This dissertation deals with new approaches in cardiac optical mapping. The principle of cardiac optical mapping as well as the current research in this field was described. The new measurement system was developed and its characteristics presented. The system design allows epicardial and endocardial mapping, employing new ratiometric techniques in 2D acquisition and simultaneous electrical and optical mapping. The measured characteristics of fluorescent dye di-4-ANEPPSS were presented. The relation between movement and consequent motion artifacts is well described. Several new approaches in signal processing were proposed, including new ratiometry technique and using image registration to suppress motion artifacts. The algorithm for elastic image registration of optical signals and innovative method for verification of registration process were presented. Application of all proposed approaches and its results are included and discussed.