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Excitation wavelengths for autofluorescence microscopy

 
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Pau
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PostPosted: Fri Sep 04, 2015 3:48 am    Post subject: Excitation wavelengths for autofluorescence microscopy Reply with quote

What are the most useful wavelengths to induce visible autofluorescence in biological specimens?

I also will be interested in simple staining like calcofluor and I have access to typical brightfield microscopy dyes like hematoxilin, eosin and so at work but I’ll not do the jump to expensive and toxic typical fluorescence dyes.

I have jointed some fluorescence equipment for my Zeiss Standard and I’m planning to make a LED illuminator

I’ve searched for info on the subject with very little success aside the physical background.

Any help and references will be highly appreciated.
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Ichthyophthirius



Joined: 07 Mar 2013
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PostPosted: Fri Sep 04, 2015 5:39 am    Post subject: Reply with quote

Hi Pau,

You can search the excitation and emission spectra of your preferred dyes on a number of online tools (search "fluorescence spectra") like this one http://www.spectra.arizona.edu/

However, it's even more useful to search for each molecule individually and see what other microscopist have done.

Limit yourself to a few dyes/fluorophores to start with rather than trying to set up everything in one go.

For example:

Acridine orange: Ex. blue Em. green (DNA), orange (lysosomes), red (RNA)
Carotene: Ex. blue Em. green/yellow
Chlorophyll: Ex. blue Em. red (quite far red for consumer cameras with IR filter)
Eosin: Ex. green Em. yellow
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Pau
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PostPosted: Fri Sep 04, 2015 8:00 am    Post subject: Reply with quote

Thanks Ichthy,

I've visited several websites but no one was so complete easy and clear than Arizona University at your link
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Charles Krebs



Joined: 01 Aug 2006
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PostPosted: Fri Sep 04, 2015 12:27 pm    Post subject: Reply with quote

LedEngin makes a good variety of UV and violet leds.

Be careful of your eyes when using some of these especially the UV ones!


http://www.ledengin.com/files/products/LZ1/LZ1-00UV00.pdf
http://www.ledengin.com/files/products/LZ1/LZ1-00UB00.pdf
http://www.ledengin.com/files/products/LZ4/LZ4-04UV00.pdf
http://www.ledengin.com/files/products/LZ4/LZ4-00UA00.pdf
http://www.ledengin.com/files/products/LZ4/LZ4-00U600.pdf
http://www.ledengin.com/files/products/LZC/LZC-00UA00.pdf
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RogelioMoreno



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PostPosted: Sat Sep 05, 2015 7:38 am    Post subject: Reply with quote

For autofluorescence the cube that I love is the following:

https://www.microscopyu.com/articles/fluorescence/filtercubes/ultraviolet/uv2b/uv2bindex.html


The second is similar to the following:

https://www.microscopyu.com/articles/fluorescence/filtercubes/blueviolet/bv2b/bv2bindex.html


Maybe in the future I will try with fluorescence dyes.

Rogelio
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Pau
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PostPosted: Sat Sep 05, 2015 8:33 am    Post subject: Reply with quote

Charles, thank you for the links:
Charles Krebs wrote:

Be careful of your eyes when using some of these especially the UV ones!

I'm well aware, for example, I've mounted an UV shield even before having any UV source.

Rogelio, thanks, your exctation filter spectrum is like a 350nm LED
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g4lab



Joined: 23 May 2008
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PostPosted: Sat Sep 05, 2015 6:40 pm    Post subject: Reply with quote

http://www.nightsea.com/area-of-use/in-the-lab/
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NileRed



Joined: 16 Jul 2010
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Location: Moscow

PostPosted: Tue Nov 17, 2015 1:51 pm    Post subject: Reply with quote

Hi Pau,

Concerning "amateur fluorescence microscopy", what I really love, my answers:

Quote:
What are the most useful wavelengths to induce visible autofluorescence in biological specimens?


