The overall goal of these procedures is to adapt specific methodologies for the determination of mechanical properties such as, stiffness, viscosity and adhesion to the specific case of embryos and developing organisms. The methods that we introduce in this paper can answer questions in the mechanobiology field, such as how their properties may respond to genetic or physical input during development or a pinch in the regulation of cellular activities. The many advantages of this technique is that they allow retrieval of crucial information with minimal manipulation, addressing questions that are inoperable by other procedures.
These methods provide insight into understanding the role of the yolk cell during a and then they can be applied to different domains in the embryo to different developmental status and even to other model organisms. Visual demonstration of this methods is critical as embryo mounting and handling are difficult to learn. To begin, collect zebra fish embryos and grow them at 28 degrees Celsius in E3 embryo medium.
Stage the embryos according to morphology. Manually decurionate stage zebra fish embryos at different ages, according to individual interest under a dissecting stereo microscope with an adjustable range in magnification between eight times and 50 times. Remove the chorions with two thin and sharp forceps.
Grip the chorion using one set of forceps and make a tear in it with the other set of forceps, then, holding the chorion in a region opposite of that of the tear, push the embryo gently through the opening. To mount the embryos for AFM, first fill a 35 millimeter petri dish with 2%agarose and embryo medium and let it solidify. Then, make small holes of approximately 1.5 millimeter in diameter and approximately 350 microns depth with thin forceps in the agarose bed.
Place the embryos in the pokes make in the agarose layer. Then, pour a solution of 0.5%low melting agarose and embryo medium, just around the embryos to secure them in the holes. Before the low melting point agarose solidifies at room temperature, rotate the embryos in such a way that the region of interest to be probed by AFM will face upwards.
Examine the embryos. Locate and image them in the petri dish, employing a 20 times objective in an inverted optical microscope coupled with the atomic force microscope at room temperature. Probing the yolk cell surface at the right angle and at the right amplitude of oscillation and frequency is essential to retrieve those data.
Different tests must be performed to optimize this parameters. Probe the yolk cell surface of the casted embryos, employing spherical polystyrene beads to 4.5 micron in diameter, attached to a cantilever with a nominal spring constant of 0.01 Newtons per meter. Set the peak to peak amplitude of cantilever oscillation to five microns and it's frequency to one Hertz.
Collect data for each region to be tested in different positions and in several embryos as a routine for data averaging. To perform particle microinjection into the yolk cell fabricate tailored microneedles using a vertical micropipette puller in borosilicate capillary glass. Prepare microneedles that have an outer diameter or 1.00 millimeter, an inner diameter or 0.58 millimeters and a length of 10 centimeters.
Prepare an injection petri dish plate by creating straight indentation lines in a 2%agarose and embryo medium bed employing custom made molds. Turn the molds upside down and place them on top of the liquid agarose gel. Remove the molds once the gel has solidified.
Then, pipette the embryos into the grooves made by the molds in the agarose, under dissecting stereo microscope at 1.2 times magnification. Before injection, almost completely remove the medium. The surface tension prevents the embryo and chorion from sticking to the needle, when removing it after injection.
Injection of the correct amount of piece is critical and will facilitate their dispersion within the yolk, afterwards and also, it's important for the retrieval of them in square displacement. Use florescent nano particles diluted in water for microinjection into the vegetal part of the embryo yolk cell. Adjust the micropipette with precision micromanipulators and use an automatic microinjector with time and pressure controls.
Set the pressure between 10 and 20 psi. Employ a magnification of 1.6 times in the dissecting stereo microscope to visualize the embryos during the injections with are performed at room temperature. To assess the viscoelastic behavior of the yolk, first decurionate the microinjected embryos, as before.
Then, embed the embryos in a 0.5%low melting point agarose and embryo medium solution at 30 degrees Celsius. While the agarose is still liquid, transfer the embryos to glass bottom plates. Orient and push them towards the cover slip.
Place the embryos mounted in the glass bottom pates on the stage of a confocal inverted microscope two hours after microinjection. Capture images of the nano particles for 26 seconds at a sampling rate of 25 Hertz, with a 63 times objective at room temperature in an inverted confocal microscope, employing standard commercial microscope software. Force indentation curves were retrieved from individual AFM measurements at a distance of 10 microns from each other in different embryos.
On average, force curves reflect a linear increase in deflection upon contact with the embryo yolk cell surface. And accurate fitting of the data with a liquid balloon model let's to infer cortical tension values, which increased gradually towards the vegetal pole and with time. Further, the observed differences between the approaching and retracting deflection values retrieved at AFM at each point in the force displacement curves, indicates the viscoelastic character of the cortex.
This viscoelastic character of the yolk cell cortex can be characterized by multi-frequency oscillations AFM measurements to retrieve it's effective complex modulus in Pascals. The elastic modulus is at least four times higher than the viscous modulus at all tested frequencies. On the other hand, the yolk mechanics was probed with microparticle rhealogy.
The mean squared displacement of individual nanoparticles injected in the yolk cell exhibited an approximately linear dependence with time lag. The ensemble average of the MSD's of the nanoparticle's population exhibits predominantly viscous character and diffusive properties. From these data, the viscosity of the yolk was calculated to be 129 millipascal seconds.
Once mastered, AFM can be done in a few hours if it's performed properly. Following this procedure, additional questions, like the role of a specific genes or mechanical inputs and tensional parameters or material properties can be studied. After watching this video, you should have a good understanding of how to determine the mechanical properties of the yolk cell of the zebrafish embryo during epiboly When attempting a particle injection rheological data retrieval, it is important to remember to properly stage the embryo and be precise on the quantities of media to be injected as not to perturb embryo development.
After this development, this technique will help exploration of the mechanobiology of model organisms in development and possibly on pathological conditions.
