Abstract
Indocyanine green (ICG) fluorescent dye has been approved by the FDA for use in medical diagnostics. Recently, we demonstrated that ICG dye has voltage-sensitive properties with a dual-component (fast and slow) response in the Langendorff-perfused rabbit heart. Here, we extended our studies by showing the different spectral properties of both components for analysis of the fractional change in ICG fluorescence in response to voltage changes. We used light from four LEDs to obtain excitation; emission was measured using an EMCCD camera with band-pass filters and a spectrometer. We applied a graphical model with Gaussian functions to construct and evaluate the individual emission curves and calculated the voltage-sensitive portion of each component of the ICG fluorescence in the rabbit heart. The results revealed that each isolated component (fast and slow) emanates from a unique ICG pool in a different environment within the cell membrane and that each component is also composed of two constituents (ICG-monomeric and ICG-aggregated). We propose the existence of different voltage-sensitive mechanisms for the components: (I) electrochromism and field-induced reorientation for the fast component; and (II) field-induced dye squeezing that amplifies intermolecular interactions, resulting in self-quenching of the dye fluorescence, for the slow component.
Highlights
More clarity would be obtained through further exploration of both the excitation and emission spectra of the dye, and this will be our future direction
We believe that this report represents an important step that may help provide a future basis for introducing the optical mapping of cardiac electrical activity in the clinic
One limitation of our study is that faithful and precise detection of the voltage-sensitive mechanisms of the dye could be obtained only when both excitation and emission spectra were recorded. Another limitation may be related to the calculation of OSs; in the analysis, signals were taken not from an area of 5 × 5 pixels, which would permit evaluation of the kinetics of the AP3, but from a much larger area (15 × 40 pixels) corresponding to about 10 ms of propagation time
Summary
The use of both an EMCCD camera with an appropriate set of emission filters and a spectrometer in the same experiment made it possible to determine the fluorescence spectrum of the ICG dye and evaluate the sensitivity of the dye’s spectral properties to voltage in a simple and straightforward manner. The ICG fluorescence obtained experimentally using a fast EMCCD camera was used to calculate the time course of a voltage-sensitive portion of that signal by subtracting the background fluorescence value from the whole optical signal (i.e., the OS at rest was set to zero). Our initial analysis (not shown) indicates that the two skew Gaussian spectra, one each for the fast and slow components, are not ideal for evaluating changes in the emitted fluorescence of the ICG dye over the entire fluorescence spectrum range, for voltage-sensitive OS at red tail. The significance level was set at p < 0.05
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