Intracellular calcium has ubiquitous functional presence in every cell type. With each new biological process being unfolded through scientific research, it has become critical to understand the roles and spatiotemporal distribution of calcium ions in various cellular compartments. Additionally, intracellular calcium levels have been observed to be differentially modulated in disease conditions like , when compared to normal cells. Such dysregulated or diseased cellular states commonly exhibit hijacking of various proteins involved in transport of calcium ions between neighboring cells and within organelles of an individual cell type. In earlier studies, the use of calcium-binding fluorescent sensors paired with microscopy methods remarkably unmasked the irregularities in calcium signaling and distribution during abnormal cellular states. As the understanding of the regulatory machinery for intracellular calcium grew, so did the need to acquire better ways to capture such processes in real time. Qualitative and quantitative fluorescent indicators for calcium are utilized due to their relative ease of use and broad range of calcium affinities showcased for recording changes in the pattern of cellular calcium distribution. However, these sensors display numerous limitations, the most serious being the lack of ability to measure calcium at subcellular levels. Recent developments in this area, including genetically encoded fluorescent calcium sensors, systems designed for high throughput measurements, optimized imaging techniques for visualizing calcium dynamics of 3D cell cultures, or paired measurement of fluctuation in levels of calcium and associated proteins like IP3, have provided a new and improved set of tools to study subcellular calcium signaling.