High-resolution assessment of multidimensional cellular mechanics using label-free refractive-index traction force microscopy.

A critical requirement for studying cell mechanics is three-dimensional assessment of cellular shapes and forces with high spatiotemporal resolution. Traction force microscopy with fluorescence imaging enables the measurement of cellular forces, but it is limited by photobleaching and a slow acquisition speed. Here, we present refractive-index traction force microscopy (RI-TFM), which simultaneously quantifies the volumetric morphology and traction force of cells using a high-speed illumination scheme with 0.5-Hz temporal resolution. Without labelling, our method enables quantitative analyses of dry-mass distributions and shear (in-plane) and normal (out-of-plane) tractions of single cells on the extracellular matrix. When combined with a constrained total variation-based deconvolution algorithm, it provides 0.55-Pa shear and 1.59-Pa normal traction sensitivity for a 1-kPa hydrogel substrate. We demonstrate its utility by assessing the effects of compromised intracellular stress and capturing the rapid dynamics of cellular junction formation in the spatiotemporal changes in non-planar traction components.

Hybrid machine-learning framework for volumetric segmentation and quantification of vacuoles in individual yeast cells using holotomography.

The precise, quantitative evaluation of intracellular organelles in three-dimensional (3D) imaging data poses a significant challenge due to the inherent constraints of traditional microscopy techniques, the requirements of the use of exogenous labeling agents, and existing computational methods. To counter these challenges, we present a hybrid machine-learning framework exploiting correlative imaging of 3D quantitative phase imaging with 3D fluorescence imaging of labeled cells. The algorithm, which synergistically integrates a random-forest classifier with a deep neural network, is trained using the correlative imaging data set, and the trained network is then applied to 3D quantitative phase imaging of cell data. We applied this method to live budding yeast cells. The results revealed precise segmentation of vacuoles inside individual yeast cells, and also provided quantitative evaluations of biophysical parameters, including volumes, concentration, and dry masses of automatically segmented vacuoles.

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.

Surviving, navigating and innovating through a pandemic: A review of research on school leadership during COVID-19, 2020-2021.

This article contributes to knowledge and understanding about leading schools during the COVID-19 pandemic crisis by reviewing 21 articles published during the immediate period of the pandemic (during 2020-2021). Key findings include the value of leaders supporting and connecting the school community with a view to establishing a more resilient and responsive style of leadership during a period of major crisis. Furthermore, supporting and connecting all members of the school community to address equity through alternate strategies and digital technologies provides opportunities for leaders to build capacity in staff and students to respond to further changes. Implications and recommendations are discussed in the light of these findings.

Quantification of structural heterogeneity in H&E stained clear cell renal cell carcinoma using refractive index tomography.

Clear cell renal cell carcinoma (ccRCC) is a common histopathological subtype of renal cancer and is notorious for its poor prognosis. Its accurate diagnosis by histopathology, which relies on manual microscopic inspection of stained slides, is challenging. Here, we present a correlative approach to utilize stained images and refractive index (RI) tomography and demonstrate quantitative assessments of the structural heterogeneities of ccRCC slides obtained from human patients. Machine-learning-assisted segmentation of nuclei and cytoplasm enabled the quantification at the subcellular level. Compared to benign regions, malignant regions exhibited a considerable increase in structural heterogeneities. The results demonstrate that RI tomography provides quantitative information in synergy with stained images on the structural heterogeneities in ccRCC.

Long-term label-free assessments of individual bacteria using three-dimensional quantitative phase imaging and hydrogel-based immobilization.

Three-dimensional (3D) quantitative phase imaging (QPI) enables long-term label-free tomographic imaging and quantitative analysis of live individual bacteria. However, the Brownian motion or motility of bacteria in a liquid medium produces motion artifacts during 3D measurements and hinders precise cell imaging and analysis. Meanwhile, existing cell immobilization methods produce noisy backgrounds and even alter cellular physiology. Here, we introduce a protocol that utilizes hydrogels for high-quality 3D QPI of live bacteria maintaining bacterial physiology. We demonstrate long-term high-resolution quantitative imaging and analysis of individual bacteria, including measuring the biophysical parameters of bacteria and responses to antibiotic treatments.

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.

Revisiting the Faure report: Contemporary legacy and challenged legitimacy.

Since its publication in 1972, the Faure report has been regarded as a foundational text on the subject of lifelong learning, offering a plethora of ideas and repertoires. This article contemplates why and how the notions of self-fulfilment and self-learning are interrelated and profoundly important in understanding contemporary lifelong learning discourses, and how both have been appropriated by subsequent policy texts embedded in neoliberal thinking. The author argues that pursuing lifelong learning for self-fulfilment becomes voluntary self-exploitation as the individual's desire to learn unwittingly becomes driven by the instinct to survive and thrive in neoliberal socio-political environments. He also demonstrates that the ideas and repertoires provided in the Faure report function as a fertile ground for lifelong learning discourses, even though the abundant mix of ideas and propositions make it difficult to view the report as an ideologically coherent and conceptually tight-knit blueprint for the future of education. Nonetheless, the author argues that the legacy of the Faure report is still valid beyond its historical specificity. He points out that when read within the context of the unprecedented worldwide experience of COVID-19, the Faure report's proposition and reservations regarding mass media and cybernetics can shed light on the potential for contemporary technologies to strengthen emancipatory experiences of lifelong learning. Reflecting on this, he suggests that it is necessary to think collectively about how we can appreciate and harness technological innovation as an emancipatory tool to liberate ourselves from ignorance and prejudice through borderless and limitless connections to others, and to learn how to live with them.

