0000000000306074
AUTHOR
Chrysanthe Preza
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 desi…
Improvement of two-dimensional structured illumination microscopy with an incoherent illumination pattern of tunable frequency.
In two-dimensional structured illumination microscopy (2D-SIM), high-resolution images with optimal optical sectioning (OS) cannot be obtained simultaneously. This tradeoff can be overcome by using a tunable-frequency 2D-SIM system and a proper reconstruction method. The goal of this work is twofold. First, we present a computational approach to reconstruct optical-sectioned images with super-resolution enhancement (OS-SR) by using a tunable SIM system. Second, we propose an incoherent tunable-frequency 2D-SIM system based on a Fresnel biprism implementation. Integration of the proposed computational method with this tunable structured illumination (SI) system results in a new 2D-SIM system…
Investigating the performance of reconstruction methods used in structured illumination microscopy as a function of the illumination pattern's modulation frequency
Surpassing the resolution of optical microscopy defined by the Abbe diffraction limit, while simultaneously achieving optical sectioning, is a challenging problem particularly for live cell imaging of thick samples. Among a few developing techniques, structured illumination microscopy (SIM) addresses this challenge by imposing higher frequency information into the observable frequency band confined by the optical transfer function (OTF) of a conventional microscope either doubling the spatial resolution or filling the missing cone based on the spatial frequency of the pattern when the patterned illumination is two-dimensional. Standard reconstruction methods for SIM decompose the low and hi…
Optimal design of incoherent tunable-frequency structured illumination microscope scheme
Structured illumination microscopy (SIM) improves resolution and optical sectioning capability compared to conventional widefield techniques. The main idea of this method is the illumination of the sample with a structured pattern of fixed spatial modulation frequency. Previously, a Fresnel biprism has been implemented in a structured illumination (SI) device providing tunable-frequency sinusoidal patterns. However, the use of this SI system introduces a tradeoff between the visibility and field of view of the illumination fringes. In this contribution, we analyze theoretically this tradeoff and propose the optimal design for the Fresnel biprism-based SIM system.
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 i…
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 pointspread 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 a…
Tunable structured illumination system based on a Wollaston prism
Experimental verification of a simple illumination system to generate a 1D structured pattern with tunable modulation frequency is shown based on a Wollaston prism illuminated by the diffracted field of an incoherent linear source.
Tunable-frequency three-dimensional structured illumination microscopy with reduced data-acquisition
The performance of a tunable three-dimensional (3D) structured illumination microscope (SIM) system and its ability to provide simultaneously super-resolution (SR) and optical-sectioning (OS) capabilities are investigated. Numerical results show that the performance of our 3D-SIM system is comparable with the one provided by a three-wave interference SIM, while requiring 40% fewer images for the reconstruction and providing frequency tunability in a cost-effective implementation. The performance of the system has been validated experimentally with images from test samples, which were also imaged with a commercial SIM based on incoherent-grid projection for comparison. Restored images from d…
Optical transfer function engineering for a tunable 3D structured illumination microscope
Two important features of three-dimensional structured illumination microscopy (3D-SIM) are its optical sectioning (OS) and super-resolution (SR) capabilities. Previous works on 3D-SIM systems show that these features are coupled. We demonstrate that a 3D-SIM system using a Fresnel biprism illuminated by multiple linear incoherent sources provides a structured illumination pattern whose lateral and axial modulation frequencies can be tuned separately. Therefore, the compact support of the synthetic optical transfer function (OTF) can be engineered to achieve the highest OS and SR capabilities for a particular imaging application. Theoretical performance of our engineered system based on the…
Evaluation of the use of wavefront encoding to reduce depth-induced aberration in structured-illumination microscopy
Three-dimensional imaging is affected by depth-induced spherical aberration (SA) when imaging deep into an optically thick sample. In this work, we evaluate the impact of SA on the performance of incoherent grating-projection structured illumination microscopy (SIM). In particular, we analyze the reduction of the contrast in the structured pattern and compare the reconstructed SIM images for different amounts of SA. In order to mitigate the impact of SA, we implement and evaluate in SIM a wavefront encoded imaging system using a square cubic (SQUBIC) phase mask, an approach shown previously to be successful in conventional microscopy.
Recent Advances in 3D Structured Illumination Microscopy
In structured illumination microscopy (SIM) the sample under investigation is illuminated using a structured illumination (SI) pattern. This SI pattern encodes high spatial frequencies of fine features within the sample, which usually are not transferred by the conventional three-dimensional (3D) optical transfer function (OTF) of the imaging system and fills the missing cone of frequencies in the OTF for better discrimination of the out-of-focus light. Thereby, SIM provides super-resolution (SR) performance beyond the diffraction limit and optical-sectioning (OS) capability with the use of data post-processing approaches. 3D structured patterns that include lateral and axial variations in …