Monte Carlo simulation of 3D mapping of cardiac electrical activity with spinning slit confocal optics

Optical techniques used to map transmembrane potential and intracellular Ca2+ activities of intact hearts are restricted to the surface and cannot resolve activity in deeper layers due to the lack of depth resolution. The recent development of spinning slit confocal optics offers advantages of depth resolution as well as high-speed confocal imaging which are necessary for millisecond-scale, depth-resolved mapping of membrane potential and/or intracellular Ca2+ concentration. Here, we show simulated confocal optics derived from confocal slits on a high-speed spinning disk using Monte Carlo method with a numerical heart tissue model and find that depth-resolved optical mapping is feasible down to around 800 microm below the surface using 670-nm excitation light. The numerical model shows that (1) a minimum slit separation, which is found to be a function of depth of the focal plane and the numerical aperture of the objective lens, for minimum background noise exists and (2) narrower slit widths result in slightly greater depth resolution but has a negative impact in significantly lower overall fluorescence intensity. An experimental test of this optics has been performed by imaging two overlapping layers of fluorescent beads and the result confirms the expected depth resolution in non-scattering medium. These results will be able to serve as a benchmark on how a 3D-imaging system can be expected to perform and what kind of theoretical depth-resolution can be expected from it.