Characterization of a three-dimensional double-helix point-spread function for fluorescence microscopy in the presence of spherical aberration.

We characterize the three-dimensional (3-D) double-helix (DH) point-spread function (PSF) for depth-variant fluorescence microscopy imaging motivated by our interest to integrate the DH-PSF in computational optical sectioning microscopy (COSM) imaging. Physical parameters, such as refractive index and thickness variability of imaging layers encountered in 3-D microscopy give rise to depth-induced spherical aberration (SA) that change the shape of the PSF at different focusing depths and render computational approaches less practical. Theoretical and experimental studies performed to characterize the DH-PSF under varying imaging conditions are presented. Results show reasonable agreement between theoretical and experimental DH-PSFs suggesting that our model can predict the main features of the data. The depth-variability of the DH-PSF due to SA, quantified using a normalized mean square error, shows that the DH-PSF is more robust to SA than the conventional PSF. This result is also supported by the frequency analysis of the DH-PSF shown. Our studies suggest that further investigation of the DH-PSF's use in COSM is warranted, and that particle localization accuracy using the DH-PSF calibration curve in the presence of SA can be improved by accounting for the axial shift due to SA. (C) 2013 Society of Photo-Optical Instrumentation Engineers (SPIE)