Image restoration approach to address reduced modulation contrast in structured illumination microscopy.

The performance of structured illumination microscopy (SIM) is hampered in many biological applications due to the inability to modulate the light when imaging deep into the sample. This is in part because sample-induced aberration reduces the modulation contrast of the structured pattern. In this paper, we present an image restoration approach suitable for processing raw incoherent-grid-projection SIM data with a low fringe contrast. Restoration results from simulated and experimental ApoTome SIM data show results with improved signal-to-noise ratio (SNR) and optical sectioning compared to the results obtained from existing methods, such as 2D demodulation and 3D SIM deconvolution. Our proposed method provides satisfactory results (quantified by the achieved SNR and normalized mean square error) even when the modulation contrast of the illumination pattern is as low as 7%.

Experimental validation of a customized phase mask designed to enable efficient computational optical sectioning microscopy through wavefront encoding.

In this paper, wavefront-encoded (WFE) computational optical sectioning microscopy (COSM) using a fabricated square cubic (SQUBIC) phase mask, designed to render the system less sensitive to depth-induced aberration, is investigated. The WFE-COSM system is characterized by a point spread function (PSF) that does not vary as rapidly with imaging depth compared to the conventional system. Thus, in WFE-COSM, image restoration from large volumes can be achieved using computationally efficient space-invariant (SI) algorithms, thereby avoiding the use of depth-variant algorithms. The fabricated SQUBIC phase mask was first evaluated and found to have a 75% fidelity compared to the theoretical design; it was then integrated in a commercial wide-field microscope to implement a WFE-COSM system. Evaluation of the WFE-COSM system is demonstrated with comparative studies of theoretical and experimental PSFs and simulated and measured images of spherical shells located at different depths in a test sample. These comparisons show that PSF and imaging models capture major trends in experimental data with a 99% correlation between forward image intensity distribution in experimental and simulated images of spherical shells. Our experimental SI restoration results demonstrate that the WFE-COSM system achieves more than a twofold performance improvement over the conventional system of up to a 65 µm depth below the coverslip investigated in this study.

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

In this work, a wavefront encoded (WFE) imaging system built using a squared cubic phase mask, designed to reduce the sensitivity of the imaging system to spherical aberration, is investigated. The proposed system allows the use of a space-invariant image restoration algorithm, which uses a single PSF, to restore intensity distribution in images suffering aberration, such as sample-induced aberration in thick tissue. This provides a computational advantage over depth-variant image restoration algorithms developed previously to address this aberration. Simulated PSFs of the proposed system are shown to change up to 25% compared to the 0 µm depth PSF (quantified by the structural similarity index) over a 100 µm depth range, while the conventional system PSFs change up to 84%. Results from experimental test-sample images show that restoration error is reduced by 29% when the proposed WFE system is used instead of the conventional system over a 30 µm depth range.

Image restoration for three-dimensional fluorescence microscopy using an orthonormal basis for efficient representation of depth-variant point-spread functions.

A depth-variant (DV) image restoration algorithm for wide field fluorescence microscopy, using an orthonormal basis decomposition of DV point-spread functions (PSFs), is investigated in this study. The efficient PSF representation is based on a previously developed principal component analysis (PCA), which is computationally intensive. We present an approach developed to reduce the number of DV PSFs required for the PCA computation, thereby making the PCA-based approach computationally tractable for thick samples. Restoration results from both synthetic and experimental images show consistency and that the proposed algorithm addresses efficiently depth-induced aberration using a small number of principal components. Comparison of the PCA-based algorithm with a previously-developed strata-based DV restoration algorithm demonstrates that the proposed method improves performance by 50% in terms of accuracy and simultaneously reduces the processing time by 64% using comparable computational resources.

Spatial light modulator phase mask implementation of wavefront encoded 3D computational-optical microscopy.

Spatial light modulator (SLM) implementation of wavefront encoding enables various types of engineered point-spread functions (PSFs), including the generalized-cubic and squared-cubic phase mask wavefront encoded (WFE) PSFs, shown to reduce the impact of sample-induced spherical aberration in fluorescence microscopy. This investigation validates dynamic experimental parameter variation of these WFE-PSFs. We find that particular design parameter bounds exist, within which the divergence of computed and experimental WFE-PSFs is of the same order of magnitude as that of computed and experimental conventional PSFs, such that model-based approaches for solving the inverse imaging problem can be applied to a wide range of SLM-WFE systems. Interferometric measurements were obtained to evaluate the SLM implementation of the numeric mask. Agreement between experiment and theory in terms of a wrapped phase, 0-2π, validates the phase mask implementation and allows characterization of the SLM response. These measurements substantiate experimental practice of computational-optical microscope imaging with an SLM-engineered PSF.