B. Murienne - Master Project Thesis - Infoscience - EPFL

2.5 Optical mapping
2.5.1 Generalities
For our purpose, optical mapping includes the use of an ion-specific or voltage-sensitive
dye to track specific phenomena optically. This imaging technique is based on light-tissue
interactions, such as photon scattering, absorption, fluorescence, which are dependent on the light
wavelength and thus limit the spatiotemporal resolution of the images.
2.5.2 Staining
In cardiac studies, fluorescent probes are usually used because they yield higher fractional
changes in signal per each voltage variation than others. Longer wavelengths are generally
preferred in case of optical recordings from deep inside the myocardial wall, as light absorption
and scattering decreases with longer wavelengths. A classification of voltage-sensitive dyes into
two groups, the fast and slow dyes, based on their response time and molecular mechanism of
voltage sensitivity, was introduced by Cohen and Salzberg in 1978. Only the fast probes are used
in cardiac studies as they allow one to detect voltage changes on a time scale of microseconds.
One of the most important families of dyes is the styryl dye family. In case of action potentials
recordings, the styryl dyes di-4-ANEPPS, di-8-ANEPPS and RH-237, which can be excited using
visible light, are widely used [14].
Voltage-sensitive ANEP dyes have very interesting properties. They modify their
electronic structure and thus fluorescence spectra in response to changes in the surrounding
electric field. Their optical response is fast enough to detect transient potential changes in
excitable cells including cardiac cells and tissue preparations. They also show a potential-
dependent shift in excitation spectra allowing the quantization of membrane potential using
excitation ratio measurements. Ratiometric measurements are usually used to correct unequal dye
loading, bleaching and focal-plane shift, as the ratio of two fluorescent signals does not depend
on their absolute intensities. Their absorption and fluorescence spectra are highly dependent on
their environment and they are essentially non-fluorescent in water and become strongly
fluorescent when binding to membranes. Di-4-ANEPPS and di-8-ANEPPS are voltage-sensitive
dyes which are commonly used in cardiac studies. The di-4-ANEPPS dye has a uniform 10 % per
100 mV change in fluorescence intensity as well as the most consistent potentiometric response in
different cell and tissue type among the other ANEP dyes. The di-8-ANEPPS dye, which spectra
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