Paramagnetic beads manipulation in living Zebrafish embryos

Nicolas Beuchat1, A.J. Kabla2 & R.J. Adams3

Contact: nicolas.beuchat@epfl.ch1Swiss Federal Institute of Technology Lausanne (EPFL), Switzerland

2Dept. of Engineering, Cambridge, UK3Dept. of Physiology, Development & Neuroscience, Cambridge, UK

RNA & beads microinjectionThe first step is to inject a mRNA coding for a membrane-targeted version of GFP in order to fluorescently label our embryos. A pressure microinjection system coupled with a micromanipulator is used to inject RNA into the yolk at the 1 to 4-cell stage. Using a spinning-disk, the cell membrane are clearly visible (Fig.1).

Fig.1 – Confocal images of (a) the anterior part of a 3-days old embryos and (b) the eye of a .

The paramagnetic beads injection is similar to the mRNA injection. The difference lies in the large size of the injected material. The larger the beads diameter, the harder it is to inject without damage to the embryo. I made micropipettes with borosilicate capillary tubes pulled with a laser-puller in order to have the desired shape. The tip is then broken according to the diameter of the beads being injected. A high beads-concentration in a 37.5% Glycerol solution in DEPC is loaded in the micropipette. The beads concentration is chosen so that around one bead is injected for a an injected volume of 4 nl. Contrary to the RNA injection, the beads are directly injected in the blastomere, inside or in-between cells, at the 4-cell stage (1hpf) to the high stage (3.3hpf) taking advantage of the “bulb-shape” of the blastomere.

While imaging, it is easy to find the beads using the brightfield channel of the spinning-disk (Fig.2a). On the GFP channel, beads appear as a black hole (Fig.2b).

Fig.2 – (a) 10.6 µm bead detection in the tail of a 3-days old embryo using the brightfield channel and (b) confocal image of the blastula with a 22.9 µm bead.

ElectromagnetIn order to apply a force on the beads, we need to have a strong magnetic field and, as the beads are paramagnetic, a large magnetic field gradient. In addition, we would like to control the force magnitude and to be able switch our system off. For this purpose, an electromagnet has been designed as follow:

Four solenoids in parallel made of insulated copper wire Separating zinc washer between the coil Iron core with a mechanically sharpened tip

The coils are in parallel in order to circumvent the voltage limitation of the generator. In addition, for the same amount of current (maximum current of 1A) passing through each wire, the resistance would be lower and therefore, the wire would overheat less quickly. The wire length of each coil is the same so that the resistance is also equal. This would ensure us that we have the same current in each coil.

The tip is mechanically sharpened so that we obtain a large magnetic field gradient. However, if the tip is too sharp, the gradient will be very

high near the tip but would quickly decrease with the distance. As we are working with embryos, we would like to have forces over distances of a few hundreds micrometers.

Fig 3 – Our custom electromagnet is made of four solenoids branched in parallel each having a resistance of ~16 Ω. The first coil is thinner and longer than the others because of space limitation with the spinning-disk.

Fig.4 – Forces measurement using particle image velocimetry (PIV). Making movies of a pool of beads in glycerol-water solution allowed me to manually track several particles using ImageJ software. Therefore, knowing the velocity versus the distance to the tip, the force can be found using Stokes formula for low Reynold number particle: F = 6 π η r v

Embryo mountingIn order to image our Zebrafish embryos, it is necessary to fix them in a low-gelling temperature agarose. This is normally done in my lab using metallic ring chambers in which the embryo is trapped between two coverslips. However, we need to come as close as possible near the embryo to generate a sufficiently high force. Thus, we obviously needed to design a custom mounting system that :

Can fit under our microscope Have an open side to come with the electromagnet tip Have enough space for the large size of the electromagnet Has a non-magnetizable structure (aluminium here)

Using TurboCAD software (CAD = Computer Assisted Design), 2D and 3D plans could be done. Engineers at the Dept. of Engineering could therefore build our mounting system in the workshops.

Fig.5 – 2D plan of our custom mouting setup. Exporting the file as a dxf file allows engineers to cut the main shape with a sand jet machine.

