L's Approach: Illumination Pattern Estimation Technique for Optical Microscopy

SummaryWe propose a novel solution to the correction of illumination nonuniformity without removing the imaging sample. Calibration of the spatial illumination pattern in reflectance microscopy is challenging due to the fact that the illumination source is colocated with the objective lens and therefore cannot be observed directly. Our proposed methodology overcomes this by collecting three spatially translated images in a strategic way. We prove that ‘log‐illumination pattern’ recovery can be reformulated as a deconvolution of log‐ratio of captured images, and develop an efficient, noise‐robust implementation. Experiments with simulated and reflectance microscopy data verify the effectiveness of our proposed approach.Lay DescriptionThe proposed technique overcomes a common problem of nonuniform illumination in microscopy. For the case of reflectance microscopy, we show that a few images with large amounts of overlap can be used to recover the illumination pattern. In conjunction with an image formation model, the recovered illumination pattern may be used to correct the images captured under similar illumination conditions.