Quantifying spatial localization of optical mapping using Monte Carlo simulations.

Optical mapping techniques used to study spatial distributions of cardiac activity can be divided into two categories. 1) Broad-field excitation method, in which hearts stained with voltage or calcium sensitive dyes are illuminated with broad-field excitation light and fluorescence is collected by image or photodiode arrays. 2) Laser scanning method, in which illumination uses a scanning laser and fluorescence is collected with a photomultiplier tube. The spatial localization of the fluorescence signal for these two methods is unknown and may depend upon light absorption and scattering at both excitation and emission wavelengths. We measured the absorption coefficients (micro a), scattering coefficients (micro s), and scattering anisotropy coefficients (g) at representative excitation and emission wavelengths in rabbit heart tissue stained with di-4-ANEPPS or co-stained with both Rh237 and Oregon Green 488 BAPTA 1. Monte Carlo models were then used to simulate absorption and scattering of excitation light and fluorescence emission light for both broad-field and laser methods in three-dimensional tissue. Contributions of local emissions throughout the tissue to fluorescence collected from the tissue surface were determined for both methods. Our results show that spatial localization depends on the light absorption and scattering in tissue and on the optical mapping method that is used. A tissue region larger than the laser beam or collecting area of the array element contributes to the optical recordings.