Spectra of Commonly Used Fluorophores
One of the most important concerns for biologists imaging immuno-stained samples is bleed-through, or detection of signal from two fluorophores at the same time. In almost all cases this can be avoided by properly adjusting the laser intensity used to excite the fluorophore as well as the filters used in the configuration. Also imaging sequentially instead of simulaneously can minimize bleed through. The following links and graphs are provided for your convenience to aid in identifying the optimal laser for fluorophore excitation and to ensure there is no chance that two fluorophores can be excited and detected in the same track of a configuration. A short example of how to use spectra is also included.
Additional spectra, charts and references can be found online through several databases at:
405 laser line (UV/DAPI/Hoescht fluorescence)
488 laser line (GFP/YFP/FITC/Alexa488/green)
GFP/YFP/CFP
from http://dwb.unl.edu/Teacher/NSF/C08/C08Links/pps99.cryst.bbk.ac.uk/projects/gmocz/gfp.htm
543 laser line (Cy3, Alexa 568, red)
Cy3 dye
from http://www.jacksonimmuno.com/technical/f-cy3-5.asp
594 laser line (Texas red, orange dyes)
633 laser line (Alexa 633, far red)
Cy5 (see graph above under 543 laser line)
A short example how to use excitation/emission spectra:
If you are imaging samples stained with DAPI, Alexa488, Cy3 and Cy5, you can generate the following excitation (below left) and emission (below right) spectra. From the excitation graph, you can see that the 405 laser will excite DAPI and the very tail of the 488 spectrum (the two spectra overlap between 400-425). The 488 laser line will excite both Alexa488 and Cy3. The 543 laser will excite Cy3 and the tail of the Cy5 spectrum. The 633 laser will only excite Cy5. So how can we distinguish between fluorophores that can be excited by the same laser?
This is where properly adjusting the bandpass, short pass and long pass filters for detection in the configuration is important. If we look at the spectrum on the right, we see that DAPI emits light from ~400-650 nm. Alexa488 emits light from ~480-675. So, if we set the filters to allow only emission light between 405-480 to reach the detector, we can be sure when exciting with the 405 laser that we detect only the DAPI signal. What about the 488 laser line that excites both Alexa488 and Cy3? You can see that selecting a bandpass filter to limit the emission spectrum to 505-550 captures the majority of the emission from Alexa488, while cutting-off detection of the majority of the Cy3 spectrum. In this case, there is a small amount of overlap that can be minimized by using a low laser intensity or completely unmixed using digital software (the Meta system or digital unmixing) if entirely necessary. The 543 line only very weakly excites Cy5, and a band-pass filter from 550-630 cuts-off Cy5 detection almost entirely, leaving a pure Cy3 signal. The 633 laser line can only excite the Cy5 fluorophore in this case, thus bleed-through from the other fluorophores can not occur.
