Accurate Image Registration for Simultaneous Optical Mapping of Membrane Potential and Calcium Intracellular

Abstract

The present research describes the measurement system that optically records with high spatial resolution simultaneously membrane potential (Vm) and calcium intracellular (Cai2+) signals, from the same pixel location, in the surface of the Langendorff-perfused rabbit heart, which is stained with voltage and calcium sensitive dyes. Optical System configuration consists of 2 high speed CMOS digital cameras with temporal resolution from 60 fps to 500 fps and spatial resolution of 512×512 pixel resolution and each will correspond to an element of about 17 µmm × 17 µmm2 pixel, as well as a customized LED ring light that was used as excitation light source. The fluorescence from the stained heart was collected through an optical configuration system that separates membrane potential and intracellular calcium signals using a specific Dichroic mirror and optical filters. In order to mapping accurately both signals from the same pixel area, a new method for registration of two images consists of point based registration and adjustment of image registration using mutual Information criteria was developed. The system was validated in vivo experiment and was able to record changes in membrane potential and intracellular calcium at the same location of a Langendorff-perfused rabbit heart.