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Lung cancer is the commonest cause of cancer-related deaths worldwide. Early detection has an impact on survival outcomes. Lung cancer screening with low-dose computed-tomography reduces lung cancer mortality, however, this approach also identifies considerable numbers of incidental solitary pulmonary nodules (SPNs). SPNs cause clinical and diagnostic uncertainty - whilst the majority are benign, some will represent early treatable lung cancer. Confirmatory histological diagnosis is recommended prior to commencing radical treatment of lung cancer, however, the diagnostic yield of existing sampling techniques is adversely affected by smaller lesion size. Fibre-based optical imaging techniques, which are deliverable via bronchoscopic, transthoracic and robotic platforms, enable high-resolution imaging of distal lung alveolar structure through elastin autofluorescence. Label-free optical techniques, such as fluorescence-lifetime imaging microscopy (FLIM) and Raman spectroscopy, which measure endogenous fluorophore decay rates and shifts in molecular vibrational states respectively, have the capability to optimise diagnostic accuracy and deliver in situ diagnostics in patients with suspected lung cancer. As a step towards future translation, a proof-of-concept study of fibre-based optical fingerprinting techniques was conducted in freshly resected ex vivo human non-small cell lung cancer (NSCLC) and SPNs. Lung cancer was characterised by disrupted alveolar elastin autofluorescence structure, significantly shorter fluorescence lifetime and elevated protein: phospholipids Raman peak intensity ratio (1124: 1728 cm-1), compared with non-cancerous lung tissue. Furthermore, benign SPNs displayed altered fluorescence images with a marked reduction in elastin fibre density, and significantly shorter fluorescence lifetime, compared with malignant SPNs. Our novel high-resolution fibre-based FLIM system discriminated lung cancer, from adjacent healthy tissue ex vivo, with high sensitivity and good specificity. Therefore, this minimally invasive technique, which permits access to distally-located lesions via existing diagnostic platforms, has potential as a real-time biopsy guidance tool, capable of aiding identification of optimal sampling sites and avoiding repeated procedures in patients with early-stage lung cancer. The mechanisms responsible for alterations in lung cancer fluorescence lifetime signature are not fully understood. Metabolic reprogramming is a hallmark feature of cancer cells, characterised by the preferential switch from oxidative phosphorylation to aerobic glycolysis (known as the Warburg effect). Human bronchial epithelial cell perturbation studies were undertaken to evaluate whether alterations in glucose metabolism account for changes in endogenous fluorophore lifetimes. In 16-human bronchial epithelial cells, NADH and FAD fluorescence lifetimes were shown to decrease with cobalt chloride treatment and increase with 3-bromopyruvate treatment, which enhanced and inhibited glycolytic ATP production respectively. Identical perturbation studies conducted on fresh ex vivo human lung tissue microsections demonstrated that metabolic pathway inhibitors selectively alter endogenous fluorophore lifetimes in lung cancer. Ex vivo human lung cancer microsections displayed a significant increase in fluorescence lifetime following treatment with cobalt chloride, which stabilises hypoxia-inducible factor-1α and inhibits pyruvate dehydrogenase (PDH) complex. Enhanced PDH-dependent tricarboxylic acid flux in NSCLC may play a role in the reduction in fluorescence lifetime associated with lung cancer, due to an increase in the relative contribution of the short lifetime component related to α-lipoamide dehydrogenase enzyme within PDH complex, which is a major contributor to flavoprotein autofluorescence. The application of benchtop FLIM in fixed unstained human lung tissue sections enables evaluation of fluorescence lifetime characteristics at the cellular level. Formaldehyde-fixation was associated with a reduction in fluorescence lifetime of human lung tissue. The use of multiple excitation wavelengths and long exposure times to evaluate fixed unstained lung tissue enabled lifetime visualisation and calculation of red blood cells (RBC), which were inherently poorly fluorescent and did not contribute to fluorescence lifetime signatures in fresh ex vivo lung tissue. Fixed unstained non-cancerous lung sections have significantly higher amounts of RBC, compared with lung cancer, which accounted for the disproportionate reduction in non-cancer fluorescence lifetimes following tissue fixation. High-resolution FLIM imaging of fixed unstained benign lung pathologies demonstrated RBC and macrophages, which display very short fluorescence lifetime values, likely contribute to fluorescence lifetime signatures in non-malignant human lung tissue. Elastin fibres, which are a major contributor to autofluorescence signal in human lung tissue, demonstrated no significant difference in fluorescence lifetime with respect to underlying lung pathology. With regards fixed unstained NSCLC tissue sections, cancer cell subtype influenced fluorescence lifetime signatures, with adenocarcinoma cells displaying significantly lower fluorescence lifetimes compared with squamous cell carcinoma cells, and the proportion of cancer cells within fixed unstained histological sections may also contribute to the overall fluorescence lifetime signature of human lung cancer.