A method for determining a critical angle of total reflection based upon images captured at different angles of incidence of a light beam includes illuminating a sample with an excitation light beam, capturing images of at least part of the sample at a plurality of different angles of incidence of the excitation light beam, and determining a critical angle of total reflection at an interface of the sample based upon analysis of the images. An apparatus for determining a critical angle of total reflection at an interface of a sample includes a light source arrangement to illuminate a sample with an angle of incidence, an image capturing arrangement to capture an image of the sample, and a processing arrangement to determine the critical angle of total reflection at an interface of the sample on the basis of an analysis of images captured at a plurality of different angles of incidence.