Fluorescence is a technology where a fluorophore is excited by light at a specific wavelength and light is emitted at a longer wavelength and can be detected. This has been applied for imaging in surgery because in the near infra-red spectrum, light can penetrate through overlying tissue and provide information to the surgeon about underlying structures. Two important applications of fluorescence include identification of critical anatomical structures to prevent injury and guide dissection, and identification of tumours to aid diagnostics. During colorectal surgery, it is essential for surgeons to be aware of the location of the structures in the urinary tract. The ureter is an essential landmark of the correct dissection plane as well as its injury being a devastating complication. To date, fluorescence identification of the ureter has only been described in technical papers. This thesis explores ureteric fluorescence further with a large cohort of patients using methylene blue and a first in man study assessing IRDye 800BK (Chapter 3). Urethral identification with NIR light is also explored from proof of principle to in vivo assessment using methylene blue, ICG and a NIR light emitting device (Chapter 4). During oesophagectomy, the thoracic duct can be injured complicating the post-operative course with chylothorax. Fluorescence of the duct for prevention and identification of intraoperative injuries is, for the first time, described (Chapter 5). Finally, WFA as a target for sugars in the mucus is explored to highlight cancer and dysplasia during colonoscopy and TEMS. Autofluorescence in the colon provides difficulties in fluorescent imaging and is explored as well as the struggles with utilising mucus as a target with WFA. However, an additional potential marker, GPA33, could provide a future for in vivo colonic imaging.