DIY Epifluorescence

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kwesi
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Joined: Tue Aug 10, 2021 7:08 pm

DIY Epifluorescence

#1 Post by kwesi » Wed Sep 14, 2022 7:05 pm

Apologies if this has been posted to this forum before; I looked around but didn't see it posted anywhere.

I'm researching affordable epifluorescence microscopy set-ups for viewing autofluorescence in fungi, chloroplasts, plastics, etc.
I found this article published by the American Chemical Society that models a few DIY solutions that seem to work for them. I think I'm going to buy a used filter cube, build the PVC filter drawer and mount it to the frame of my compound microscope. I'd omit the eyepiece though and have it fit my DSLR reduction optics. I think the hard part will be getting the 160mm tube distance correct and making sure the holes are all aligned properly.

This build appeals to me over some of the other DIY solutions I've seen; since it's modular it seems very straightforward to setup once it's built and I wouldn't have to worry about protecting myself or others from stray UV light like in some other setups. I also like the idea of using a manufactured filter cube since the filters are pre-matched for minimal crossover between excitation and emission wavelengths. If this works well I'd also like to show it to my students as I'm running some plant histology workshops and fluorescence could be interesting to them (it would be great if it helped make the arbuscules in mycorrhizal fungi more prominent).

If anyone has any tips for viewing autofluorescence in plants/fungi/microbes I'm all ears! I've done a fair amount of research but most of the papers and videos I've turned up are aimed at reducing or filtering out autofluorescence rather than getting the most out of it, and a lot of the data on fluorophores seems to be focused on stains rather than endogenous compounds. Since I want to view autofluorescence in a range of specimens, I'm thinking I should find a filter cube that uses wide band blue or UV excitation and a long pass barrier filter. Does that seem like a good way to go, or would it be better to get a couple of cubes with different bandpass barrier filters for viewing different samples, since bandpass filters have higher signal-to-noise ratios?

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Scarodactyl
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Joined: Sat Mar 03, 2018 9:09 pm

Re: DIY Epifluorescence

#2 Post by Scarodactyl » Wed Sep 14, 2022 7:27 pm

It might be even easier to find an old fluoreacence illuminator and replace the (likely missing or nonfunctional) mercury lamphouse with a diy LED one. I took this approach for a friend's lab which needed a fluorescence setup on the cheap. I keep meaning to write that up, it just needs a bit more work.

kwesi
Posts: 28
Joined: Tue Aug 10, 2021 7:08 pm

Re: DIY Epifluorescence

#3 Post by kwesi » Wed Sep 14, 2022 11:19 pm

Scarodactyl wrote:
Wed Sep 14, 2022 7:27 pm
It might be even easier to find an old fluoreacence illuminator and replace the (likely missing or nonfunctional) mercury lamphouse with a diy LED one.
Would it not cost a fair bit more to buy an illuminator + filters than to just buy a filter cube and build a drawer for it? If building the parts is a barrier the site I linked to also has CAD and STL files so you can get a website to 3D print them for you, should only be about $100.
Also maybe I'm misunderstanding but wouldn't adding a component between the turret and head of a microscope only work for those with infinity optics?

Scarodactyl
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Re: DIY Epifluorescence

#4 Post by Scarodactyl » Wed Sep 14, 2022 11:27 pm

Sometimes turrets can be found cheap. I got a leitz biomed with a ploem and three prisms for like 220 dollars a while back. The finite ones have lenses that compensate for the extra space.
You can certainly DIY the whole thing, but with this sort of thing it's sometimes nice to reduce the variables involved.

Hobbyst46
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Re: DIY Epifluorescence

#5 Post by Hobbyst46 » Thu Sep 15, 2022 7:03 pm

For fluorescence, it would be important and cost-effective to know the expected fluorophores ahead of purchase or any DIY project.

The name "auto fluorescence" does not mean much. In a green plant part (including algae) the main fluorophore are likely chlorophylls and similar molecules. For these, excitation peak is 400-430nm whereas emission is >600nm. Namely, a huge Stokes shift, so an excitation filter plus a dichroic mirror (shortpass) with a cutoff at anywhere in the 450-500nm range (or even wider) and an emission filter (longpass) of >690nm (say) will do the job nicely. Without UV.

However plants contain other fluorophores, Vitamin-E like for example that fluoresce at ~550nm, and are better excited with UV (365-390nm). What fungi contain I do not know.

And a cube containing high quality interference filters (including the said mirror) is expensive ! around 200-300$ I think.
Monochromatic LEDs are more convenient and safer in use than a mercury or xenon lamp. In addition, One can omit the excitation filter, so the cube becomes less expensive (by 20% maybe).

A few years ago, on the photomacrography forum there was a nice description of a DIY multiLED epi-fluorescence accessory. Can't remember the name of the author, but it was impressive.