Fig.6 – 3D representation of our custom mounting system. (left) Holding system and (right) mounting chamber (not to scale). Embryos are fixed in the mounting chamber between two coverslips in a low-gelling temperature agarose solution.

Fig.7 – Mounting, imaging and force producing system.

Beads manipulation in Zebrafish embryosEmbryos at different developmental stages were imaged. After the blastula, regardless of the beads diameter chosen, the injected bead did not move while turning on our system. The force we had were too low for the stiffness of the tissue.However, at the blastula stage, and using the 22.9 µm beads, it has been possible to record beads movement toward the tip of the electromagnet (Fig.7).

Fig.8 – (a) to (d) Two 22.9 µm beads in an embryo at the blastula stage 7 seconds before applying a magnetic field (a), when the current is turned on (b), 7 seconds after (c) and 14 seconds after (d). (e) and (f) Pixels standard deviation of two image sequences (7 seconds and 20 frames each), before (e) and after (f) turning on the current. We notice no beads displacement without magnetic field and a 9 µm displacement with the field.

IntroductionZebrafish embryonic development involved many morphogenetic change that are consequence of intrinsic cell behaviour, meaning that the tissue morphology is modified by the “action” of its own cells, or extrinsic cell behaviour, meaning that the tissue is passively deformed by adjacent cells or tissues. Using 4D confocal imaging, it is possible to retrieve many informations from those morphological process as cell shape change, strain rates, cell identity, etc. However, it is not possible to directly characterize forces using imaging. The long-term aim of this project is to apply small mechanical perturbation on developing tissue. It is hope that this procedure may allow us to characterize

forces actually taking place during morphogenesis. Therefore, it could be possible to have a measurable distinction between extrinsic and intrinsic cell behaviour in a tissue. Others interesting tissue properties could eventually be studied as the stiffness, elasticity, etc.Paramagnetic beads ranging from 4.35 to 22.9 µm in diameter would eventually be injected in living Zebrafish embryos. Using an electromagnet, it would therefore be possible to apply forces on those beads and hopefully to see a displacement of the bead or a response of the tissue. Thus, the goal of this pilot study is to see if it is feasible to achieve sufficiently high forces with this method to manipulate the bead inside the embryo.

AcknowledgementI would like to thank my supervisor Richard Adams and my collaborating physicist Alexandre Kabla.I would also like to warmly thank Samantha England and Stephen Young, for helping me learning many techniques and protocols, and every other members of the lab especially Will Deacon and Guy Blanchard. Many thanks to the Amgen Foundation for funding this project over the summer.Finally, I would like to thank Fiona Russell, Andrea Kells and all the Cambridge Amgen Scholars with whom I had an unforgettable experience.

References[1] deVries, A. H. B. et al. Micro Magnetic Tweezers for Nanomanipulation Inside Live Cells, Biophysical Journal 88:2137-2144 (2005)

[2] Matthews B. D. et al. Electromagnetic needls with submicron pole tip radii for nanomanipulation of biomolecules and living cells, Applied Physics Letters (2004)

[3] Blanchard G. B. et al. Tissue tectonics: morphogenetic strain rates, cell shape change and intercalation, Nature Methods (2009)

Conclusion & further workThe system designed during this project allow us to apply forces in a unidirectional and linear way with limited control on the y and z-axis. Nevertheless, this project has shown that it is possible to apply forces (up to 500 pN) inside a living embryos using paramagnetic beads even though those forces were only sufficient for very young embryos. Different things could be done to improve the system:

Design a cooling system to be able to apply forces on a longer time scale (hours) as the electromagnet has a tendency to overheat after a few seconds. Improve the z-axis positioning of the tip relative to the embryo so that distance between the two would be greatly reduced. Sharpened the electromagnet tip if it is possible to get closer to the embryo.

Use smaller but more numerous solenoids in parallel with a generator not limited in current. Try different materials for the core (more magnetizable) and the wire (less resistive). Find a less invasive and more accurate injection method.

In the future, if the proposed improvements for the unidirectional and linear system proposed here go well, it could be possible to imagine a 4 to 6 pole system capable of generating forces in any direction in the third dimension. It would allow a fine and precise way of applying mechanical perturbation in living embryos. Some interesting properties of tissue and cell behaviour might therefore be unravelled.