Cardiologists and computer scientists from universities in the US and UK have colluded to prepare a comprehensive document detailing procedures for the preparation, handling, and successful optical mapping of cardiac tissue slices. The goal of their study was to refine experimental conditions for efficient steps toward performing optical mapping and semi-automated analysis of the data obtained from cardiac tissue slices, which are a unique 2D model for studies in electrophysiology, allowing for observation of various key functional parameters including transmembrane potential (V_m), calcium transients (CaT), and their relationship with cellular substrates. These optical mapping procedures could be better implemented into cardiac research with the potential to uncover new paths in pharmacological research and development. The article chooses to report how to prepare for cardiac optical mapping using two models, rabbit and guinea pig, to represent similar heart tissues others may use and have the opportunity to demonstrate various materials and methods others may modify and apply to their own projects. Following procedures detailing isolation of the hearts, the article specifies the steps and solutions used in loading the hearts with voltage and calcium indicators: rabbit with di-4-ANBDQPQ and Rhod-2 AM, and guinea pig with di-4-ANBDQBS and Cal-520^® AM. After sectioning the hearts into slices, the voltage and calcium indicators permit convenient pseudo-simultaneous monitoring of V_m, action potential duration (APD), and CaT during the fluorescence imaging protocols of the article. The acquired data could later be analyzed for potential relationships among the parameter leading to further insights on electro-mechanical coupling and mechano-electric feedback in cardiac tissue.

Within the protocols, the researchers specifically demonstrated the uses of two calcium indicators, the red Rhod-2 and green Cal-520^®, in their two heart models. The researchers described in their discussion that both dyes exhibited relatively high calcium affinities, which assists with quick loading and calcium binding but needs to be considered when reading calcium amplitude data. Rhod-2 AM is one of the most commonly used red calcium dyes amid fluorescent research tools, known for its easy applicability to many experiments, with its max Ex/Em at 549/578nm. If increased fluorescence is needed, Rhod-4 is also available as a brighter and more sensitive option. In the introduction of Cal-520^®, the article describes the dye as having "a spectrum very similar to Fluo-4, with an improved signal-to-noise ratio". Additionally, in the dye loading protocol of this study, while all other dyes used in the experiment were specified to be paired with Pluronic-127 or probenecid "to prevent dye-leakage from the cytoplasmic space into the extracellular medium", Cal-520^® didn't need to be loaded with such extra reagents. This is consistent with Cal-520^®'s enhanced intracellular retention properties, which lead to brighter signals and conveniently less reagent used upon loading target cells.