Atrial fibrillation (AF) is one of the most common arrhythmias, but its mechanisms remain unclear because of the complex human atrial structure and pathology of this disease.1,2 High-resolution optical mapping3 and 3-dimensional (3D) structural imaging of the atria in ex vivo animal models4 have provided a wealth of information for better understanding of AF.1 However, these high-resolution techniques cannot currently be performed in patients to directly uncover the important role of atrial anatomic substrates in pathophysiological conduction.3 For the first time in the intact human heart, this study integrates functional data collected by high-resolution near infrared optical mapping with the 3D atrial structure of the same heart obtained by novel micro-computed tomographic (CT) imaging to investigate the structural basis for conduction during sinus rhythm, atrial pacing, and sustained atrial flutter (AFL) and AF. An intact explanted human heart (unused donor, 63-year-old woman, chronic hypertension, heart weight 608 g) from the Lifeline of Ohio Organ Procurement Organization was obtained in the operating room at the time of cross-clamp and immediately preserved with cold cardioplegic solution (1–3°C) in accordance with The Ohio State University Institutional Review Board. Whole intact atria were dissected from ventricles and coronary-perfused with oxygenated Tyrode solution at constant pressure (55–60 mm Hg) and temperature (37°C). Subepicardial optical mapping of the whole coronary-perfused atria was conducted with near-infrared voltage-sensitive dye di-4-ANBDQBS3 to detect and map atrial activations during sinus rhythm, posterior left atrium pacing, and pacing-induced sustained AFL and AF (Figures 1–3). A high spatial (100×100 pixels, 1.16×1.16 mm2) and temporal (1 frame/ms) resolution Ultima-L CMOS camera (SciMedia, Japan) was focused on both atria from the epicardial surface (Figure 1A). After functional mapping, the human atria was formalin-fixed for 48 hours, and then washed with PBS and incubated at 4°C …