Holographic approach for optical poration and trapping of developing embryos

Abstract

Summary form only given. Optical techniques such as poration and trapping can be viable tools toward micromanipulation of living organisms. In embryos, there are two major technical issues that need to be addressed. Injection of fluorophores or genetic constructs into a single cell of an embryo will enable cell-fate mapping and determine timing dependent protein functionality during its development. Furthermore, a non-contact method for orienting the embryo at its later stage of development is desirable for high magnification imaging. In this work, we present a holographic based system that can be utilized for both approaches as shown in our previous work [1]. Using a pulsed Ti:Sapphire femtosecond (fs) laser and a spatial light modulator (SLM), we can demonstrate successful injection of impermeable molecules such as fluorescently labeled dextran molecules and Propidium iodide into Pomatoceros lamarckii embryos. By using the continuous wave output of a Ti:Sapphire laser, we can perform optical trapping of embryos using low numerical aperture (NA) objectives.We demonstrate optical poration of developing embryos in its early stages of development with dextran molecules of varying molecular weights, from 3 kDa to 500 kDa. A single blastomere can be optoinjected with a tightly focused pulsed Ti:Sapphire fs laser shown in Fig.1(A). We characterized the optical poration efficiency by bathing the embryos in a sea water solution mixed with 10 μM of Propidium iodide. This dye was chosen so as to avoid the necessity of washing the embryos after optical poration. The efficiency ranged from 44% to 55% for early-stage to more developed embryos by sequential delivery of fs pulses targeting at three different spatial positions as demonstrated in our previous work [2] using laser power of 65 mW and exposure time of 30 ms. The holographic system presented can also be used to optically orient or trap a developing embryo. By wavefront modulation of the incident beam at the Fourier plane, the beam can be positioned laterally and displaced axially. We show that we can optically trap an embryo using a low NA, 20x objective as shown in Fig.1(B). The embryo rotates about an axis during trapping. By producing a dual focus trap using the SLM created by superposing overlapping foci, 36 μm apart, we can stably trap and fixed the orientation of a singlecell embryo.