Emission crosstalk of Alexa Fluor 488 and 546
The excitation efficiency of the two fluorochromes is determined
by the point of intersection between the laser line used and the
excitation spectrum (dotted line).
Accordingly, Alexa Fluor 488 is excited to about 80 %,
Alexa Fluor 546 to about 60 %.
At a level of about 5%, the excitation spectrum of Alexa
Fluor 546 is also intersected by the 488 nm laser line (arrow).
Theoretically, this constitutes excitation crosstalk, as one line
excites both markers, but it is inefficient enough to be negligible.
Contrary to this, the emission spectra of the two dyes overlap
significantly.
The red area marks the emission crosstalk of Alexa Fluor 488
occurring if Alexa Fluor 546 is detected to the right of the
543 nm laser line.
Combined excitation and emission crosstalk
If GFP is used together with YFP, the emission spectra will
overlap considerably.
The red area marks the emission crosstalk between GFP and
YFP occurring if YFP is detected to the right of the 514 nm laser line.
In addition, there will be a pronounced excitation crosstalk.
The 488 nm line excites not only GFP but also YFP to an
efficiency of about 30 % (arrow).
Source: http://home.ncifcrf.gov/ccr/flowcore/welcome.htm;
modified
13
Relative fluorescence
488 nm
laser line
543 nm
laser line
488 nm
laser line
543 nm
laser line
Relative fluorescence
350
400
450
500
550
600
650
700
350
400
450
500
550
600
650
700
Wavelength in nm
Wavelength in nm
