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Simple Summary  In spite of significant improvements in diagnosis and treatment options, cancer treatments still suffer from late detection and patient-specific multidrug resistance causing limited efficacy of systemic drug therapies. This review delineates the pioneering works on current advances in Raman spectroscopy and fluorescence lifetime imaging microscopy and their non-invasive application in in vitro and in vivo cancer identification, early-stage monitoring, drug screening, and metabolic analysis. In addition, the remaining challenges and limitations of the methods will be discussed and important aspects of their application in the clinic, enabling them to be a real asset to clinicians in the future and complementing current gold standard methods, will be explained.    Abstract  Hurdles for effective tumor therapy are delayed detection and limited effectiveness of systemic drug therapies by patient-specific multidrug resistance. Non-invasive bioimaging tools such as fluorescence lifetime imaging microscopy (FLIM) and Raman-microspectroscopy have evolved over the last decade, providing the potential to be translated into clinics for early-stage disease detection, in vitro drug screening, and drug efficacy studies in personalized medicine. Accessing tissue- and cell-specific spectral signatures, Raman microspectroscopy has emerged as a diagnostic tool to identify precancerous lesions, cancer stages, or cell malignancy. In vivo Raman measurements have been enabled by recent technological advances in Raman endoscopy and signal-enhancing setups such as coherent anti-stokes Raman spectroscopy or surface-enhanced Raman spectroscopy. FLIM enables in situ investigations of metabolic processes such as glycolysis, oxidative stress, or mitochondrial activity by using the autofluorescence of co-enzymes NADH and FAD, which are associated with intrinsic proteins as a direct measure of tumor metabolism, cell death stages and drug efficacy. The combination of non-invasive and molecular-sensitive in situ techniques and advanced 3D tumor models such as patient-derived organoids or microtumors allows the recapitulation of tumor physiology and metabolism in vitro and facilitates the screening for patient-individualized drug treatment options.

Raman Imaging and Fluorescence Lifetime Imaging Microscopy for Diagnosis of Cancer State and Metabolic Monitoring