Réétudier le rapport Faure : un héritage contemporain et une légitimité remise en question ­ Depuis sa publication en 1972, le rapport Faure fait figure de texte fondateur sur l'apprentissage tout au long de la vie au sujet duquel il offre pléthore d'idées et de répertoires. Le présent article examine non seulement pourquoi et comment les notions d'épanouissement personnel et d'autoapprentissage sont interdépendantes et profondément essentielles pour comprendre les discours sur l'apprentissage tout au long de la vie, mais aussi comment les textes politiques ultérieurs ancrés dans la pensée néolibérale se les sont appropriées. L'auteur affirme qu'apprendre tout au long de la vie dans une optique d'épanouissement personnel devient une autoexploitation volontaire étant donné que le souhait de la personne d'apprendre incidemment est mu par l'instinct de survie et de réussite dans des environnements sociopolitiques néolibéraux. Il démontre aussi que les idées et répertoires présentés dans le rapport Faure servent de terreau fertile aux discours sur l'apprentissage tout au long de la vie bien que la profusion d'idées et propositions rendent difficile de le considérer pour l'avenir de l'éducation comme un plan cohérent sur le plan idéologique et rigoureux du point de vue conceptuel. Néanmoins, l'auteur affirme que l'héritage du rapport Faure conserve sa validité au-delà de sa spécificité historique. Il indique que lu dans le contexte de la covid-19, une expérience sans précédent dans le monde entier, la proposition et les réserves du rapport Faure concernant les médias de masse et la cybernétique peuvent fournir un éclairage sur ce que les technologies contemporaines sont susceptibles d'apporter pour renforcer les expériences émancipatrices de l'apprentissage tout au long de la vie. En se penchant sur la question, il indique qu'il est nécessaire de réfléchir collectivement à la façon d'apprécier et d'exploiter les innovations technologiques en tant qu'outils émancipateurs pour nous affranchir de l'ignorance et des préjugés en créant des liens sans frontières et illimités avec les autres, et en apprenant comment vivre avec eux.

High-fidelity optical diffraction tomography of live organisms using iodixanol refractive index matching.

Optical diffraction tomography (ODT) enables the three-dimensional (3D) refractive index (RI) reconstruction. However, when the RI difference between a sample and a medium increases, the effects of light scattering become significant, preventing the acquisition of high-quality and accurate RI reconstructions. Herein, we present a method for high-fidelity ODT by introducing non-toxic RI matching media. Optimally reducing the RI contrast enhances the fidelity and accuracy of 3D RI reconstruction, enabling visualization of the morphology and intra-organization of live biological samples without producing toxic effects. We validate our method using various biological organisms, including C. albicans and C. elegans.

Roadmap on Digital Holography-Based Quantitative Phase Imaging.

Quantitative Phase Imaging (QPI) provides unique means for the imaging of biological or technical microstructures, merging beneficial features identified with microscopy, interferometry, holography, and numerical computations. This roadmap article reviews several digital holography-based QPI approaches developed by prominent research groups. It also briefly discusses the present and future perspectives of 2D and 3D QPI research based on digital holographic microscopy, holographic tomography, and their applications.

Label-free multiplexed microtomography of endogenous subcellular dynamics using generalizable deep learning.

Simultaneous imaging of various facets of intact biological systems across multiple spatiotemporal scales is a long-standing goal in biology and medicine, for which progress is hindered by limits of conventional imaging modalities. Here we propose using the refractive index (RI), an intrinsic quantity governing light-matter interaction, as a means for such measurement. We show that major endogenous subcellular structures, which are conventionally accessed via exogenous fluorescence labelling, are encoded in three-dimensional (3D) RI tomograms. We decode this information in a data-driven manner, with a deep learning-based model that infers multiple 3D fluorescence tomograms from RI measurements of the corresponding subcellular targets, thereby achieving multiplexed microtomography. This approach, called RI2FL for refractive index to fluorescence, inherits the advantages of both high-specificity fluorescence imaging and label-free RI imaging. Importantly, full 3D modelling of absolute and unbiased RI improves generalization, such that the approach is applicable to a broad range of new samples without retraining to facilitate immediate applicability. The performance, reliability and scalability of this technology are extensively characterized, and its various applications within single-cell profiling at unprecedented scales (which can generate new experimentally testable hypotheses) are demonstrated.

Isotropically resolved label-free tomographic imaging based on tomographic moulds for optical trapping.

