When choosing the right fluorescent conjugate, you need to ask yourself a few
- What filters/lasers are on your microscope?
First you need to know what filters and/or lasers you have on your microscope.
They will determine what fluorescent proteins you will be able to excite and
- How many fluorophores do you plan to use on the one sample?
If you are using more than one fluorescent label in your sample, then you will
need to carefully choose your other fluorescent dye(s) so as to minimize
You can do this by looking at the emission and excitation spectra for your
fluorescent dyes, and choosing pairings that reduce how much overlap the
emission and excitation spectra have with others dyes.
If you are using a laser to excite your fluorophore, you only need to make sure
that your laser (ie. 488 nm) is not exciting more than 2 dyes at the same time.
If the laser is hitting the leading edge of your second fluorescent dye
excitation spectra, then you may end up with a slight excitation of that
However if the filter that you are using to capture the emission spectra of that
dye does not pick up any of the emission spectra of the second dye, then you
will not see any spectral overlap while capturing your images.