title graphic AIF graphic
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 (l is the emission color):

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, DsRed2
Near infra red, approximately 670nm Cy5, Alexa 633, Alexa 647 DRAQ5, APC


The following table shows the filters we have for Olympus microscopes:
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 and there may be more filters available since this web page was last updated.

For questions about GFP, mCherry, etc.
Please visit the Fluorescent Protein Resource Center.

N.b.  FITC, GFP and other green labels cannot be imaged with DsRed!

Probes for BioRad Radiance 2000 Confocal Microscopy:
For the BioRad confocal microscope, you may not use any of the stains from group blue which require excitation at 360 nm.  You may use any of the dyes from the green, red, or near infra red categories.   The confocal microscope has lasers that excite dyes at 488 nm, 568 nm and 637 nm only.  [Example triple label.


Probes for Leica AOBS Confocal Microscopy:
The Leica AOBS uses variable spectral detection instead of traditional emission filters.  There are a few laser lines for excitation and the dyes are limited by these.  Any dye that is excited at one of these following wavelengths can be used.
405; 458; 476; 488; 514; 543; and 633 nm.
N.b. that there is no line at 568 which is the best line for reds similar to rhodamine.


An often asked question is, What is good for labeling nucleic acids other than DAPI?
This is an important question because Dapi & Hoechst cannot be used with the confocal or with CFP.
The first answer we provide is propidium iodide in the red range because it is so easy to use.
But there is a new family of nucleic acid stains by Molecular Probes called Syto.   Nobody has used them in the AIF so we can't recommend them, but we'd love somebody to try and show us the results.  The most promising Syto dyes appear to be Syto 13 or Syto 16 in the green range or Syto 60 in the infra red range


Suggestions for work with live cells:
When FITC is excited, it produces free radicals which can be very harmful to live cells.   Also, FITC is pH sensitive such that its intensity changes depending on the pH.   And FITC bleaches very quickly.  For these reasons, we recommend more stable and brighter Alexa 488 or Cy2 instead of FITC.  Or, if you can, switch to a red probe such as Alexa 568, Cy3, Rhodamine, or Texas Red.

On the digital stations, any custom filters can be built for any wavelenths between 360 nm and 700+ nm.

For instance, we have special filters on Digital Station #1 for double labeling and time lapse imaging of cyanFP and yellowFP. Digital Station #3 has a few special filters, for instance for chromosome painting.  And the Leica AOBS confocal can image any part of the visible spectrum without using filters.

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

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