We demonstrate large scale high sensitivity optical diffraction tomography (ODT) imaging of zebrafish. We make this possible by three improvements. First, we optimize the field of view by using a high magnification over numerical aperture ratio ODT set-up with phase stepping. Second, we decrease the noise in the reconstructed images by off-axis sample placement, numerical focus tracking, and acquisition of a large number of projections. Third, we optimize the tissue clearing procedure to prevent scattering and refraction. We demonstrate our technique by imaging a zebrafish over a 4.1x4.1x5.5 mm3 volume with 4 micrometer spatial resolution. In addition, we demonstrate with the same set-up combined phase and polarization contrast optical diffraction tomography imaging. We use the phase and amplitude of the digital hologram to reconstruct the refractive index and (scaled) birefringence, respectively. Birefringence contrast imaging is demonstrated on zebrafish and shows high contrast images of the muscle tissue, something that is not well visible in conventional phase-based optical diffraction tomography.
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.
We use digital holography to quantify surface topography of rough objects in full-field. We calculate the variance of the intensity image as a focus metric over a set of reconstruction distances for each pixel, which results in a focus metric curve. The distance where the variance peaks is an estimate for the depth. First we analyze the lateral resolution of this method using the Talbot effect and argue that sub-mm axial resolution is feasible. Then, using a Michelson setup without magnifying optics or lateral scanning we experimentally demonstrate that sub-mm FWHM width of the focus curve can be achieved. This is significantly better than what was previously reported using digital holography and could make this technique useful for characterising objects in art and machine vision.