32 4 Confocal imaging of intracellular calcium cycling in the intact heart Neha Singh, Manvinder Kumar, James E. Kelly, Gary L. Aistrup, and J. Andrew Wasserstrom Northwestern University Feinberg School of Medicine, Chicago, IL, USA Introduction Laser scanning confocal imaging has allowed for the investigation of cellular cardiac function with both high temporal and spatial resolution for nearly 20 years. Nearly all of the studies of cellular cardiac function to date have been performed in isolated cardiac myocytes, giving enormous insights into cardiac cellular electrical properties and intracellular Ca 2+ cycling. Recently, however, a number of studies have used this technique to measure Ca 2+ cycling in myocytes of intact heart in situ [1–3], often giving dramatically different results from those obtained from isolated myocytes, even from the same species. The focus of this chapter is to describe how confocal microscopy can be applied to measure intracellular Ca 2+ cycling in intact hearts, particularly from rodents, although we have also used this approach successfully in atrial and ventricular preparations from larger animals as well. The advantages of this approach are the same as those in isolated myocytes: very high scan rates allow high-resolution temporal measurements of rapid Ca 2+ cycling and selective line placement allows for high-resolution spatial measurements within cardiac myocytes but now in the intact heart. Furthermore, recordings made in intact hearts allow for measurements of Ca 2+ cycling in many cells simultaneously so that the native het- erogeneities in cellular function can be explored, which is especially important during rapid pacing and cardiac arrhythmias. Even higher spatial resolu- tion is now becoming available with the use of rapid two-dimensional (2-D) imaging (e.g., the Zeiss 5/7Live confocal microscope), which permits imaging of microscopic Ca 2+ events at micron resolution on a millisecond timescale. The applications of this form of Ca 2+ imaging are only now being explored. So far they range from high-resolution Ca 2+ imaging during physiological and pharmacological manipu- lations (altered heart rates, arrhythmia induction, ischemia and acidosis, drug effects on Ca 2+ cycling, among others) to the determination of the extent of integration of exogenous cells transplanted in normal and injured hearts in vivo. No other currently avail- able approach can provide deinitive answers to the question of whether or not transplanted cells actually integrate into surrounding myocardium. In addition, there is some evidence suggesting that imaging of transmembrane voltage, perhaps even simultaneously with Ca 2+ imaging, may be accomplished in intact hearts using confocal microscopy. However, this approach is dificult and falls outside the scope of the current discussion, which is focused on the use of confocal microscopy for high-resolution Ca 2+ imaging in the whole heart. The future development of combined voltage–Ca 2+ imaging could provide an enormous advance in the imaging of cellular function in the intact heart. Manual of Research Techniques in Cardiovascular Medicine, First Edition. Edited by Hossein Ardehali, Roberto Bolli, and Douglas W. Losordo. © 2014 John Wiley & Sons, Ltd. Published 2014 by John Wiley & Sons, Ltd.