Fourier space aberration correction for high resolution refractive index imaging using incoherent light.

An aberration correction method is introduced for 3D phase deconvolution microscopy. Our technique capitalizes on multiple illumination patterns to iteratively extract Fourier space aberrations, utilizing the overlapping information inherent in these patterns. By refining the point spread function based on the retrieved aberration data, we significantly improve the precision of refractive index deconvolution. We validate the effectiveness of our method on both synthetic and biological three-dimensional samples, achieving notable enhancements in resolution and measurement accuracy. The method's reliability in aberration retrieval is further confirmed through controlled experiments with intentionally induced spherical aberrations, underscoring its potential for wide-ranging applications in microscopy and biomedicine.

Vectorial inverse scattering for dielectric tensor tomography: overcoming challenges of reconstruction of highly scattering birefringent samples.

Many important microscopy samples, such as liquid crystals, biological tissue, or starches, are birefringent in nature. They scatter light differently depending on the polarization of the light and the orientation of the molecules. The complete characterization of a birefringent sample is a challenging task because its 3 × 3 dielectric tensor must be reconstructed at every three-dimensional position. Moreover, obtaining a birefringent tomogram is more arduous for thick samples, where multiple light scattering should also be considered. In this study, we developed a new dielectric tensor tomography algorithm that enables full characterization of highly scattering birefringent samples by solving the vectoral inverse scattering problem while accounting for multiple light scattering. We proposed a discrete image-processing theory to compute the error backpropagation of vectorially diffracting light. Finally, our theory was experimentally demonstrated using both synthetic and biologically birefringent samples.

Non-interferometric stand-alone single-shot holographic camera using reciprocal diffractive imaging.

An ideal holographic camera measures the amplitude and phase of the light field so that the focus can be numerically adjusted after the acquisition, and depth information about an imaged object can be deduced. The performance of holographic cameras based on reference-assisted holography is significantly limited owing to their vulnerability to vibration and complex optical configurations. Non-interferometric holographic cameras can resolve these issues. However, existing methods require constraints on an object or measurement of multiple-intensity images. In this paper, we present a holographic image sensor that reconstructs the complex amplitude of scattered light from a single-intensity image using reciprocal diffractive imaging. We experimentally demonstrate holographic imaging of three-dimensional diffusive objects and suggest its potential applications by imaging a variety of samples under both static and dynamic conditions.

Label-free histological analysis of retrieved thrombi in acute ischemic stroke using optical diffraction tomography and deep learning.

For patients with acute ischemic stroke, histological quantification of thrombus composition provides evidence for determining appropriate treatment. However, the traditional manual segmentation of stained thrombi is laborious and inconsistent. In this study, we propose a label-free method that combines optical diffraction tomography (ODT) and deep learning (DL) to automate the histological quantification process. The DL model classifies ODT image patches with 95% accuracy, and the collective prediction generates a whole-slide map of red blood cells and fibrin. The resulting whole-slide composition displays an average error of 1.1% and does not experience staining variability, facilitating faster analysis with reduced labor. The present approach will enable rapid and quantitative evaluation of blood clot composition, expediting the preclinical research and diagnosis of cardiovascular diseases.

Single-shot refractive index slice imaging using spectrally multiplexed optical transfer function reshaping.

The refractive index (RI) of cells and tissues is crucial in pathophysiology as a noninvasive and quantitative imaging contrast. Although its measurements have been demonstrated using three-dimensional quantitative phase imaging methods, these methods often require bulky interferometric setups or multiple measurements, which limits the measurement sensitivity and speed. Here, we present a single-shot RI imaging method that visualizes the RI of the in-focus region of a sample. By exploiting spectral multiplexing and optical transfer function engineering, three color-coded intensity images of a sample with three optimized illuminations were simultaneously obtained in a single-shot measurement. The measured intensity images were then deconvoluted to obtain the RI image of the in-focus slice of the sample. As a proof of concept, a setup was built using Fresnel lenses and a liquid-crystal display. For validation purposes, we measured microspheres of known RI and cross-validated the results with simulated results. Various static and highly dynamic biological cells were imaged to demonstrate that the proposed method can conduct single-shot RI slice imaging of biological samples with subcellular resolution.

