Biology is not static - it is dynamic. Most biological functions are in the nanosecond time domain. Investigation of any small sensitive changes can be detectable or monitored using fluorescence lifetime imaging microscopy (FLIM) techniques. Any intensity artefacts involved in intensity-based imaging can be avoided using FLIM. FLIM’s sensitivity in monitoring biological events in real time is based on its profound sensing of environmental changes. The intensity-based seminal work of Brittan Chance in the 1960’s exploited the auto-fluorescent properties of the coenzymes NADH (reduced form of non-fluorescent NAD+) and FAD (oxidized form of non-fluorescent FADH2) [1] to investigate disease process. Instead, we developed a FLIRR (fluorescence lifetime redox ratio) microscopy technique, which is based entirely on lifetime measurements, unaffected by intensity artefacts, using isolated individually segmented cell data which can separate data of intra-cellular OXPHOS from glycolysis [2-4]. FLIM assays are of particular interest in measuring responses to treatment in various cancer pathologies, cancer being a metabolically heterogeneous pathology, which generates energy by oxidative phosphorylation (OXPHOS) and (often preferentially) by glycolysis. Two-photon fluorescence lifetime imaging microscopy (FLIM) is widely used to capture auto-fluorescence signals from cellular components to investigate dynamic physiological changes in live cells and tissues. Furthermore, using whole-cell average data for redox levels does not capture subtle and changing differences between heterogeneous individual cells, between cytosolic glycolysis and mitochondrial OXPHOS and intracellular gradients The newly developed FLIRR approach has been shown to monitor drug response in live prostate cancer (PCa) cells. Traditional FLIM imaging for NAD(P)H, FAD applies a sequential multi-wavelength excitation (740 nm & 890 nm) to avoid spectral bleed-through (SBT). This sequential imaging complicates image acquisition, may introduce mitochondrial motion artifacts, and reduce temporal resolution. We have demonstrated a FLIM imaging protocol using a single wavelength excitation (800 nm) for prostate cancer (PCa) cells, allowing simultaneous image acquisition of NADH and FAD [5] to monitor the drug response. This single wavelength excitation simplifies FLIM imaging, data analysis using artificial intelligence (machine learning), decreasing the total imaging time, avoids motion artifacts and increases temporal resolution. Then, the question arises how this non-invasive, translational technique can advance early detection of PCa.