Large scale high sensitivity optical diffraction tomography of zebrafish

Abstract

In this work we demonstrate large-scale high-sensitivity optical diffraction tomography (ODT) of zebrafish. We make this possible by three improvements. First, we obtain a large field of view while still maintaining a high resolution by using a high magnification over numerical aperture ratio digital holography setup. With the inclusion of phase shifting we operate close to the optimum magnification over numerical aperture ratio. Second, we decrease the noise in the reconstructed images by implementing off-axis sample placement and numerical focus tracking in combination with the acquisition of a large number of projections. Although both techniques lead to an increase in sensitivity independently, we show that combining them is necessary in order to make optimal use of the potential gain offered by each respective method and obtain a refractive index (RI) sensitivity of 8 • 10 −5. Third, we optimize the optical clearing procedure to prevent scattering and refraction to occur. We demonstrate our technique by imaging a zebrafish larva over 13 mm 3 field of view with 4 micrometer resolution. Finally, we demonstrate a clinical application of our technique by imaging an entire adult cryoinjured zebrafish heart.

Challenges and advances in optical 3D mesoscale imaging

“…However, label‐free techniques are also increasingly used by themselves to directly access the structure of mesoscale samples. Examples include optical diffraction tomography and quantitative phase imaging approaches used for zebrafish and C. elegans imaging 29,30 . In addition, Optical Coherence Tomography (OCT) can be used to image non‐invasively organs like the eye and the brain, but also increasingly in developmental biology, as for the vascularisation of Xenopus 31,32 …”

“…Examples include optical diffraction tomography and quantitative phase imaging approaches used for zebrafish and C. elegans imaging. 29,30 In addition, Optical Coherence Tomography (OCT) can be used to image non-invasively organs like the eye and the brain, but also increasingly in developmental biology, as for the vascularisation of Xenopus. 31,32 In contrast to current label-free techniques, the flexibility of the current fluorescence toolbox allows the study of structures with high specificity.…”

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

“…To improve the axial resolution of 3D microscopy, various methods of sample rotation have been demonstrated. A straightforward approach is the rotation of a sample loaded in a microcapillary 8,9 or a rotating tip 10,11 . However, the instrumentation for loading the specimen requires customised sample stages which reduce throughput and sample fixation which limits live-cell imaging.…”