Regularization of dielectric tensor tomography.

Dielectric tensor tomography reconstructs the three-dimensional dielectric tensors of microscopic objects and provides information about the crystalline structure orientations and principal refractive indices. Because dielectric tensor tomography is based on transmission measurement, it suffers from the missing cone problem, which causes poor axial resolution, underestimation of the refractive index, and halo artifacts. In this study, we study the application of total variation and positive semi-definiteness regularization to three-dimensional tensor distributions. In particular, we demonstrate the reduction of artifacts when applied to dielectric tensor tomography.

Optical trapping with holographically structured light for single-cell studies.

A groundbreaking work in 1970 by Arthur Ashkin paved the way for developing various optical trapping techniques. Optical tweezers have become an established method for the manipulation of biological objects, due to their noninvasiveness and precise controllability. Recent innovations are accelerating and now enable single-cell manipulation through holographic light structuring. In this review, we provide an overview of recent advances in optical tweezer techniques for studies at the individual cell level. Our review focuses on holographic optical tweezers that utilize active spatial light modulators to noninvasively manipulate live cells. The versatility of the technology has led to valuable integrations with microscopy, microfluidics, and biotechnological techniques for various single-cell studies. We aim to recapitulate the basic principles of holographic optical tweezers, highlight trends in their biophysical applications, and discuss challenges and future prospects.

Quantitative phase and refractive index imaging of 3D objects via optical transfer function reshaping.

Deconvolution phase microscopy enables high-contrast visualization of transparent samples through reconstructions of their transmitted phases or refractive indexes. Herein, we propose a method to extend 2D deconvolution phase microscopy to thick 3D samples. The refractive index distribution of a sample can be obtained at a specific axial plane by measuring only four intensity images obtained under optimized illumination patterns. Also, the optical phase delay of a sample can be measured using different illumination patterns.

Tomographic measurement of dielectric tensors at optical frequency.

The dielectric tensor is a physical descriptor of fundamental light-matter interactions, characterizing anisotropic materials with principal refractive indices and optic axes. Despite its importance in scientific and industrial applications ranging from material science to soft matter physics, the direct measurement of the three-dimensional dielectric tensor has been limited by the vectorial and inhomogeneous nature of light scattering from anisotropic materials. Here, we present a dielectric tensor tomographic approach to directly measure dielectric tensors of anisotropic structures including the spatial variations of principal refractive indices and directors. The anisotropic structure is illuminated with a polarized plane wave with various angles and polarization states. Then, the scattered fields are holographically measured and converted into vectorial diffracted field components. Finally, by inversely solving a vectorial wave equation, the three-dimensional dielectric tensor is reconstructed. Using this approach, we demonstrate quantitative tomographic measurements of various nematic liquid-crystal structures and their fast three-dimensional non-equilibrium dynamics.

Single-shot wide-field topography measurement using spectrally multiplexed reflection intensity holography via space-domain Kramers-Kronig relations.

Surface topology measurements of micro- or nanostructures are essential for both scientific and industrial applications. However, high-throughput measurements remain challenging in surface metrology. We present single-shot full-field surface topography measurement using Kramers-Kronig holographic imaging and spectral multiplexing. Three different intensity images at different incident angles were simultaneously measured with three different colors, from which a quantitative phase image was retrieved using spatial Kramers-Kronig relations. A high-resolution topographic image of the sample was then reconstructed using synthetic aperture holography. Various patterned structures at the nanometer scale were measured and cross-validated using atomic force microscopy.

Spatially multiplexed dielectric tensor tomography.

Dielectric tensor tomography (DTT) enables the reconstruction of three-dimensional (3D) dielectric tensors, which provides a physical measure of 3D optical anisotropy. Herein, we present a cost-effective and robust method of DTT using spatial multiplexing. Exploiting two orthogonally polarized reference beams with different angles in an off-axis interferometer, two polarization-sensitive interferograms were multiplexed and recorded using a single camera. Then, the two interferograms were demultiplexed in the Fourier domain. By measuring the polarization-sensitive fields for various illumination angles, 3D dielectric tensor tomograms were reconstructed. The proposed method was experimentally demonstrated by reconstructing the 3D dielectric tensors of various liquid-crystal (LC) particles with radial and bipolar orientational configurations.

