2020/08/17 Dynamic full-field optical coherence tomography: 3D live-imaging of retinal organoids

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2020/08/17 Dynamic full-field optical coherence tomography: 3D live-imaging of retinal organoids

Jules SchollerKassandra GrouxOlivier GoureauJosé-Alain SahelMathias FinkSacha ReichmanClaude Boccara & Kate Grieve

Abstract

Optical coherence tomography offers astounding opportunities to image the complex structure of living tissue but lacks functional information. We present dynamic full-field optical coherence tomography as a technique to noninvasively image living human induced pluripotent stem cell-derived retinal organoids. Coloured images with an endogenous contrast linked to organelle motility are generated, with submicrometre spatial resolution and millisecond temporal resolution, creating a way to identify specific cell types in living tissue via their function.

The comprehension of the human body and its mechanisms at the subcellular scale is still an open area of research. During the seventeenth century, the first examinations of life under the microscope were conducted directly on humans, animals and bacteria1. Then, at the end of the nineteenth century, cell culture studies began to replace in vivo studies, as this allows the creation of in vitro models beneficial for the comprehension of biological phenomena in different environments2,3. Because of the two-dimensional nature of early cell cultures, the possibilities of understanding tissues and organs as a whole were limited. Recently, three-dimensional (3D) cultures have been developed from stem cells to generate organoids that mimic a variety of tissues and serve as models of human development4 and disease studies5,6,7. Organoids could also serve as sources of human tissues for transplantation and as platforms for drug screening8,9,10. These self-organizing structures develop cellular compositions and architectures similar to in vivo tissues, thereby replicating biologically relevant intercellular phenomena in vitro11,12.

For each biological trend, optical imaging devices have been developed and optimized to image tissues, cell cultures and, recently, organoids, which are one of the most fundamental tools in biology, clinical pathology and medical diagnosis13. There are many challenges in imaging 3D structures: due to their relatively transparent nature, it is difficult to obtain contrast on specific structures without staining. Moreover, 3D samples require optical sectioning to discriminate the layer in focus from out-of-focus layers. In this study, we present dynamic full-field optical coherence tomography (D-FFOCT) as a technique to image retinal organoids derived from human induced pluripotent stem cells (hiPSCs)14, which are a major breakthrough in the study of the retina and retinal diseases15,16,17. These hiPSC-derived retinal organoids are routinely imaged with various techniques (see Supplementary Table 1). However, each of the existing methods presents major drawbacks, such as the need for fixation or mechanical sectioning, rendering the study of dynamic phenomena impossible; the need for labelling, requiring cumbersome and costly preparation; or a lack of functional contrast, indicating only cell presence and not cell health or behaviour18,19,20. Optical coherence tomography (OCT) is commonly used in biology and medicine to obtain 3D images of microstructures in tissue. OCT contrast arises from the local endogenous optical backscattering level21. The main drawback of traditional OCT is the trade-off between imaging depth and resolution. To increase the lateral resolution, the numerical aperture of the system must be increased. As a consequence, the depth of field decreases, and only a small layer of the sample can be imaged. Current OCT systems therefore have a lateral resolution on the order of 10 µm, which is insufficient to resolve cell structures laterally. Using an incoherent light source and a camera, time domain full-field OCT (FFOCT) is an en face variant of OCT with a higher spatial and temporal resolution than OCT in the en face plane22. As FFOCT acquires an entire en face plane rather than a line in the depth dimension, the numerical aperture can be arbitrarily increased without any imaging depth trade-off. Using the FFOCT experimental setup shown in Fig. 1(a) and detailed in the Methods section, a novel contrast mechanism has recently been exploited by measuring temporal fluctuations of back-scattered light in a technique called dynamic FFOCT (D-FFOCT)23. By revealing sub-cellular structures that have very weak back-scattering, these dynamic measurements provide contrast based on local intra-cellular motility24,25 with sub-micrometre resolution, and can achieve millisecond temporal resolution to study fast phenomena.

figure1
Fig. 1: Experimental setup and post-processing schematic.

Full Publication: https://www.nature.com/articles/s41377-020-00375-8/figures/1

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