. 2016 Mar 25;310(11):H1388–H1401. doi: 10.1152/ajpheart.00665.2015

Table 3.

Examples of important aspects to consider when imaging Ca_i²⁺

	Isolated Cardiomyocytes and Monolayers	Myocardium Cytosolic Ca²⁺ Imaging	Myocardium SR Ca²⁺ Imaging
Probe compartmentalization	Compartmentalization is condition-dependent. Mitochondria retain rhod 2 (14, 99) and indo 1 (89). Fluo 3 localizes to lysosomes (99). Fluo 5N (84) and mag-fura 2 (33) are retained in the SR after cytosolic washout.	Rhod 2 compartmentalization depends upon experimental conditions. Although rhod 2 may localize to the mitochondria (14), it is often used for myocardium cytosolic Ca²⁺ imaging (49, 70, 72) and is compatible with other modes of fluorescence imaging (58).	Mag-fluo 4 (45, 104) and fluo 5N (111) are low-affinity dyes that have been used to image SR Ca²⁺. For SR localization, cytosolic washout at 37°C follows long-term loading at room temperature (45, 111).
Probe dissociation constants	Ca²⁺ transients from isolated cells and monolayers are typically imaged with high-affinity probes such as fluo 4 (95), which may prolong CaD (26, 27).	Fluo 4 is a higher affinity probe than rhod 2, and causes artifacts in the Ca²⁺ transient, as shown for swine LV at long cycle lengths (43).	Very-low-affinity Ca²⁺ dyes (K_d >22 μM) are typically used to image SR Ca²⁺.
Measurements derived from Ca²⁺ transients	Transient amplitudes are typically measured (76). Ratiometry provides Ca_i²⁺ concentration and kinetic measurements (22, 90).	Ca²⁺ kinetics usually measured (39, 52, 70) but not amplitudes. Ratiometry of fura 2 (106, 121), indo 1 (15, 52, 83), or fura red (119) has been used to measure amplitudes.	Nonratiometric imaging provides kinetic measurements and short-term relative assessments of SR Ca²⁺ release amplitudes (45, 104, 111).
Imaging approach	Confocal imaging at rates usually >16 frames/s for monolayers (1, 4). Photomultipliers can provide 1,000 samples/s for isolated cell measurements (92). Photodiode array imaging >500 samples/s (118) and CCD imaging >100 frames/s (1, 11).	Photodiode array systems (20, 52) and CCD/CMOS camera systems (49, 58, 85) are used to image myocardial Ca²⁺ fluorescence. Frame rates are typically >250 frames/s, but most conventional mapping systems use cameras that image >1,000 frames/s (49).	SR Ca²⁺ (fluo 5N fluorescence) has been imaged from the epicardium of perfused rabbit hearts at frame rates between 500 and 1,000 frames/s using CMOS cameras (111).

CaD, intracellular calcium transient duration; LV, left ventricular.