Reducing depth induced spherical aberration in 3D widefield fluorescence microscopy by wavefront coding using the SQUBIC phase mask

Nurmohammed Patwary; Ana Doblas; Sharon V. King; Chrysanthe Preza

doi:10.1117/12.2040191

12 March 2014 Reducing depth induced spherical aberration in 3D widefield fluorescence microscopy by wavefront coding using the SQUBIC phase mask

Nurmohammed Patwary, Ana Doblas, Sharon V. King, Chrysanthe Preza

Author Affiliations +

Proceedings Volume 8949, Three-Dimensional and Multidimensional Microscopy: Image Acquisition and Processing XXI; 894911 (2014) https://doi.org/10.1117/12.2040191
Event: SPIE BiOS, 2014, San Francisco, California, United States

Abstract

Imaging thick biological samples introduces spherical aberration (SA) due to refractive index (RI) mismatch between specimen and imaging lens immersion medium. SA increases with the increase of either depth or RI mismatch. Therefore, it is difficult to find a static compensator for SA¹. Different wavefront coding methods^2,3 have been studied to find an optimal way of static wavefront correction to reduce depth-induced SA. Inspired by a recent design of a radially symmetric squared cubic (SQUBIC) phase mask that was tested for scanning confocal microscopy¹ we have modified the pupil using the SQUBIC mask to engineer the point spread function (PSF) of a wide field fluorescence microscope. In this study, simulated images of a thick test object were generated using a wavefront encoded engineered PSF (WFEPSF) and were restored using space-invariant (SI) and depth-variant (DV) expectation maximization (EM) algorithms implemented in the COSMOS software⁴. Quantitative comparisons between restorations obtained with both the conventional and WFE PSFs are presented. Simulations show that, in the presence of SA, the use of the SIEM algorithm and a single SQUBIC encoded WFE-PSF can yield adequate image restoration. In addition, in the presence of a large amount of SA, it is possible to get adequate results using the DVEM with fewer DV-PSFs than would typically be required for processing images acquired with a clear circular aperture (CCA) PSF. This result implies that modification of a widefield system with the SQUBIC mask renders the system less sensitive to depth-induced SA and suitable for imaging samples at larger optical depths.

Citation Download Citation

Nurmohammed Patwary, Nurmohammed Patwary, Ana Doblas, Ana Doblas, Sharon V. King, Sharon V. King, Chrysanthe Preza, Chrysanthe Preza, "Reducing depth induced spherical aberration in 3D widefield fluorescence microscopy by wavefront coding using the SQUBIC phase mask", Proc. SPIE 8949, Three-Dimensional and Multidimensional Microscopy: Image Acquisition and Processing XXI, 894911 (12 March 2014); doi: 10.1117/12.2040191; https://doi.org/10.1117/12.2040191

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