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This paper introduces a novel approach to initialize an iterative 3D refractive-index reconstruction process. This approach requires no new data to be collected, and enables faster and more stable convergence of the iteration process.

We present a computational imaging pipeline implementing a multiple scattering algorithm with cloud computing, to recover a complex object’s 3D refractive index. Preliminary results show promise in 3D visualization of a Drosophila melanogaster (fruit fly) embryo.

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Supporting Information 1

This publisher’s note reports typographical corrections to Optica 7, 394 (2020)OPTIC82334-253610.1364/OPTICA.383030.

We present a microscope system for high-resolution complex-field tomography from intensity-only measurements with illumination angle scanning. Preliminary results using a multi-slice framework show promise in visualizing the 3D structure of a cluster of 3 µm transparent microspheres.

Though structured illumination (SI) microscopy is a popular imaging technique conventionally associated with fluorescent super-resolution, recent works have suggested its applicability towards sub-diffraction coherent imaging with quantitative endogenous biological contrast. Here, we demonstrate that SI can efficiently integrate the principles of fluorescent super-resolution and coherent synthetic aperture to achieve 3D dual-modality sub-diffraction resolution, fluorescence and refractive-index (RI) visualizations of biological samples. Such a demonstration can enable future biological studies to synergistically explore molecular and biophysical/biochemical mechanisms at sub-diffraction resolutions. We experimentally demonstrate this framework by introducing a SI microscope capable of 3D sub-diffraction fluorescence and RI imaging, and verify its biological visualization capabilities by experimentally reconstructing 3D RI/fluorescence visualizations of fluorescent calibration microspheres as well as alveolar basal epithelial adenocarcinoma (A549) and human colorectal adenocarcinmoa (HT-29) cells, fluorescently stained for F-actin., 24 pages, 6 figures in main text, 3 figures in supplementary text

Refractive-index (RI) is an inherent optical property of materials that can provide important biochemical and biophysical information about a biological sample. Optical-diffraction-tomography (ODT) is a current standard to obtain quantitative three-dimensional RI distributions, by measuring optical fields diffracted from the sample by rotated illumination beams. This method for ODT also synthetically enlarges the microscope’s lateral spatial-frequency support, and thus images the RI distribution with lateral resolution beyond the microscope's coherent diffraction limit. Fluorescence microscopy offers a complementary set of biological insights by offering imaging capabilities with molecular specificity. Analogous to ODT, super-resolution fluorescence techniques can offer these insights at spatial resolutions beyond the microscope's incoherent diffraction limit. Unfortunately, such super-resolution techniques are generally incompatible with ODT and a generalized sub-diffraction technique has been difficult to find, which hinders a cohesive, high-resolution, multimodal analysis of biological samples. We experimentally introduce, for the first time to our knowledge, a novel, high resolution, optical system that uses structured illumination (SI) to enable 3D sub-diffraction resolution imaging for both fluorescence and RI. We demonstrate sub-diffraction resolution, multimodal SI imaging of HT29 and MCF7 cells fluorescently stained for F-actin, such that the 3D RI and fluorescent distributions may offer unique, but complementary, insights into the biological samples.

This work introduces a novel reinterpretation of structured illumination (SI) microscopy for coherent imaging that allows three-dimensional imaging of complex refractive index (RI). To do so, we show that coherent SI is mathematically equivalent to a superposition of angled illuminations. It follows that raw acquisitions for standard SI-enhanced quantitative-phase images can be processed into complex electric-field maps describing sample diffraction under angled illuminations. Standard diffraction tomography (DT) computation can then be used to reconstruct the sample 3D RI distribution at sub-diffraction resolutions. We demonstrate this concept by using a SI-quantitative-phase imaging system to computationally reconstruct 3D RI distributions of human breast (MCF-7) and colorectal (HT-29) adenocarcinoma cells. Our experimental setup uses a spatial light modulator to generate structured patterns at the sample and collects angle-dependent sample diffraction using a common-path, off-axis interference configuration with no moving components. Furthermore, this technique holds promise for easy pairing with SI fluorescence microscopy, and important future extensions may include multimodal, sub-diffraction resolution, 3D RI and fluorescent visualizations., Comment: 19 total pages (including references), 8 figures

Sub-diffraction resolution imaging has played a pivotal role in biological research by visualizing key, but previously unresolvable, sub-cellular structures. Unfortunately, applications of far-field sub-diffraction resolution are currently divided between fluorescent and coherent-diffraction regimes, and a multimodal sub-diffraction technique that bridges this gap has not yet been demonstrated. Here we report that structured illumination (SI) allows multimodal sub-diffraction imaging of both coherent quantitative-phase (QP) and fluorescence. Due to the conventional fluorescent applications of SI, we first demonstrated the principle of SI-enabled three-dimensional (3D) QP sub-diffraction imaging with calibration microspheres. Image analysis confirmed enhanced lateral and axial resolutions over diffraction-limited QP imaging, and established striking parallels between coherent SI and conventional optical diffraction tomography. We next introduce an optical system utilizing SI to achieve 3D sub-diffraction, multimodal QP/fluorescent visualization of A549 biological cells fluorescently tagged for F-actin. Our results suggest that SI has unique utility in studying biological phenomena with significant molecular, biophysical, and biochemical components., Comment: 15 pages of main text (including Figures), 13 pages of Supplementary Information (including Figures), 4 Figures in main text, 9 Figures in Supplementary Information

