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. Compared to previous work the scale and sensitivity are enhanced by the following steps. First, we obtain a large field of view while still maintaining a high image resolution by using a high magnification over numerical aperture ratio ODT set-up. With the inclusion of phase shifting we demonstrate that 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. In this way, high RI sensitivity can be achieved that is necessary for phase tomography of optically cleared tissue structures, which we can identify for features with RI down to 6•10^-4. Third, we optimize the optical clearing procedure to prevent scattering and refraction to deteriorate our large scale images. We demonstrate our technique by imaging a 3 day old zebrafish and an adult cryoinjured zebrafish heart in a large 5.5 x 5.5 x 4.1 mm³ volume with 4 micrometer resolution. Various tissue structures can be clearly identified. The volume of the cryoinjured heart is segmented and quantified based on the refractive index distribution.