Single camera system for multi-wavelength fruorescent imaging in the heart

Optical mapping has been a powerful method to measure the cardiac electrophysiological phenomenon such as membrane potential(V_m), intracellular calcium(Ca²⁺), and the other electrophysiological parameters. To measure two parameters simultaneously, the dual mapping system using two cameras is often used. However, the method to measure more than three parameters does not exist. To exploit the full potential of fluorescence imaging, an innovative method to measure multiple, more than three parameters is needed. In this study, we present a new optical mapping system which records multiple parameters using a single camera. Our system consists of one camera, custom-made optical lens units, and a custom-made filter wheel. The optical lens units is designed to focus the fluorescence light at filter position, and form an image on camera´s sensor. To obtain optical signals with high quality, efficiency of light collection was carefully discussed in designing the optical system. The developed optical system has object space numerical aperture(NA) 0.1, and image space NA 0.23. The filter wheel was rotated by a motor, which allows filter switching corresponding with needed fluorescence wavelength. The camera exposure and filter switching were synchronized by phase locked loop, which allow this system to record multiple fluorescent signals frame by frame alternately. To validate the performance of this system, we performed experiments to observe V_m and Ca²⁺ dynamics simultaneously (frame rate: 125fps) with Langendorff perfused rabbit heart. Firstly, we applied basic stimuli to the heart base (cycle length: 500ms), and observed planer wave. The waveforms of V_m and Ca²⁺ show the same upstroke synchronized with cycle length of pacing. In addition, we recorded V_m and Ca²⁺ signals during ventricular fibrillation induced by burst pacing. According to these experiments, we showed the efficacy and a- ailability of our method for cardiac electrophysiological research.