Mountants for fluorescence microscopy

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MicroBob
Posts: 1493
Joined: Sun Dec 25, 2016 9:11 am

Mountants for fluorescence microscopy

#1 Post by MicroBob » Mon Oct 21, 2019 7:08 pm

Hi together,
I've just started to experiment with Epi fluorescence microscopy:viewtopic.php?f=6&t=8206&sid=235a0e88a4 ... 4d8ecc7191
One thing I became aware of immediately was that my preferred mountant for botanical slides, Euparal, shows a lot of autofluorescence itself. I got a green background when I wanted black.
I have now compared a couple of mountants I have for their fluorescence properties: Equal conditions, at first just slide and cover slip, then the different mountants in uniform thickness. LOCA is UV cured, the rest still includes the solvents so will change a bit when drying. Exposure was 2 to 4 times a long as for a normal object, so the effect is exaggerated.

Bob
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Hobbyst46
Posts: 2157
Joined: Mon Aug 21, 2017 9:02 pm

Re: Mountants for fluorescence microscopy

#2 Post by Hobbyst46 » Mon Oct 21, 2019 9:27 pm

Bob, that is a very appropriate test.
What were the wavelengths in this test ? I would check favorite candidates at several wavelength ranges; although expected auto fluorescence from colorless mountants is probably excited in the UV and violet and blue light, not higher than 500nm (and the respective appropriate cutoff and long pass settings).
I see Aroclor on your list. High refractive diatom mountants are most probably fluorescent. On the other hand, clean diatoms are not, so there is hardly motivation to test Pleurax, Naphrax etc (but I think that it is anyway known that these mountants are auto-fluorescent).
Zeiss Standard GFL+Canon EOS-M10, Olympus VMZ stereo

MicroBob
Posts: 1493
Joined: Sun Dec 25, 2016 9:11 am

Re: Mountants for fluorescence microscopy

#3 Post by MicroBob » Tue Oct 22, 2019 8:09 am

Hi Doron,
I tested with the excitation filter combination that blocks above ca. 460nm.
I included the Caedax-Aroclor mix as I have it and the r.i. would be not extremely high. I don't know the r.i. of this mixture and I don't know the typ and concentration of the Aroclor. It probably was mixed as a medium high mountant for radiolaria. Given todays availability of Caedax and Aroclo probably nobody would mix them without carefully noting what it is.
My Dammar resin is soluted it https://www.sigmaaldrich.com/catalog/se ... gIgrvD_BwE
Do you know which process could be suitable after staining in water based stains?
I was suggested to use glycerol as mountant, with something solidifying around it. What I'm not sure is how the glycerol will work with the water based stains - they may leak out of the specimen. Another Idea could be a drop of paraffine oil after fully removing the water with xylene.

My LOCA TP 2500 mounts out of isopropanol look good so far but I don't know how well LOCA, isopropanol and stains interact in the long term.

Bob

Hobbyst46
Posts: 2157
Joined: Mon Aug 21, 2017 9:02 pm

Re: Mountants for fluorescence microscopy

#4 Post by Hobbyst46 » Tue Oct 22, 2019 12:52 pm

MicroBob wrote:
Tue Oct 22, 2019 8:09 am
Do you know which process could be suitable after staining in water based stains?
I was suggested to use glycerol as mountant, with something solidifying around it. What I'm not sure is how the glycerol will work with the water based stains - they may leak out of the specimen. Another Idea could be a drop of paraffine oil after fully removing the water with xylene.
The commercial mountant I mentioned, named Fluoromount, is water based. Your specimen can be very slightly wet or near dry (like if you remove the water by absorption on tissue/filter paper), you put a drop or two of the mountant on the specimen, cover slip and let dry (more than an hour and less than 24h). It is supposed to be a long-term mountant, but I do not know how good it is for botanical sections.

Glycerol never solidifies. Worse, it leaches stains out from plant cells, chlorophyll from algae, etc. I would not use it on plant specimens.
A glycerol-water mixture is not better than pure glycerol.

Paraffin is questionable IMO. I believe that hydrophobic stains, like Rhodamine B, dissolve in paraffin to some extent.
You showed that the solvent of Gum Dammar is benzyl alcohol. This stuff probably fluoresces somewhat when excited with UV, so I am guessing that it will be weakly auto-fluorescent somewhare between the UV and violet; if this mountant is important, real data can certainly be found in the literature. Your Gum Dammar slide appears to be free of auto-fluorescence.
Zeiss Standard GFL+Canon EOS-M10, Olympus VMZ stereo

MicroBob
Posts: 1493
Joined: Sun Dec 25, 2016 9:11 am

Re: Mountants for fluorescence microscopy

#5 Post by MicroBob » Tue Oct 22, 2019 2:43 pm

From what I found about dammar and autofluorescence was that it should have some. My own test showed that it has some, but very very little at a layer of ca. 0.17mm thickness. I will try to get some Benzyl alcohol or dry the varnish and resolve in xylene. I hope that the different solvent doesn't change the autofluorescence of the dammar resin. Also it is quite runny now and the autofluorescence might be increaded after drying. I will report what I will find out. Dammar resin has been in use for a long time and my expectation is that it would give durable slides. Since I won't use it all the time I'm not worried about the xylene.

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