3D Fluorescence Imaging of Late-Stage Zebrafish Embryo with Acoustic Tweezers

This paper presents trapping, micromanipulation, and fluorescent imaging of a single late-stage zebrafish embryo under Light Sheet Microscope™ with underwater acoustic tweezers, in a completely-liquid environment without any embryo-growth-limiting gel (such as agarose) commonly required to hold the embryo and suspend it in a desired orientation. This new method of trapping can provide a more favorable optical environment for imaging compared to gel as well as providing a better growth environment, with less mechanical stress compared to agarose gel trapping, for the embryo. The trapping and manipulation of such large living organisms in liquid are studied using two different acoustic tweezers operating at frequencies of 0.524 and 2.10 MHz. These tweezers are designed and fabricated with a wavelength comparable to the embryo’s size [1]. Precise and stable micromanipulation of the acoustically trapped zebrafish embryo within the limited space under the microscope is obtained through integrating acoustic absorbers with engravings, which allow the absorbers to fit the objectives of the microscope, thus limiting the reflection-inducing interfaces in the microscope. Slice-by-slice fluorescence images obtained by performing Z-stack imaging of the stably trapped zebrafish embryo allow the 3D reconstruction of the Lateral Plate Mesoderm (LPM).