A major challenge in three-dimensional (3D) microscopy is to obtain accurate spatial information while simultaneously keeping the microscopic samples in their native states. In conventional 3D microscopy, axial resolution is inferior to spatial resolution due to the inaccessibility to side scattering signals. In this study, we demonstrate the isotropic microtomography of free-floating samples by optically rotating a sample. Contrary to previous approaches using optical tweezers with multiple foci which are only applicable to simple shapes, we exploited 3D structured light traps that can stably rotate freestanding complex-shaped microscopic specimens, and side scattering information is measured at various sample orientations to achieve isotropic resolution. The proposed method yields an isotropic resolution of 230 nm and captures structural details of colloidal multimers and live red blood cells, which are inaccessible using conventional tomographic microscopy. We envision that the proposed approach can be deployed for solving diverse imaging problems that are beyond the examples shown here.

Holotomography: Refractive Index as an Intrinsic Imaging Contrast for 3-D Label-Free Live Cell Imaging.

Live cell imaging provides essential information in the investigation of cell biology and related pathophysiology. Refractive index (RI) can serve as intrinsic optical imaging contrast for 3-D label-free and quantitative live cell imaging, and provide invaluable information to understand various dynamics of cells and tissues for the study of numerous fields. Recently significant advances have been made in imaging methods and analysis approaches utilizing RI, which are now being transferred to biological and medical research fields, providing novel approaches to investigate the pathophysiology of cells. To provide insight into how RI can be used as an imaging contrast for imaging of biological specimens, here we provide the basic principle of RI-based imaging techniques and summarize recent progress on applications, ranging from microbiology, hematology, infectious diseases, hematology, and histopathology.

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.

Deep-learning-based three-dimensional label-free tracking and analysis of immunological synapses of CAR-T cells.

The immunological synapse (IS) is a cell-cell junction between a T cell and a professional antigen-presenting cell. Since the IS formation is a critical step for the initiation of an antigen-specific immune response, various live-cell imaging techniques, most of which rely on fluorescence microscopy, have been used to study the dynamics of IS. However, the inherent limitations associated with the fluorescence-based imaging, such as photo-bleaching and photo-toxicity, prevent the long-term assessment of dynamic changes of IS with high frequency. Here, we propose and experimentally validate a label-free, volumetric, and automated assessment method for IS dynamics using a combinational approach of optical diffraction tomography and deep learning-based segmentation. The proposed method enables an automatic and quantitative spatiotemporal analysis of IS kinetics of morphological and biochemical parameters associated with IS dynamics, providing a new option for immunological research.

Three-dimensional label-free observation of individual bacteria upon antibiotic treatment using optical diffraction tomography.

Measuring alterations in bacteria upon antibiotic application is important for basic studies in microbiology, drug discovery, clinical diagnosis, and disease treatment. However, imaging and 3D time-lapse response analysis of individual bacteria upon antibiotic application remain largely unexplored mainly due to limitations in imaging techniques. Here, we present a method to systematically investigate the alterations in individual bacteria in 3D and quantitatively analyze the effects of antibiotics. Using optical diffraction tomography, in-situ responses of Escherichia coli and Bacillus subtilis to various concentrations of ampicillin were investigated in a label-free and quantitative manner. The presented method reconstructs the dynamic changes in the 3D refractive-index distributions of living bacteria in response to antibiotics at sub-micrometer spatial resolution.

Measurements of three-dimensional refractive index tomography and membrane deformability of live erythrocytes from Pelophylax nigromaculatus.

Unlike mammalian erythrocytes, amphibian erythrocytes have distinct morphological features including large cell sizes and the presence of nuclei. The sizes of the cytoplasm and nuclei of erythrocytes vary significantly over different species, their environments, or pathophysiology, which makes hematological studies important for investigating amphibian species. Here, we present a label-free three-dimensional optical quantification of individual amphibian erythrocytes from frogs Pelophylax nigromaculatus (Rana nigromaculata). Using optical diffraction tomography, we measured three-dimensional refractive index (RI) tomograms of the cells, which clearly distinguished the cytoplasm and nuclei of the erythrocytes. From the measured RI tomograms, we extracted the relevant biochemical parameters of the cells, including hemoglobin contents and hemoglobin concentrations. Furthermore, we measured dynamic membrane fluctuations and investigated the mechanical properties of the cell membrane. From the statistical and correlative analysis of these retrieved parameters, we investigated interspecific differences between frogs and previously studied mammals.

Label-free non-invasive quantitative measurement of lipid contents in individual microalgal cells using refractive index tomography.

Microalgae are promising candidates for biofuel production due to their high lipid content. To facilitate utilization of the microalgae for biofuel, rapid quantification of the lipid contents in microalgae is necessary. However, conventional methods based on the chemical extraction of lipids require a time-consuming destructive extraction process. Here, we demonstrate label-free, non-invasive, rapid quantification of the lipid contents in individual micro-algal cells measuring the three-dimensional refractive index tomograms. We measure three-dimensional refractive index distributions within Nannochloropsis oculata cells and find that lipid droplets are identifiable in tomograms by their high refractive index. In addition, we alter N. oculata under nitrogen deficiency by measuring the volume, lipid weight, and dry cell weight of individual cells. Characterization of individual cells allows correlative analysis between the lipid content and size of individual cells.