The outcome is that I think protein/dye/stain fluorophore-based fluorescence stuff is for the lab rats, not me. However...

Autofluorescence looks a lot more interesting now I know it's not just UV illumination (excitation). Plenty of visible wavelengths work for excitation too, but usually need narrow band emission filters to get a clean image. Happily, some filters fitted to this scope match a few of the more useful plant emission wavelengths too (from lignin, chlorophyll etc) so it seems I gained a pretty significant "something new" to experiment with as well. Diatoms first though...

Random interesting paper on the topic: Vital Autofluorescence: Application to the Study of Plant Living Cells
https://www.hindawi.com/journals/ijs/2012/124672/

Beatsy
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Re: Amateur uses for fluorescence microscopy?

Post by Beatsy »

pbraub wrote:
Thu Dec 15, 2022 1:56 pm
Hi,

this looks like a standard setup for in situ hybridisation (e.g. for genetics or pathology).

DAPI is used as a counterstain in many fixed pathological/biological samples.

Spectrum green and spectrum orange are proprietary fluorophores used by abbott. These filter sets are very "narrow" and give good contrast with these probes.
(spectrum green captures some of FITC/Cy2 and orange some of TRITC/Cy3)

The B/G Filter set is most likely a dual-band filter for observation of the green and orange channels combined (e.g. to evaluate co-localisation of the orange and green signal, or to count ratios between orange and green...)

FITC is similar to spectrum green but should be wider. this gives you more light (for both excitation and emission) at the cost of less contrast for special purpose probes but covers more fluorophores (like FITC, Alexa488 or Cy2)

Texas Red is a fluorophore with green ex and red emission. Should be well above spectrum orange (wavelength wise).
Thanks for this. Three days ago, I'd have not understood a word of it - but after my googlings and readings of the past few days, I do (understand a word or two :D )
For hobbyist purpose one of the things to try would be to exchange the emission filter of the DAPI cube to a longpass (LP 400 or higher).
The green and dual band filters also give some interesting autofluorescence in some subjects. But just try around.
Yes, auto-fluorescence does look best for "less-serious" tinkering, and the most interesting - to me. The bandpass filters will add options so it's definitely worth having them - as a "comes with for free" option, anyway.
All in all I like the BX61. How are you controlling yours? I'm not totally happy with mine (controlled by micro-manager).
I'm just using the handset. I assume PC control requires windows 7 plus some no-longer-supported software from Olympus. But I didn't bother looking for it as I had no intent to hook the BX to a PC for diatom imaging. Mine came with a peltier-cooled, mono, CCD camera too (which does pixel-shift). But that looks like it has a firewire interface and needs drivers etc. I didn't look hard for any s/w for that either - I use Sony mirrorless cameras. Is it feasible to get this stuff working under PC control now? I do have an old redundant PC fossilising under a pile of boxes, somewhere...

pbraub
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Re: Amateur uses for fluorescence microscopy?

Post by pbraub »

You can get pretty far with micro-manager (https://micro-manager.org/) its a free and open source control software for microscopes (everything from hobby usb cameras to fancy custom build setups). chances are good that you can get the fw camera working. it has a broad range of drivers (also industrial cameras often work)

You can connect to the BX-UCB via a normal serial connection. I use a dedicated card, but there are also USB to serial connector cables (which I used until they suddenly decided to change the com port on every boot which screwed up the micromanager (MM) configuration). The MM unfortunately cannot control the Top Lens of the UCD8A (if you happen to have one).

If you are into programming there should even be some possibility to trigger the Sony camera from MM (there is an Arduino interface for MM). This would be one of my dream projects - however I don't have time at the moment.

So in short - its totally feasible to get the BX61 working under windows without investing into Olympus software. They ask pretty steep prices for automated control. MM and BX61 are no dream match, in some cases its pretty slow.


Have fun with your new BX61.
I'm off to bed, have to stare through a B/G filter tomorrow morning to evaluate a lot of FISH, yay.
Peter

Lou Jost
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Re: Amateur uses for fluorescence microscopy?

Post by Lou Jost »

Beatsy, I like UV excitation much more than visible-light excitation, because the fluorescence is always (under our conditions) of a lower wavelength than the excitation light (a consequence of conservation of energy). This means UV fluorescence can be any color, but green excitation can only produce yellow or red tones. Many plant parts have multicolored fluorescence under UV, but monotonous colors under green excitation.

Beatsy
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Re: Amateur uses for fluorescence microscopy?

Post by Beatsy »

Lou Jost wrote:
Thu Dec 15, 2022 6:27 pm
Beatsy, I like UV excitation much more than visible-light excitation, because the fluorescence is always (under our conditions) of a lower wavelength than the excitation light (a consequence of conservation of energy). This means UV fluorescence can be any color, but green excitation can only produce yellow or red tones. Many plant parts have multicolored fluorescence under UV, but monotonous colors under green excitation.
I know what you mean Lou, and it would have probably been my preference, not least because (until recently) I thought UV was the only way. But the other excitation wavelengths can reach parts that UV can't. There are proteins and "stuff" that don't fluoresce under UV, but do at longer wavelengths and this can reveal different structures or processes or reactions etc. So while I'm certainly pleased to have UV excitation, and will almost certainly use it most, I'm also interested in tinkering to find "other results" too. Even with that, the multicoloured aspect is easily restored with simple post processing to layer images taken under different wavelengths. In an imaging/arty context, there's no rule that (say) green emissions have to be rendered green either. I think (hope) there's scope to reveal new and interesting features in old familiar subjects...
Last edited by Beatsy on Fri Dec 16, 2022 2:00 am, edited 1 time in total.

Beatsy
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Re: Amateur uses for fluorescence microscopy?

Post by Beatsy »

pbraub wrote:
Thu Dec 15, 2022 2:44 pm
...
So in short - its totally feasible to get the BX61 working under windows without investing into Olympus software. They ask pretty steep prices for automated control. MM and BX61 are no dream match, in some cases its pretty slow.
...
Thanks for that Peter. I will take a look at it when I start getting UV (brightfield) imaging up and running. If I could use the peltier cooled camera, it would save me the difficult decision of which Sony camera to convert to full spectrum mono. I have A1, A7riv, A9 and A7rii. Currently, with the way the camera is attached using C-Mount, the A7riv looks best. It has about the right image scale with 3.75 micron pixels and supports pixel-shift. But that's my highest resolution camera at 61 megapixels - next highest is A1 at 50 megapixels (and no-way would I convert that). A7rii is a candidate - but lacks pixel-shift. A9 looks pretty good as well with near 6 micron pixels, but they don't grab all the detail at lower powers. I'd have to use a projection lens to increase image scale there, but it would be good for low light. It also lacks pixel shift though. Still agonising over this...

Pau
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Re: Amateur uses for fluorescence microscopy?

Post by Pau »

Lou Jost wrote:
Thu Dec 15, 2022 6:27 pm
... because the fluorescence is always (under our conditions) of a lower wavelength than the excitation light (a consequence of conservation of energy).
Of course the First Law of Thermodynamics must be accomplished but IMO what explains this phenomenon (Stokes shift*) is the Second Law of Thermodynamics that also must be obeyed

* https://en.wikipedia.org/wiki/Stokes_shift
Pau

Lou Jost
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Re: Amateur uses for fluorescence microscopy?

Post by Lou Jost »

I agree. Conservation of energy is enough to explain why the fluorescence light cannot have a shorter wavelength than the excitation light (for single-photon transitons) but the second law is needed to explain why there is a shift in wavelength between incoming and outgoing light.

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