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Since the early 1990s, single-molecule detection in solution at room temperature has enabled direct observation of single biomolecules at work in real time and under physiological conditions, providing insights into complex biological systems that the traditional ensemble methods cannot offer. In particular, recent advances in single-molecule tracking techniques allow researchers to follow individual biomolecules in their native environments for a timescale of seconds to minutes, revealing not only the distinct pathways these biomolecules take for downstream signaling but also their roles in supporting life. In this review, we discuss various single-molecule tracking and imaging techniques developed to date, with an emphasis on advanced three-dimensional (3D) tracking systems that not only achieve ultrahigh spatiotemporal resolution but also provide sufficient working depths suitable for tracking single molecules in 3D tissue models. We then summarize the observables that can be extracted from the trajectory data. Methods to perform single-molecule clustering analysis and future directions are also discussed. 

Abstract Fluorescence lifetime imaging microscopy (FLIM) is a powerful tool to quantify molecular compositions and study molecular states in complex cellular environment as the lifetime readings are not biased by fluorophore concentration or excitation power. However, the current methods to generate FLIM images are either computationally intensive or unreliable when the number of photons acquired at each pixel is low. Here we introduce a new deep learning-based method termedflimGANE(fluorescencelifetimeimaging based onGenerativeAdversarialNetworkEstimation) that can rapidly generate accurate and high-quality FLIM images even in the photon-starved conditions. We demonstrated our model is up to 2,800 times faster than the gold standard time-domain maximum likelihood estimation (TD_MLE) and thatflimGANEprovides a more accurate analysis of low-photon-count histograms in barcode identification, cellular structure visualization, Förster resonance energy transfer characterization, and metabolic state analysis in live cells. With its advantages in speed and reliability,flimGANEis particularly useful in fundamental biological research and clinical applications, where high-speed analysis is critical.