I use the Action-Potential (AP) dye molecule Di-4-ANEPPS for atria optical mapping.
I try to understand the mechanism of action of this molecule.
So I understood that Di-4 is fixed in the plasma membrane and don't move. Only the electron cloud will change during Action Potential. The modification of the electron cloud induce a shift of the excitation and emission spectrum : it is the stark effect.
The goal is to observe a modification of fluorescence according to the Action Potential so the best is to have an emission filter in one of the 2 slopes of the emission spectrum (between 600 and 670 nm or 700 and 800 nm). In this case a very small modification of the AP lead a high modification of the emission.
In a lot of papers the excitation wavelength is around 530nm (green light). My question is why use the excitation wavelength in the slope of excitation spectrum and not at the maximum for Di-4 (480nm)?
We can excite at 480nm in order to have the max of molecules excited and use an emission filter in the emission slope in order to detect well the changes in AP.
Or maybe I understand nothing and it's another mechanism.
Dear Alexandre,
at first please give us (The Answerers) more infos about your atria optical mapping (p.e. left or right or left and right/ Animals or humans.....).
The above cited dye (Di-4-ANEPPS) is not fixed but interact with the side of the membrane and environment.
This dye has an inner salt (Pyridin part) and the naphtalenyl part has a basic dibutylamino group. It has no reactive group required for a fixation. Fixed is a bad word Bound is better.
How do you introduce the dye in the atria and in was for a solvent.
Zwitterionic dyes exhibit good potentiometric response to different cells and tissues but the spectral properties (e.g. Em ) of the bound dye are dependant on membrane environment.
You can try the Ex with 465 nm and the Em will be + /- 635 nm . Molecular Probes
tested this dye in the past with a phospholipid membranes model and found 465 nm/ 635 nm.
Concerning your question and the variation of A.P. you can see the sensitivity of the dye with the environment.
Atria mapping involves a lot of physicochemical and physiological processes. please investigate the transmembrane potential towards the K+, Na+ and Cl- concentration gradients for active transport processes.
Good luck
JRG
Dear Alexandre,
the idea is that, while the membrane depolarizes, both the excitation and emission spectra move toward shorter wavelenghts, so with both filters on the slopes of the spectras you have the maximum variations in fluorescence during the action potential.
With 480 nm excitation you would get a lot more raw fluorescence signal, but very small variations during APs.
I hope i have been useful
Paolo
Dear Alexandre,
Another perspective on the choice of wavelength of excitation or emission is related to the depth penetration of light. Ansorption and photon scattering is greater for short wavelengths meaning that light can not penetrate as deeply in the preparation if using 485 nm rather than 530 nm. This relationship also extends in to the near-infrared. See Walton RD et al 2010 Heart Rhythm 7(12):1843.
A second consideration is a compatibility issue with blebbuststin (the preferred mechanical inhibitor with the least known side effects). Blebbistatin is infarct light sensitive and breaks down rapidly with exposure to blue or UV light. Therefore green (530 nm) is more compatible.
I hope that this was useful. Best of luck.
Richard Walton
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