3D microscope imaging robust to restoration artifacts introduced by optically thick specimens

Abstract

We demonstrate 3D microscope imaging using computational optical sectioning microscopy (COSM) with an engineered point-spread function (PSF) robust to depth-induced spherical aberration (SA). Earlier we demonstrated that wavefront encoding (WFE) using a squared cubic (SQUBIC) phase mask reduces the PSF depth-variance in the presence of SA and that space-invariant (SI) restoration of simulated images using a single WFE-PSF does not lead to artifacts as in the conventional case. In this study, we show experimental verification of our WFE COSM approach. The WFE system used is a commercial microscope with a modified side port imaging path, where a spatial light modulator projects the SQUBIC phase mask on the back focal plane of the imaging lens. High resolution images of a test sample with 6 µm in diameter microspheres embedded in UV-cured optical cement (RI = 1.47) were captured using both the engineered and the conventional imaging paths of the system. The acquired images were restored using a regularized SI expectation maximization algorithm based on Tikhonov-Miller regularization with a roughness penalty. A comparative study quantified in terms of the correlation coefficients between the XZ medial sections of the restored images, from experimental data, shows an 11% reduction in depth sensitivity in the SQUBIC system compared to the conventional system.

Reducing effects of aberration in 3D fluorescence imaging using wavefront coding with a radially symmetric phase mask

“…The design of the SQUBIC phase mask depends on the microscope objective (MO) lens' numerical aperture (NA) and the refractive index (RI) of the immersion medium [14]; therefore, this study investigates system performance with two types of objective lenses: a 20X/0.8 NA dry and a 63X/1.4 NA oil immersion, and quantifies performance for different imaging conditions and different amounts of SA. Preliminary results from the study were presented in conference publications [19][20][21]. Simulated SQUBIC-WFE images of a 3 µm in diameter solid sphere at different depths within the embedding medium are restored using a single PSF computed at depth 30 µm.…”