In-silico clearing approach for deep refractive index tomography by partial reconstruction and wave-backpropagation

Abstract

Refractive index (RI) is considered to be a fundamental physical and biophysical parameter in biological imaging, as it governs light-matter interactions and light propagation while reflecting cellular properties. RI tomography enables volumetric visualization of RI distribution, allowing biologically relevant analysis of a sample. However, multiple scattering (MS) and sample-induced aberration (SIA) caused by the inhomogeneity in RI distribution of a thick sample make its visualization challenging. This paper proposes a deep RI tomographic approach to overcome MS and SIA and allow the enhanced reconstruction of thick samples compared to that enabled by conventional linear-model-based RI tomography. The proposed approach consists of partial RI reconstruction using multiple holograms acquired with angular diversity and their backpropagation using the reconstructed partial RI map, which unambiguously reconstructs the next partial volume. Repeating this operation efficiently reconstructs the entire RI tomogram while suppressing MS and SIA. We visualized a multicellular spheroid of diameter 140 µm within minutes of reconstruction, thereby demonstrating the enhanced deep visualization capability and computational efficiency of the proposed method compared to those of conventional RI tomography. Furthermore, we quantified the high-RI structures and morphological changes inside multicellular spheroids, indicating that the proposed method can retrieve biologically relevant information from the RI distribution. Benefitting from the excellent biological interpretability of RI distributions, the label-free deep visualization capability of the proposed method facilitates a noninvasive understanding of the architecture and time-course morphological changes of thick multicellular specimens.

Quantitative phase imaging by gradient retardance optical microscopy

“…By deploying a single optical element in the illumination path, our approach greatly simplifies related hardware configurations and incurs no energy losses in alternative detection modes. Compared to laser-based QPI techniques 29 , 30 , GROM operates independently of any need to characterize or make assumptions about the scattering medium, though it may exhibit lower sensitivity. To address this limitation, we have developed a method that involves capturing additional images at finer differences in retardance (see Fig.…”

“…As such, performing QPI in multiply scattering environments requires specific strategies, such as combining laser-based tomography 26 , 27 with dedicated reconstruction strategies 28 . In similar embodiments, exceptional QPI outcomes in thick specimens were reported through either the backpropagation of partial reconstructions from holograms captured at various angles or the application of specific optical diffraction tomography and image-stitching algorithms that take optical scattering into account 29 , 30 . Alternative strategies rely on temporally incoherent illumination that—additionally—suppress speckle noise and enable high resolution imaging 31 .…”

Recent Advances and Current Trends in Transmission Tomographic Diffraction Microscopy

“…Finally, precise measurements of the electromagnetic field resulting from light-sample interaction not only allow for numerically recreating image formation in any type of optical microscope (using same illumination/detection numerical aperture), but also permit us to numerically manipulate the optical properties of the sample itself. In this view, the so-called in silico clearing of the observed sample has been recently proposed to image highly scattering spheroids [ 136 , 137 ]. In this technique, layer-by-layer sample reconstruction allows for suppressing multiple scattering and sample-induced aberration from one layer in order to reconstruct the next layer, providing a numerical equivalent of chemical tissue clearing [ 138 ].…”

Non-invasive Visualization and Characterization of Bile Canaliculus Formation Using Refractive Index Tomography

“…[4][5][6][7][8][9][10][11] However, accurately assessing the 3D architecture of the BCs within thick samples is challenging because the imaging depth is limited by multiplescattering light originating from them. 12,13) Second, intact samples should be evaluated to address the native behavior of BCs. For example, the BC undergoes cycles of expansion and contraction to secrete bile acids efficiently, 14) suggesting the importance of addressing BC morphology in the context of its dynamics.…”