Summary An algorithm to produce a uniformly focused image in digital acquisition of high magnification light microscopy images is presented. In very high magnification microscopic imaging the specimen surface cannot be considered ideally flat so that capturing a single image frame is usually not sufficient to capture an image that is focused everywhere. An image formation model for light microscopic images is presented, and based on this model an algorithm to construct a uniformly focused image is presented. The algorithm requires that multiple frames of the image at different focal planes be processed to combine their information to obtain an estimated of the desired image which is more completely focused than any of the individual frames. Experimental results show that the proposed algorithm is very effective in approximating the desired image in high magnification microscopic imaging and highly robust comparing to the gradient method.
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Wu et al. (1996) studied this question.