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Multiphoton fluorescence lifetime imaging microscopy (FLIM) is used to collect label-free metabolic information from biological samples via autofluorescence lifetime imaging of reduced nicotinamide adenine dinucleotide and nicotinamide adenine dinucleotide phosphate (NAD(P)H). However, FLIM has traditionally been limited by slow acquisition due to the limited bandwidth of analog electronics that perform photon counting and time-tagging. This slow acquisition has restricted the applicability of multiphoton FLIM of NAD(P)H by impeding the ability to accurately study biological problems that require characterization of fast dynamics. Faster image acquisition can be achieved by directly digitizing the amplified output of a hybrid photodetector and computationally determining photon counts via the Single- and multi-photon PEak Event Detection (SPEED) algorithm. This method, bypassing the limited-bandwidth analog electronics used for photon counting and time-tagging of photons in traditional FLIM, enables fast photon counting capabilities which are well suited for fast, high-dynamic range biological processes such as metabolic changes during apoptosis. Here, we utilize this technology to examine fast dynamics of apoptosis in 2D culture of normal and cancerous human breast cell lines, rat mammary tumor tissue-derived organoids, and in vivo rat mammary tumors. Results indicate that apoptosis-related metabolic dynamics are biological model-dependent and based on local pharmacokinetics, with tumor derived organoids in Matrigel showing a significantly slower response than in vivo or in vitro 2D cell models. Future work should carefully consider these implications when determining which tumor model to use for experimentation and should improve tumor models to better represent in vivo tumor apoptosis dynamics.
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