Sino-atrial (SA) and AV nodes are integral parts of an anatomically extended atrial pacemaker and conduction system (APCS) that encompasses the entire region between superior and inferior venae cavas and extends into the triangle of Koch. Heart rate and atrio-ventricular (AV) delay are orchestrated by autonomic nervous system within the APCS, which cannot be fully understood without combining both reductionist and integrative approaches that focus on investigation of ionic and structural mechanisms, respectively. We have developed a methodology of multimodal biophotonic imaging that allows structure/function investigation of the rabbit, canine and human APCS. We applied high-resolution fast fluorescent imaging with voltage sensitive dye di-4-ANEPPS to optically map (OM) action potentials in APCS in the human, canine, and rabbit perfused preparations during normal heart rate, electrical stimulation, and during adrenergic and cholinergic stimulation. Optical coherence tomography (OCT) was applied to map 3D intact fiber structure of the APCS with spatial resolution of 10um and depth of penetration of 1-2 mm. Immunohistochemistry was used for mapping protein expression in functionally and structurally characterized preparations. Optical signals integrate electrical activity from 1-2mm of depth into tissue and carry 3D information about the genesis and conduction of the action potential within APCS. In both SA and AV nodal regions we have identified optical signatures from different layers of APCS structure and reconstructed the 3D pattern of excitation in multiple layers. In the canine SA node we have confirmed our earlier finding in the rabbit that a functional block zone (FBZ) exists that electrically uncouples SA node from the septum and thus facilitates source-sink matching. Adrenergic and cholinergic stimulation results in significant anatomic migration of the leading pacemaker on a beat-to-beat basis. Area of migration is variable from preparation to preparation and includes the entire intercaval region and the triangle of Koch. However, the isolated triangle of Koch shows remarkable lack of migration of the leading pacemaker that is located in the inferior AV nodal extension that also serves as the slow pathway input into the AV node. Thus, in contrast to the SA nodal region, the AV nodal pacemaker responds to both adrenergic and cholinergic stimulation without anatomic migration. Anatomic 3D reconstruction of this region in the human offers a venous pathway for novel therapeutic approaches. Cholinergic stimulation of the FBZ that is located between the SA node and the septum provides a functional substrate for reentry that drives atrial flutter or atrial fibrillation in the canine and rabbit. Perfusion with acetylcholine (1-10uM) allowed induction of reentry around the SA node and FBZ in 15 rabbit and 5 canine preparations. Multimodal biophotonic imaging of the rabbit and human AV junction revealed that AV conduction may bypass the compact AV node under certain physiological conditions. The lower nodal bundle expressing a high density of Cx43 exists below the compact AV node. Our data suggest that slow and fast pathway excitation leads to different patterns of engagement of the compact AV node and the bundle of His. Fast pathway excitation leads into the compact AV node followed by superior His excitation. Slow pathway excitation leads into the lower nodal bundle and inferior His excitation. Differing AV delays and distinct His electrogram morphologies confirm different patterns of excitation. Slow pathway stimulation allows AV conduction without AV delay, which suggests a bypass of the compact AV node via the lower nodal bundle. APCS is an anatomically and functionally vast structure that includes tissues within the entire intercaval region of the right atrium and the triangle of Koch. Control of heart rate and AV delay is achieved via precise modulation of the APCS and cannot be understood within purely reductionist paradigm. Multimodal biophotonic imaging allows comprehensive investigation of control mechanisms of the heart rate and AV delay.