Double helix PSF engineering for computational fluorescence microscopy imaging

Sreya Ghosh; Sean A. Quirin; Ginni Grover; Rafael Piestun; Chrysanthe Preza

doi:10.1117/12.909757

2 February 2012 Double helix PSF engineering for computational fluorescence microscopy imaging

Sreya Ghosh, Sean A. Quirin, Ginni Grover, Rafael Piestun, Chrysanthe Preza

Proceedings Volume 8227, Three-Dimensional and Multidimensional Microscopy: Image Acquisition and Processing XIX; 82270F (2012) https://doi.org/10.1117/12.909757
Event: SPIE BiOS, 2012, San Francisco, California, United States

Abstract

Point spread function engineering with a double helix (DH) phase mask has been recently used in a joint computationaloptical approach for the determination of depth and intensity information from fluorescence images. In this study, theoretically determined DH-PSFs computed from a model that incorporates different amounts of depth-induced spherical aberration (SA) due to refractive-index mismatch in the three-dimensional imaging layers, are evaluated through a comparison to empirically determined DH-PSFs measured from quantum dots. The theoretically-determined DH-PSFs show a trend that captures the main effects observed in the empirically-determined DH-PSFs. Calibration curves computed from these DH-PSFs show that SA slows down the rate of rotation observed in a DH-PSF which results in: 1) an extended range of rotation; and 2) asymmetric rotation ranges as the focus is moved in opposite directions. Thus, for accurate particle localization different calibration curves need to be known for different amounts of SA. Results also show that the DH-PSF is less sensitive to SA than the conventional PSF. Based on this result, it is expected that fewer depth-variant (DV) DH-PSFs will be required for 3D computational microscopy imaging in the presence of SA compared to the required number of conventional DV PSFs.

Citation Download Citation

Sreya Ghosh, Sean A. Quirin, Ginni Grover, Rafael Piestun, and Chrysanthe Preza "Double helix PSF engineering for computational fluorescence microscopy imaging", Proc. SPIE 8227, Three-Dimensional and Multidimensional Microscopy: Image Acquisition and Processing XIX, 82270F (2 February 2012); https://doi.org/10.1117/12.909757

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