365 nm - excitation (365.5 nm - is on of the most intensive emission peak of high pressure mercury bulb, so this is a reason). Actually for autofluorescence you can use even green excitation, but in such case you will observe only yellow or red autofluorescence, thus 365 nm allow you to excite maximum cell components with autofluorescence from violet to red.

Quote:
I also will be interested in simple staining like calcofluor and I have access to typical brightfield microscopy dyes like hematoxilin, eosin and so at work but I’ll not do the jump to expensive and toxic typical fluorescence dyes.


It is very important to understand that many fluorescent dyes are not proper for fluorescent staining e.g. fluoresceine is not used directly, only in the form of conjugates with antibodies or other targeting molecules - it is not your case, such compounds are very expensive.
So, what can I recommend to try (not expensive, not extremely toxic)
Acridine family - Acridine orange, Acridine yellow, acriflavine, quinacrine (ebay, pet drug store) - one their drawback - they stain almost everything, but more intensive xyleme in plants sections and nucleas in cells. It is very important to try different buffers (e.g. PBS), because different pH (acidic, neutral and basic) will result in different fluorescence pattern. Also, living and dead cells will stain differently.
Nile blue - quite cheap dye (ebay) for both brightfield and fluorescence staining (red or yellow fluorescence) it is used for stainig of lipids, fatty components of cell and fat droplets.
Doxorubicin, Chromomycine A3 and some other related antibiotics can be used for staining of nucleus.
Aniline blue (at basic pH ~8-9, so use proper buffer!!!) - Callose, cell walls, cellulose

Nilered (my nickname from this beautiful dye), DAPI, Sytox, YOYO, TOTO, Propidium iodide - are expensive professional dyes. Ethidium bromide - very bad guy, avoid it.

So, good luck!
Roman Very Happy
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Pau
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PostPosted: Tue Nov 17, 2015 3:35 pm    Post subject: Reply with quote

Roman, thank you for all that info!

365 is high in my list.

I was convinced that Acridine Orange was a very toxic mutagen dye because it interacts with DNA. After your recommendation I've googled a bit and it doesn't seem so dangerous

I'm still in the way to desing the LED illuminator, I'm really slow!
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discomorphella



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PostPosted: Thu Nov 19, 2015 11:18 am    Post subject: Reply with quote

Pau, if you can get a copy of Steven Ruizin's book on plant microscopy and microtechnique he has a large chart of many autofluorescence ex/em wavelengths. The acridine family is not exactly harmless, all the acridine nucleus dyes are at least suspected mutagens, but good quality neoprene gloves and safety glasses should be adequate. Avoid breathing dust when you are measuring out the dyes....also, the anthracyclines like doxorubicin are incredibly toxic, they are in fact antineoplastic agents. Don't mess with those....
DAPI and the Hoechest dyes are indole derivatives and intercalators, you can probably avoid those. The rest are ok with gloves.

David
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Pau
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PostPosted: Thu Nov 19, 2015 12:05 pm    Post subject: Reply with quote

David,
Thanks for the info. About acridine orange I was surprised to do not find clear references about its mutagen (so also carcinogenic) risk in official chemical product data sheets. I vaguely remember it in the list of mutagen agents in my Genetics courses back in the late 70s.

Safety first Very Happy . I don't plan to use any of those products, I'm planning the do autofluorescence and simple fluorescence microscopy as a home project. I don't have an adequate lab, just a school lab at work.
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esotericman



Joined: 08 Jun 2012
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PostPosted: Mon Nov 23, 2015 9:47 am    Post subject: Reply with quote

405nm, 488nm, 543nm depending on what you want to image.

I just imaged some pseudoscorpions which really were bright with 488, but if you want to look at resilin, you need 405.

What are you looking at and for?
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Pau
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PostPosted: Mon Nov 23, 2015 9:59 am    Post subject: Reply with quote

Thanks for the info
Quote:
What are you looking at and for?

Nothing really specific: Plant structures (lignine, cellulose, photosyntetic pigments...) maybe some small arthropods, stained microscope slides and so.

This is wy I'm desinging a multiLED illuminator to have acces to different wavelenghts from UV to green.
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