Label-free monitoring of 3D cortical neuronal growth in vitro using optical diffraction tomography.

The highly complex central nervous systems of mammals are often studied using three-dimensional (3D) in vitro primary neuronal cultures. A coupled confocal microscopy and immunofluorescence labeling are widely utilized for visualizing the 3D structures of neurons. However, this requires fixation of the neurons and is not suitable for monitoring an identical sample at multiple time points. Thus, we propose a label-free monitoring method for 3D neuronal growth based on refractive index tomograms obtained by optical diffraction tomography. The 3D morphology of the neurons was clearly visualized, and the developmental processes of neurite outgrowth in 3D spaces were analyzed for individual neurons.

Pupil-aberration calibration with controlled illumination for quantitative phase imaging.

Quantitative phase imaging (QPI) exploits sample-induced changes in the optical field to analyze biological specimens in a label-free manner. However, the quantitative nature of QPI makes it susceptible to optical aberrations. We propose a method for calibrating pupil aberrations by imaging a sample of interest. The proposed method recovers pupil information by utilizing the cross-spectral density between optical fields at different incident angles and allows both thin and weakly scattering three-dimensional samples for calibration. We experimentally validate the proposed method by imaging various samples, including a resolution target, breast tissue, and a polystyrene bead, and demonstrate aberration-free two- and three-dimensional QPI.

Optimizing illumination in three-dimensional deconvolution microscopy for accurate refractive index tomography.

In light transmission microscopy, axial scanning does not directly provide tomographic reconstruction of specimen. Phase deconvolution microscopy can convert a raw intensity image stack into a refractive index tomogram, the intrinsic sample contrast which can be exploited for quantitative morphological analysis. However, this technique is limited by reconstruction artifacts due to unoptimized optical conditions, which leads to a sparse and non-uniform optical transfer function. Here, we propose an optimization method based on simulated annealing to systematically obtain optimal illumination schemes that enable artifact-free deconvolution. The proposed method showed precise tomographic reconstruction of unlabeled biological samples.

Label-free three-dimensional observations and quantitative characterisation of on-chip vasculogenesis using optical diffraction tomography.

Label-free, three-dimensional (3D) quantitative observations of on-chip vasculogenesis were achieved using optical diffraction tomography. Exploiting 3D refractive index maps as an intrinsic imaging contrast, the vascular structures, multicellular activities, and subcellular organelles of endothelial cells were imaged and analysed throughout vasculogenesis to characterise mature vascular networks without exogenous labelling.

Fluid-Matrix Interface Triggers a Heterogeneous Activation of Macrophages.

Spontaneous activation of macrophages in response to inflammation is a key part of innate immunity and host defense. Macrophages represent a heterogeneous population of cells with different phenotypic profiles performing distinct functions in host defense. Although a spectrum of macrophage activation stages exists in an inflamed region, the effect of local physical conditions on the heterotypic activation of macrophages is unknown. Here, we introduce an in vivo fluid-matrix interface analogous culture platform, an asymmetric microenvironment, facilitating the formation of macrophage aggregates (MAs). Macrophages were self-assembled to form MAs of ∼100 µm diameter at the collagen matrix-medium interface upon phorbol-12-myristate-13-acetate treatment. The macrophages within the half-embedded MAs into the matrix were heterogeneously activated, resulting in inhomogeneous cell-cell and cell-matrix interactions within the aggregates. Our demonstration may aid in a better understanding of the acquisition of macrophage heterogeneity in response to tissue-specific microenvironments.

Three-dimensional label-free imaging and quantification of migrating cells during wound healing.

The wound-healing assay is a simple but effective tool for studying collective cell migration (CCM) that is widely used in biophysical studies and high-throughput screening. However, conventional imaging and analysis methods only address two-dimensional (2D) properties in a wound healing assay, such as gap closure rate. This is unfortunate because biological cells are complex 3D structures, and their dynamics provide significant information about cell physiology. Here, we presented 3D label-free imaging for wound healing assays and investigated the 3D dynamics of CCM using optical diffraction tomography. High-resolution subcellular structures as well as their collective dynamics were imaged and analyzed quantitatively.