Diameter measurement from images of fluorescent cylinders embedded in tissue.

Absorption and scattering of light by tissue as well as limitations in the resolution of the optical system influence the appearance of tissue embedded objects in fluorescence images and may reduce the accuracy of measurements from these images. Although the principles of light scattering in tissue and optical resolution are well known, the interplay between the two in fluorescence imaging in an imaging cryomicrotome is not well understood. In this paper we present and investigate an image formation model in a reflection geometry, like an imaging cryomicrotome, that takes both light scattering by tissue as well as the point spread function of the lens into account. The validity of the model was investigated by comparison of diameter estimates of fluorescent cylinders as obtained from images acquired in a reflection geometry with those estimated from simulated images. The results reveal that diameter values estimated from the simulated images are in excellent agreement with the experimental estimates. Our approach in modeling the image formation process of embedded fluorescent structures allows for the prediction of accuracy of quantitative estimates from fluorescence images. The relationship between imaging parameters and bias can be applied to arrive at accurate diameter estimates of near cylindrical structures like blood vessels.