Investigation of the SQUBIC phase mask design for depth-invariant widefield microscopy point-spread function engineering

Doblas, Ana; King, Sharon V.; Patwary, Nurmohammed; Saavedra, Genaro; Martı́nez-Corral, Manuel; Preza, Chrysanthe

doi:10.1117/12.2038610

Cited by 7 publications

(19 citation statements)

References 20 publications

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“…In microscopy, phase-only elements have been placed in the back focal plane of a microscope's objective, mainly for the purpose of extending the depth of field of the microscope when imaging samples that are spread over a wide range of depths [7][8][9][10], but also for the purpose of reducing the depth of field, allowing for better optical sectioning [11].…”

Section: Introductionmentioning

confidence: 99%

Enhancing the performance of the light field microscope using wavefront coding

et al. 2014

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Light field microscopy has been proposed as a new high-speed volumetric computational imaging method that enables reconstruction of 3-D volumes from captured projections of the 4-D light field. Recently, a detailed physical optics model of the light field microscope has been derived, which led to the development of a deconvolution algorithm that reconstructs 3-D volumes with high spatial resolution. However, the spatial resolution of the reconstructions has been shown to be non-uniform across depth, with some z planes showing high resolution and others, particularly at the center of the imaged volume, showing very low resolution. In this paper, we enhance the performance of the light field microscope using wavefront coding techniques. By including phase masks in the optical path of the microscope we are able to address this non-uniform resolution limitation. We have also found that superior control over the performance of the light field microscope can be achieved by using two phase masks rather than one, placed at the objective's back focal plane and at the microscope's native image plane. We present an extended optical model for our wavefront coded light field microscope and develop a performance metric based on Fisher information, which we use to choose adequate phase masks parameters. We validate our approach using both simulated data and experimental resolution measurements of a USAF 1951 resolution target; and demonstrate the utility for biological applications with in vivo volumetric calcium imaging of larval zebrafish brain.

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Section: Introductionmentioning

confidence: 99%

Enhancing the performance of the light field microscope using wavefront coding

et al. 2014

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“…In earlier publications [5,6] the use of the SQUBIC phase mask was demonstrated only through simulation studies. Here, we compared simulated images with experimental images and restored the image of a test object (6 µm in diameter spherical shell).…”

Section: Discussionmentioning

confidence: 99%

“…The performance of the phase mask in terms of stabilizing the PSF with the increasing depth depends on the design parameter A. Theoretically, the higher value of A will produce a PSF that is less sensitive to SA over a larger depth [6]; however, the value of A is limited by a the practical limitation of the experimental setup [12] .…”

Section: Squbic Encoded Wfe-psfmentioning

confidence: 99%

“…The SQUBIC phase mask used in this study has been demonstrated in wide field microscopy simulations [5][6][7] in which theoretical and simulation studies showed its application in reducing the effect of depth induced SA. Wave front encoding (WFE) has been studied in extended depth of field microscopy where the back focal plane of the microscope objective lens is modified by adding a phase mask [8].…”

Section: Introductionmentioning

confidence: 98%

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

2015

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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.

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“…An alternative approach to overcome the challenges associated with DV imaging systems is based on the idea of engineering the PSF to be less sensitive to depth-induced spherical aberration (SA) while still suitable for COSM [10,11], so that computationally efficient SI methods can be used to reconstruct images over a large volume. During the last 5 years we have been investigating the integration of wavefront coding (traditionally used for extended depth of field microscopy [12]) to COSM [11,[13][14][15], a system we call wavefront-encoded COSM (WFE-COSM). The WFE-COSM PSF, based on a squared cubic (SQUBIC) phase mask (PM), has been shown to have reduced variability over different amounts of SA compared to the PSF of the conventional COSM system [13].…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2017

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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.

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Investigation of the SQUBIC phase mask design for depth-invariant widefield microscopy point-spread function engineering

Cited by 7 publications

References 20 publications

Enhancing the performance of the light field microscope using wavefront coding

Enhancing the performance of the light field microscope using wavefront coding

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

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

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