Excitation wavelengths for autofluorescence microscopy
Moderators: rjlittlefield, ChrisR, Chris S., Pau
Excitation wavelengths for autofluorescence microscopy
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.
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.
Pau
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- Joined: Thu Mar 07, 2013 5:24 am
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
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
- Charles Krebs
- Posts: 5865
- Joined: Tue Aug 01, 2006 8:02 pm
- Location: Issaquah, WA USA
- Contact:
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/ ... 00UV00.pdf
http://www.ledengin.com/files/products/ ... 00UB00.pdf
http://www.ledengin.com/files/products/ ... 04UV00.pdf
http://www.ledengin.com/files/products/ ... 00UA00.pdf
http://www.ledengin.com/files/products/ ... 00U600.pdf
http://www.ledengin.com/files/products/ ... 00UA00.pdf
Be careful of your eyes when using some of these especially the UV ones!
http://www.ledengin.com/files/products/ ... 00UV00.pdf
http://www.ledengin.com/files/products/ ... 00UB00.pdf
http://www.ledengin.com/files/products/ ... 04UV00.pdf
http://www.ledengin.com/files/products/ ... 00UA00.pdf
http://www.ledengin.com/files/products/ ... 00U600.pdf
http://www.ledengin.com/files/products/ ... 00UA00.pdf
-
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- Joined: Fri Nov 20, 2009 11:24 am
- Location: Panama
For autofluorescence the cube that I love is the following:
https://www.microscopyu.com/articles/fl ... index.html
The second is similar to the following:
https://www.microscopyu.com/articles/fl ... index.html
Maybe in the future I will try with fluorescence dyes.
Rogelio
https://www.microscopyu.com/articles/fl ... index.html
The second is similar to the following:
https://www.microscopyu.com/articles/fl ... index.html
Maybe in the future I will try with fluorescence dyes.
Rogelio
Hi Pau,
Concerning "amateur fluorescence microscopy", what I really love, my answers:
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
Concerning "amateur fluorescence microscopy", what I really love, my answers:
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.What are the most useful wavelengths to induce visible autofluorescence in biological specimens?
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.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.
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
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!
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!
Pau
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- Location: NW USA
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
DAPI and the Hoechest dyes are indole derivatives and intercalators, you can probably avoid those. The rest are ok with gloves.
David
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 . 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.
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 . 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.
Pau
- esotericman
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Thanks for the info
This is wy I'm desinging a multiLED illuminator to have acces to different wavelenghts from UV to green.
Nothing really specific: Plant structures (lignine, cellulose, photosyntetic pigments...) maybe some small arthropods, stained microscope slides and so.What are you looking at and for?
This is wy I'm desinging a multiLED illuminator to have acces to different wavelenghts from UV to green.
Pau