Extensive evidence implicates cation-permeable plasma membrane pores formed by oligomeric forms of β amyloid (Aβ) in cytotoxicity during Alzheimer’s disease (AD) [Ullah et al., PLoS One, 2015, 10(9); Demuro et al., J. Cell. Biol. 2011, 195(2):515-524]. We use total internal reflection fluorescence microscopy (TIRFM) to monitor the Ca2+ flux through these pores, revealing detailed information about their gating kinetics and time evolution. This massively parallel imaging technique provides simultaneous and independent recording from thousands of pores in a patch of membrane of living cells for extended time. Manual analysis of these data consisting of tens of thousands of image frames is very challenging. Thus, we developed a pipeline of computational tools to retrieve, analyze, and predict the behavior of these pores at extended timescales to shed light on their toxicity and better understand disease progression [Shah et al., Biophys. J. 2018, 115(1):9-21; Biophys. J. 2018, 114(3):291a]. Analyzing the imaging data includes detection of pores, generating their location maps, tracking their movement, retrieval of time series data for each pore, separating signal from noisy and drifting background, and extracting the key statistics about their gating kinetics and evolution. The information extracted from tens of thousands of pores is used to develop Markov chain models in order to understand and correlate their kinetics and long-term behavior with cytotoxicity. This talk will give an overview of the tools mentioned above, our current understanding of Aβ pores, and their implications for intracellular Ca2+ signaling.