At the bottom of this webpage are links to graphical interactive databases of fluorophores and filter sets.

For fluorescence microscopy one of the common questions we get is:
What two, three or four fluorescent dyes should we use for multi labeling?

For instance, let's say your have cultured cells or a vibratome section of tissue and you want to look at DNA, f-actin, your favorite protein and microtubules (tubulin) by fluorescent light microscopy. We might suggest you use respectively DAPI (binds to DNA), fluorescein (bound to phalloidin which binds to f-actin filaments), Cy3 (antibody method for your favorite protein) and Cy5 (antibody method to alpha or beta tubulin).

In general, you may use any one dye from each of the groupings below for multi-labeling -- this list is not exhaustive. Please consult with us for other special combinations, for instance dyes that excite at 490 nm but have a long Stokes shift to emit inthe red or far-red. Also, there are a lot of options for quantum dots and a few other ways of generating contrast in muti-photon.

Blue, approximately 460nm. Dapi, Hoechst Cascade Blue, Pacific Blue, FluoroBlue
Green, approximately 520nm Alexa 488, Cy2,GFP, eGFP, YFP, Fluo3 FITC (fluorescein), Oregon Green, , Bodipy, NBD, Calcium Green
Red, approximately 590nm. Alexa 568, Cy3, Propidium iodide, Mitotracker red, DiI, DiA Rhodamine, Texas red, TRITC, Alexa 594, Alexa 546, Alexa 555, DsRed2, phycoerythrin (PE)
Near infra red, approximately 670nm eFluor 660, Alexa 633, Alexa 647, Cy5, DRAQ5 APC

Most probes are excited at 405, 488, 568, and 633 nm with short Stokes shifts such that the emissions are detected in the next longer wavelength range (violat to blue, blue to green, etc.).

However, a few probes have longer Stokes shifts. For instance, look at Brilliant Violet series of probes. They all excite in the 400-420 nm range but emit at different colors all across the spectrum. One wavelength, such as a 405 nm line on the confocal, can excite multiple emitting dyes that have easily separable emissions such as blue (Brilliant Violet 421), red (Brilliant Violet 570) and near infra-red (Brilliant Violet 711).

Used in combination with the shorter Stokes shifts probes, specific labelling of more than 4 molecules can be imaged in sequence without complex spectral deconvolution.

Other useful tools:

If you have experience with these, please let us know!


You may want to try recombinant single-domain antibodies (sdAbs) derived from llamas or alpacas.
An explanation of the method is published here: A toolbox of anti–mouse and anti–rabbit IgG secondary nanobodies

Multiple rounds of staining is a tedious way to do multiple probes, but here is one method:





We occasionally get questions about Alexa 700 and similar near-IR dyes. We do not have the filter blocks for any of the widefield epifluorescent scopes. The confocals (depending which one) only detect below 700 to 740 nm. And even if they did, users would have to change focus due to chromatic aberration. Also, we do not have the correct excitation lasers.

The following table shows a few standard filters sets:
NAME OF FILTER Excitation Dichroic Emission approximate color
DHE (Dehydroergosterol) D335/20 365dclp D405/40  
Dapi 360   400 LP  
CFP 436 455 480  
FM143 480/30 570 LP 505dcxt  
FITC 460-590 (depends on exact set) 500 510+ BP or LP  
YFP 500 515 535+ BP or LP  
Rhodamine 550-580 (depends on exact block)      
Cy5 620/60 660LP 700/75  
Custom filters can be purchased, there may be more filters available since this web page was last updated, and a few of the confocals have customizable spectral detection.


This is how the author of this web page perceives the colors.

probes_excitation.jpg (22056 bytes)





< Back