Calcium imaging analysis – how far have we come? [version 2; peer review: 1 approved, 2 approved with reservations]

Techniques for calcium imaging were first demonstrated in the mid-1970s, whilst tools to analyse these markers of cellular activity are still being developed and improved today. For image analysis, custom tools were developed within labs and until relatively recently, software packages were not widely available between researchers. We will discuss some of the most popular methods for calcium imaging analysis that are now widely available and describe why these protocols are so effective. We will also describe some of the newest innovations in the field that are likely to benefit researchers, particularly as calcium imaging is often an inherently low signal-to-noise method. Although calcium imaging analysis has seen recent advances, particularly following the rise of machine learning, we will end by highlighting the outstanding requirements and questions that hinder further progress and pose the question of how far we have come in the past sixty years and what can be expected for future development in the field.