Optical diffraction tomography (ODT) reconstructs a samples volumetric refractive index (RI) to create high-contrast, quantitative 3D visualizations of biological samples. However, standard implementations of ODT use interferometric systems, and so are sensitive to phase instabilities, complex mechanical design, and coherent noise. Furthermore, their reconstruction framework is typically limited to weakly-scattering samples, and thus excludes a whole class of multiple-scattering samples. Here, we implement a new 3D RI microscopy technique that utilizes a computational multi-slice beam propagation method to invert the optical scattering process and reconstruct high-resolution (NA>1.0) 3D RI distributions of multiple-scattering samples. The method acquires intensity-only measurements from different illumination angles, and then solves a non-linear optimization problem to recover the sample 3D RI distribution. We experimentally demonstrate reconstruction of samples with varying amounts of multiple scattering: a 3T3 fibroblast cell, a cluster of C. elegans embryos, and a whole C. elegans worm, with lateral and axial resolutions of 250 nm and 900 nm, respectively., 24 pages, 8 figures

High-content biological microscopy targets high-resolution imaging across large fields-of-view, often achieved by computational imaging approaches. Previously, we demonstrated 2D multimodal high-content microscopy via structured illumination microscopy (SIM) with resolution [Formula: see text] the diffraction limit, using speckle illumination from Scotch tape. In this work, we extend the method to 3D by leveraging the fact that the speckle illumination is in fact a 3D structured pattern. We use both a coherent and an incoherent imaging model to develop algorithms for joint retrieval of the 3D super-resolved fluorescent and complex-field distributions of the sample. Our reconstructed images resolve features beyond the physical diffraction-limit set by the system’s objective and demonstrate 3D multimodal imaging with [Formula: see text] m(3) resolution over a volume of [Formula: see text] m(3).

High-content biological microscopy targets high-resolution imaging across large fields-of-view (FOVs). Recent works have demonstrated that computational imaging can provide efficient solutions for high-content microscopy. Here, we use speckle structured illumination microscopy (SIM) as a robust and cost-effective solution for high-content fluorescence microscopy with simultaneous high-content quantitative phase (QP). This multi-modal compatibility is essential for studies requiring cross-correlative biological analysis. Our method uses laterally-translated Scotch tape to generate high-resolution speckle illumination patterns across a large FOV. Custom optimization algorithms then jointly reconstruct the sample’s super-resolution fluorescent (incoherent) and QP (coherent) distributions, while digitally correcting for system imperfections such as unknown speckle illumination patterns, system aberrations and pattern translations. Beyond previous linear SIM works, we achieve resolution gains of 4× the objective’s diffraction-limited native resolution, resulting in 700 nm fluorescence and 1.2 μm QP resolution, across a FOV of [Formula: see text] , giving a space-bandwidth product (SBP) of 60 megapixels.

In the biological sciences, there is much emphasis on elucidating the functions of various biological components and processes. To do so, advances in general microscopy have yielded various imaging modalities to probe such processes under specific visualization and contrast requirements. Examples of modalities that are popularly integrated into conventional biological studies include fluorescent, dark-field, phase-contrast, and polarization-sensitive microscopies, with each modality offering unique insights into the biological function of the sample. Often times, however, a comprehensive understanding of biological phenomena requires the integration of the unique and separate visualizations of various modalities. Unfortunately, conventional microscopes typically support only one modality and rarely allow multiple modalities to be used in conjunction. Though high-end microscopes may support multimodal visualization, they often require either mechanical (and often manual) toggling, which obstruct real-time multimodal imaging, or simultaneous detection via multiple cameras, which dramatically increases the microscope’s cost. Here, we present a one-shot technique that allows multiple imaging channels, of potentially different modalities, to be simultaneously detected by a single camera. We experimentally demonstrate this method on transparent cells that have been tagged for F-actin and nuclear fluorescence. Our multimodal system consists of 2-channel fluorescence and 1-channel quantitative-phase (QP) imaging, and clearly demonstrates ability for simultaneous fluorescent and QP visualization. Though we experimentally verify our framework using dual fluorescent/QP imaging, we emphasize that our framework for single-shot, simultaneous single-camera detection is applicable to an arbitrary number of widefield imaging modalities so long as they fulfill criteria for Fourier spectra separation, SNR, and detector dynamic range

Multimodal imaging is a crucial tool when imaging biological phenomena that cannot be comprehensively captured by a single modality. Here, we introduce a theoretical framework for spatial-frequency-multiplexed microscopy via off-axis interference as a novel wide-field imaging technique that enables true simultaneous multimodal and multichannel wide-field imaging. We experimentally demonstrate this technique for single-camera, simultaneous two-channel fluorescence and one-channel quantitative-phase imaging for fluorescent microspheres and fixed cells stained for F-actin and nuclear fluorescence.