Methods and systems for facilitating assessment of blood flow in a tissue volume of a subject are disclosed. In some variations, the method may include: after a predetermined amount of a fluorescence agent has been administered to the subject, exciting the fluorescence agent in the tissue volume such that the excited fluorescence agent emits fluorescent light, acquiring fluorescence data based on the fluorescent light emitted during blood flow through the tissue volume, estimating a molar concentration of the fluorescence agent in the blood flowing through the tissue volume, and generating an assessment of blood flow in the tissue volume based at least in part on the fluorescence data and the estimated molar concentration of the fluorescence agent. The estimated molar concentration may be based on the predetermined amount of the fluorescence agent and an estimated circulating blood volume of the subject.