2622 J. Opt. Soc. Am. A/Vol. 24, No. 9/September 2007 J. W. Shaevitz and D. A. Fletcher Enhanced three-dimensional deconvolution microscopy using a measured depth-varying point-spread function Joshua W. Shaevitz1,3,* and Daniel A. Fletcher2,4 1Department of Integrative Biology, University of California, Berkeley, California 94720, USA 2Department of Bioengineering, University of California, Berkeley, California 94720, USA 3Department of Physics, Princeton University, Princeton, New Jersey 08544, USA 4E-mail: fl
[email protected] *Corresponding author:
[email protected] Received February 26, 2007; revised April 23, 2007; accepted May 1, 2007; posted May 3, 2007 (Doc. ID 80413); published July 27, 2007 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 re- constructed image of a 2 m sphere. © 2007 Optical Society of America OCIS codes: 100.3020, 100.6890, 110.0180, 140.7010, 170.6900, 180.2520. 1. INTRODUCTION mismatch between the objective lens and coupling oil can Three-dimensional (3D) deconvolution microscopy is a be severe [5,6], especially in systems using high- powerful tool for visualizing complex biological struc- numerical apertures.