Multi colour FISH assay

Use for discussion of various microscopy and imaging techniques, such as phase, fluorescence and confocal microscopy, electron microscopy, and various other imaging techniques.

Moderator: r.rosati

Multi colour FISH assay

Postby Coolguy » Feb 06 2014 1:32 am

Hi all,

I am required to develop a multi color FISH assay using the following dyes linked to DNA oligonucleotide probes:

Code: Select all
                             Excitation         Emission
1. Alexa 488                 495 nm             519 nm
2. DEAC                      426 nm             480 nm
3. 6-HEX                     535 nm             556 nm
4. Licor 800                 770 nm             786 nm

Questions:
1. Can all these dyes be used together in a single assay? If not can you suggest alternatives? I know in order to visualize all 4 colours of the dyes they should be spectrally well separated (they should appear as 4 distinct colours).
2. Is Licor800 the same as IRDye 800 RS? Does anyone have experience using this dye conjugated to a DNA probe used for locating biomarkers?
3. I was told that mercury lamps in most fluorescence microscopes do not have light output at a wavelength at 770-780nm. How do I check whether there is any light output from the lamp at this wavelength?
4. Can I use the Cy5/Far red filter to visualize Licor800 assuming there is light output by the mercury lamp at higher wavelengths?
5. Are there any special protocols to reduce autofluorescence encountered in human tissue for eg. pancreas, breast etc...?

Any suggestions or feedback would be very useful, thanks in advance.

Daniel.
Coolguy
newcomer
newcomer
 
Posts: 2
Joined: Feb 06 2014 12:57 am

Re: Multi colour FISH assay

Postby DanDraney » Feb 27 2014 2:41 pm

Hi Daniel,

I am a chemist at LI-COR. What you call "Licor 800" is probably refering to the IRDye 800 phosphoramidite dye used to make labeled oligonucleotides on a DNA synthesis machine. It is not quite the same as IRDye 800RS, which is mainly used in the NHS ester for for conjugation with amines (including oligonucleotides with amino linkers). Excitation/emission maxima are similar. Details can be found from here, including chemical structures of both dyes, if you are interested:
http://www.licor.com/bio/products/reagents/irdye/

Our instruments use diode lasers for excitation of these long wavelength dyes. I believe your source is correct: mercury lamps have little energy, if any, at 770nm. I would imagine the output spectrum of your lamp is available from the manufacturer. You may also encounter problems of weak detector response above 700nm and coatings on your optics designed to filter out NIR light. Again, the manufacturer should be able to help you in evaluating these issues.

If your system can detect Cy5, you could also use our IRDye 650 (NHS ester form available). Oligonucleotides can be ordered from various suppliers with IRDye 700 (via phosphoramidite chemistry again, abs max ca. 680nm). The autofluorescence for a "650" (red) or "700" (far red / near infrared) dye will be significantly higher than for dyes with absorption maxima in the 750-790nm range, but the shorter wavelengths will probably be more easily detected on your system.

Best,
Dan
DanDraney
newcomer
newcomer
 
Posts: 1
Joined: Feb 27 2014 2:11 pm

Re: Multi colour FISH assay

Postby Coolguy » Mar 20 2014 2:33 am

Hello Dan,
Sorry to be seeing this only now. THank you so much for your valuable response, being a chemist from LI-COR, I couldn't have got a better response with regard to the Licor 800 dye clarification.Also, thank you for suggesting other dye alternatives that I can use for my FISH assay.

Best,
Daniel.
Coolguy
newcomer
newcomer
 
Posts: 2
Joined: Feb 06 2014 12:57 am

Re: Multi colour FISH assay

Postby rjm1975 » Aug 19 2014 10:31 pm

If you are doing widefield microscopy (i.e. conventional epifluorescence microscopy with dichroics, filters, and a camera) you will have a hard time cleanly resolving the first three of your dyes, especially the Alexa 488 and HEX. It might be possible with confocal with a spectrally resolved detector, but those are not so common.

If you are using a mercury lamp, you may be able to use a set labeled with: Alexa 405 (use DAPI filter), Alexa 488 (FITC filter), Cy3, and Cy5. Those will all be excited well by the mercury lamp, and separated nicely by filters. There will be a little bleedover of the Alexa 488 into the Cy3 channel. Instead of Cy3/Cy5 you could choose (for example) Texas Red and Cy5.5. For Cy5, Cy5.5, and farther out dyes you also need a camera with good red/near infrared sensitivity.
rjm1975
technician-in-training
technician-in-training
 
Posts: 8
Joined: Aug 19 2014 9:41 pm


Return to Microscopy and Imaging Techniques

Who is online

Users browsing this forum: No registered users and 1 guest