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Transbronchial needle aspiration (TBNA), often used to sample lymph nodes for lung cancer staging, is subject to sampling error even when performed with endobronchial ultrasound. Optical coherence tomography (OCT) is a high-resolution imaging modality that rapidly generates helical cross-sectional images. We aim to determine if needle-based OCT can provide microstructural information in lymph nodes that may be used to guide TBNA, and improve sampling error.We performed ex vivo needle-based OCT on thoracic lymph nodes from patients with and without known lung cancer. OCT imaging features were compared against matched histology.OCT imaging was performed in 26 thoracic lymph nodes, including 6 lymph nodes containing metastatic carcinoma. OCT visualized lymphoid follicles, adipose tissue, pigment-laden histiocytes, and blood vessels. OCT features of metastatic carcinoma were distinct from benign lymph nodes, with microarchitectural features that reflected the morphology of the carcinoma subtype. OCT was also able to distinguish lymph node from adjacent airway wall.Our results demonstrate that OCT provides critical microstructural information that may be useful to guide TBNA lymph node sampling, as a complement to endobronchial ultrasound. In vivo studies are needed to further evaluate the clinical utility of OCT in thoracic lymph node assessment.