Enhanced three-dimensional deconvolution microscopy using a measured depth-varying point-spread function

We present a technique to systematically measure the change in the blurring function of an optical microscope with distance between the source and the coverglass (the depth) and demonstrate its utility in three-dimensional (3D) deconvolution. By controlling the axial positions of the microscope stage and an optically trapped bead independently, we can record the 3D blurring function at different depths. We find that the peak intensity collected from a single bead decreases with depth and that the width of the axial, but not the lateral, profile increases with depth. We present simple convolution and deconvolution algorithms that use the full depth-varying point-spread functions and use these to demonstrate a reduction of elongation artifacts in a reconstructed image of a 2 μm sphere.