Dynamic full-field optical coherence tomography allows live imaging of retinal pigment epithelium stress model

Abstract

Retinal degenerative diseases lead to the blindness of millions of people around the world. In case of age-related macular degeneration (AMD), the atrophy of retinal pigment epithelium (RPE) precedes neural dystrophy. But as crucial as understanding both healthy and pathological RPE cell physiology is for those diseases, no current technique allows subcellular in vivo or in vitro live observation of this critical cell layer. To fill this gap, we propose dynamic full-field OCT (D-FFOCT) as a candidate for live observation of in vitro RPE phenotype. In this way, we monitored primary porcine and human stem cell-derived RPE cells in stress model conditions by performing scratch assays. In this study, we quantified wound healing parameters on the stressed RPE, and observed different cell phenotypes, displayed by the D-FFOCT signal. In order to decipher the subcellular contributions to these dynamic profiles, we performed immunohistochemistry to identify which organelles generate the signal and found mitochondria to be the main contributor to D-FFOCT contrast. Altogether, D-FFOCT appears to be an innovative method to follow degenerative disease evolution and could be an appreciated method in the future for live patient diagnostics and to direct treatment choice.

Dynamic contrast optical coherence tomography (DyC-OCT) for label-free live cell imaging

“…Various algorithms have been developed to analyze OCT signal fluctuation patterns. Initial approaches, such as the logarithmic intensity variation (LIV) 17 – 19 and standard deviation (STD) 15 , 20 – 22 algorithms, utilize the principles in SV-OCT to measure the amplitudes of OCT signal fluctuations. These methods highlight areas of high activity in tissue structures, which exhibit greater fluctuation amplitudes, while reducing the visibility of stationary structures in DyC-OCT colormaps.…”

“…It offers a high isotropic resolution (~1 μm) to enable cellular imaging, but its penetration depth in scattering tissue is lower than that of the other two OCT modalities. As a result, FF-OCT is appropriate for samples with limited thickness (~300 μm) demanding superior resolutions, such as the retina organoids and explants 22 , 24 , 32 or sparse cells 28 , 30 .…”

Advances in Optical Coherence Tomography Imaging Technology and Techniques for Choroidal and Retinal Disorders

“…Validated by multimodal imaging that overlayed fluorescence with D-FFOCT images, the success of this method suggests an imaging system that can identify specific without the need for exogenous dyes or antibodies is on the horizon. Groux et al recently presented findings from an experiment in which porcine RPE cells and human-induced pluripotent stem cell-derived RPE were captured with live D-FFOCT imaging before and after exposure to toxic stress [ 82 ]. The authors explored the dynamics of intracellular organelles during wound healing and showcased a semi-automatic segmentation-based software (SAVE Profiler) that segmented the RPE wound and determined its dimensions.…”

Advances in non-invasive biosensing measures to monitor wound healing progression

“…Optical coherence tomography scatters low-energy, highwavelength light within a specimen to construct an image of the tissue structure and offers high-resolution, multi-sectional Frontiers in Bioengineering and Biotechnology frontiersin.org imaging. This modality has been widely used, in both experimental and clinical settings, to visualize complex microarchitecture assemblies at the micrometer level and has been extensively reviewed (Cannon et al, 2022;Groux et al, 2022;Ji et al, 2022;Sanchez et al, 2022). With regards to ECM characterization, it allows for rapid quantification of the amount of ECM that has been produced, as well as identification of factors that affect production rate such as cell proliferation and assessment of collagen microchannels within the ECM (Bagnaninchi et al, 2007).…”