Pupil phase masks for enhanced depth of field microscopy were investigated by using a spatial light modulator. The phase masks were evaluated with simulations in terms of the mean square error between in-focus and out-of-focus point spread functions. The resulting best-performing phase masks were tested for fluorosphere samples using a microscope add-on containing the SLM. First, z-stacks of fixed fluorospheres in an agarose medium were recorded in order to measure the extended depth of field. The same measurements were also performed on fluorospheres subjected to Brownian motion in an aqueous solution. The results show that with deconvolution and appropriate filtering it is possible to obtain sharp fluorosphere images with an extended depth of field of at least 10 µm.

© 2013 SPIE

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