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.

Cycle-consistent deep learning approach to coherent noise reduction in optical diffraction tomography.

We present a deep neural network to reduce coherent noise in three-dimensional quantitative phase imaging. Inspired by the cycle generative adversarial network, the denoising network was trained to learn a transform between two image domains: clean and noisy refractive index tomograms. The unique feature of this network, distinct from previous machine learning approaches employed in the optical imaging problem, is that it uses unpaired images. The learned network quantitatively demonstrated its performance and generalization capability through denoising experiments of various samples. We concluded by applying our technique to reduce the temporally changing noise emerging from focal drift in time-lapse imaging of biological cells. This reduction cannot be performed using other optical methods for denoising.

Combining Three-Dimensional Quantitative Phase Imaging and Fluorescence Microscopy for the Study of Cell Pathophysiology.

Quantitative phase imaging (QPI) has emerged as one of the powerful imaging tools for the study of live cells in a non-invasive manner. In particular, multimodal approaches combining QPI and fluorescence microscopic techniques have been recently developed for superior spatiotemporal resolution as well as high molecular specificity. In this review, we briefly summarize recent advances in three-dimensional QPI combined with fluorescence techniques for the correlative study of cell pathophysiology. Through this review, biologists and clinicians can be provided with insights on this rapidly growing field of research and may find broader applications to investigate unrevealed nature in cell physiology and related diseases.

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.

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.

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.

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.