Still, the costly cube remains.
Bandpass filters are not more "accurate" than longpass or shortpass filters, assuming all are interference filters.

kwesi
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Re: DIY Epifluorescence

#6 Post by kwesi » Fri Sep 16, 2022 6:36 pm

Hobbyst46 wrote:
Thu Sep 15, 2022 7:03 pm
For fluorescence, it would be important and cost-effective to know the expected fluorophores ahead of purchase or any DIY project.
That's what I'm hoping to figure out, but so far I haven't been able to track down that information. I've seen some tables that list the excitation/emission wavelengths for different natural fluorophores in plants, but without knowing the quantum yield extinction coefficients (which basically give the relative brightness) and the relative abundance of those compounds in different cells, that data isn't super useful to me. I'm hoping that someone with more experience in this area can fill me in on which structures exhibit autofluorescence that's bright enough to be seen with a DIY setup, and what excitation/emission ranges they fall under.

I know that arbuscular mycorrhizae exhibit autofluorescence, but I can't find what the fluorophores are or their excitation/emission spectra.
Hobbyst46 wrote:
Thu Sep 15, 2022 7:03 pm
And a cube containing high quality interference filters (including the said mirror) is expensive ! around 200-300$ I think.
Luckily Nikon ones are often around $100 used which isn't too bad. I like the idea of buying individual filters to change out but single filters seem harder to find used, and new ones seem to cost hundreds on their own. Unless you know of anywhere to get them cheaper?
Hobbyst46 wrote:
Thu Sep 15, 2022 7:03 pm
Monochromatic LEDs are more convenient and safer in use than a mercury or xenon lamp
LEDs aren't truly monochromatic, and none of the LED flashlight manufacturer's I've found publish their spectral data, which makes them less reliable than filter cube makers like Nikon who do publish those data. In theory I could wire up my own flashlight from individual diodes but to figure out how to do that properly would be a whole other project. I'd like to use a white LED flashlight that covers more of the spectrum + an excitation filter so I know for sure what wavelengths I'm getting.
Hobbyst46 wrote:
Thu Sep 15, 2022 7:03 pm
Bandpass filters are not more "accurate" than longpass or shortpass filters, assuming all are interference filters.
I didn't say they were more accurate, I said they have a better signal-to-noise ratio because they filter out unwanted wavelengths. What I'm trying to find out is which areas of the spectrum will give me the most bang for my buck so I can balance the tradeoff between specificity and general usefulness. Because the data available is pretty scant, I'm not sure which fluorophores I should be going after. I'm wondering if anyone with more experience can offer some insight!

Scarodactyl
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Re: DIY Epifluorescence

#7 Post by Scarodactyl » Fri Sep 16, 2022 7:03 pm

Spectral data is readily available for eg CREE LEDs, though you'll usually have to dig through a pdf for it.
It seems daunting, and eg thorlabs charges a ton for a simple monochrome led lamphouse, but it's actually pretty cheap and easy, especially if you have a 3d printer.

kwesi
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Re: DIY Epifluorescence

#8 Post by kwesi » Sat Sep 17, 2022 4:50 am

Scarodactyl wrote:
Fri Sep 16, 2022 7:03 pm
Spectral data is readily available for eg CREE LEDs, though you'll usually have to dig through a pdf for it.
The issue I'm having isn't with LED manufacturers not sharing their data, it's that flashlight makers rarely tell you which particular LEDs they're using.

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MasLovesMicrobes
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Re: DIY Epifluorescence

#9 Post by MasLovesMicrobes » Sun Dec 18, 2022 11:14 pm

Hey Kwesi,

I also am interested in visualising autofluorescence on a broad range of specimens. I currently have an Olympus U-MWIBA filter cube installed. I think excitation filter is 460-490 and barrier filter is 515-550. I also have a U-M41007A filter installed, I believe excitation is 530-560, cant really find emission spectra for this filter but I'm guessing around 570 because it is designed for the CY3 fluorophore. I think at least U-M41007A is specified as a narrow range cube.
I will say that I do find a lot of autofluorescence with these cubes, though mostly in archaeal and bacterial blobs. I definitely am not happy with the specificity and intensity that I get with these cubes, I would not recommend these to you. They came with the microscope when I bought it, theyre cool to play around with but I am certainly looking to exchange them for some more broad spectrum ones or maybe some specifically tailored to certain fluorophores in protozoa.
Like you I am also having trouble finding information on which fluorophores are abundant naturally in protozoa, algae and pond life. Seems there is not really much interest in autofluorescence in the scientific literature. I would love to hear from you when you do settle on a certain combination of filters and mirrors, if indeed broad spectrum is the way to go, or whether youre targeting certain abundant fluorophores with different cubes. I will try and include some pictures to show you how my specimens glow and you can draw from that as well, but my advise would be to steer clear of these ones.

Kind regards,
Mas
Beauty lies not in the eye of the beholder, it lies in the lens of a microscope.

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