Name: afm and microrheology in the zebrafish embryo yolk cell
Uploaded: 2017-11-29T12:30:00
Description: Watch this Scientific Journal Video about AFM and Microrheology in the Zebrafish Embryo Yolk Cell at JoVE.com

We thank Amayra Hern&#225;ndez-Vega and Philippe-Alexandre Pouille for participating in setting up the basis for these protocols. We also thank the Molecular Imaging Platform from the IBMB-PCB, Xavier Esteban and members of the laboratory for continuous support. The Consolidated Groups Program of the Generalitat de Catalunya and DGI and Consolider Grants from the Ministry of Economy and Competitivity of Spain to EMB and DN supported this work.

All the protocol steps described below follow the animal care guidelines of our institutions.
1. Zebrafish Culture
<ol>
	<li>Breed and maintain adult zebrafish under standard conditions. 
	NOTE: AB and TL wild type embryos were used for this study.</li>
	<li>Collect embryos and grow them at 28.5 &#176;C in E3 embryo medium24. Stage them according to morphology as previously described19.</li>
</ol>
2. Atomic Force Micr...

<ol>
	<li>Hernandez-Vega, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polarized+cortical+tension+drives+zebrafish+epiboly+movements.">Polarized cortical tension drives zebrafish epiboly movements.</a> EMBO J. 36, 25-41 (2017).</li><li>Brodland, G. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Video+force+microscopy+reveals+the+mechanics+of+ventral+furrow+invagination+in+Drosophila.">Video force microscopy reveals the mechanics of ventral furrow invagination in Drosophila.</a> Proc Natl Acad Sci U S A. 107, 22111-22116 (2010).</li><li>Grill, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growing+up+is+stressful:+biophysical+laws+of+morphogenesis.">Growing up is stressful: biophysical laws of morphogenesis.</a> Curr Opin Genet Dev. 21, 647-652 (2011).</li><li>Krieg, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tensile+forces+govern+germ-layer+organization+in+zebrafish.">Tensile forces govern germ-layer organization in zebrafish.</a> Nat Cell Biol. 10, 429-436 (2008).</li><li>Koser, D. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanosensing+is+critical+for+axon+growth+in+the+developing+brain.">Mechanosensing is critical for axon growth in the developing brain.</a> Nat Neurosci. 19, 1592-1598 (2016).</li><li>Wirtz, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Particle-tracking+microrheology+of+living+cells:+principles+and+applications.">Particle-tracking microrheology of living cells: principles and applications.</a> Annu Rev Biophys. 38, 301-326 (2009).</li><li>de Pablo, P. J., Carri&#243;n-V&#225;zquez, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+Biological+Samples+with+Atomic+Force+Microscopy.">Imaging Biological Samples with Atomic Force Microscopy.</a> Cold Spring Harbor Protocols. 2014, 167-177 (2014).</li><li>Roca-Cusachs, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rheology+of+passive+and+adhesion-activated+neutrophils+probed+by+atomic+force+microscopy.">Rheology of passive and adhesion-activated neutrophils probed by atomic force microscopy.</a> Biophys J. 91, 3508-3518 (2006).</li><li>Evans, E., Yeung, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apparent+viscosity+and+cortical+tension+of+blood+granulocytes+determined+by+micropipet+aspiration.">Apparent viscosity and cortical tension of blood granulocytes determined by micropipet aspiration.</a> Biophys J. 56, 151-160 (1989).</li><li>Fabry, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time+scale+and+other+invariants+of+integrative+mechanical+behavior+in+living+cells.">Time scale and other invariants of integrative mechanical behavior in living cells.</a> Phys Rev E Stat Nonlin Soft Matter Phys. 68, 041914 (2003).</li><li>Kashef, J., Franz, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+methods+for+analyzing+cell-cell+adhesion+in+development.">Quantitative methods for analyzing cell-cell adhesion in development.</a> Dev Biol. 401, 165-174 (2015).</li><li>Chevalier, N. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+the+micromechanical+properties+of+embryonic+tissues.">Measuring the micromechanical properties of embryonic tissues.</a> Methods. 94, 120-128 (2016).</li><li>Reichlin, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Investigating+native+coronary+artery+endothelium+in+situ+and+in+cell+culture+by+scanning+force+microscopy.">Investigating native coronary artery endothelium in situ and in cell culture by scanning force microscopy.</a> J Struct Biol. 152, 52-63 (2005).</li><li>Rho, J. Y., Tsui, T. Y., Pharr, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elastic+properties+of+osteon+and+trabecular+bone+measured+by+nanoindentation.">Elastic properties of osteon and trabecular bone measured by nanoindentation.</a> J Biomech. 31, 21 (1998).</li><li>Grant, C. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface+characterisation+and+biomechanical+analysis+of+the+sclera+by+atomic+force+microscopy.">Surface characterisation and biomechanical analysis of the sclera by atomic force microscopy.</a> J Mech Behav Biomed Mat. 4, 535-540 (2011).</li><li>Efremov, Y. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atomic+force+microscopy+of+living+and+fixed+Xenopus+laevis+embryos.">Atomic force microscopy of living and fixed Xenopus laevis embryos.</a> Micron. 42, 840-852 (2011).</li><li>Henkels, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatiotemporal+Mechanical+Variation+Reveals+Critical+Role+for+Rho+Kinase+During+Primitive+Streak+Morphogenesis.">Spatiotemporal Mechanical Variation Reveals Critical Role for Rho Kinase During Primitive Streak Morphogenesis.</a> Ann Biomed Eng. 41, 421-432 (2013).</li><li>Solnica-Krezel, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gastrulation+in+zebrafish+--+all+just+about+adhesion?.">Gastrulation in zebrafish -- all just about adhesion?.</a> Curr Opin Genet Dev. 16, 433-441 (2006).</li><li>Kimmel, C. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stages+of+embryonic+development+of+the+zebrafish.">Stages of embryonic development of the zebrafish.</a> Dev Dyn. 203, 253-310 (1995).</li><li>Rohde, L. A., Heisenberg, C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+gastrulation:+cell+movements,+signals,+and+mechanisms.">Zebrafish gastrulation: cell movements, signals, and mechanisms.</a> Int Rev Cytol. 261, 159-192 (2007).</li><li>Campinho, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tension-oriented+cell+divisions+limit+anisotropic+tissue+tension+in+epithelial+spreading+during+zebrafish+epiboly.">Tension-oriented cell divisions limit anisotropic tissue tension in epithelial spreading during zebrafish epiboly.</a> Nat Cell Biol. 15, 1405-1414 (2013).</li><li>Fischer-Friedrich, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+surface+tension+and+internal+pressure+generated+by+single+mitotic+cells.">Quantification of surface tension and internal pressure generated by single mitotic cells.</a> Sci Rep. 4, 6213 (2014).</li><li>Moeendarbary, E., Harris, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+mechanics:+principles,+practices,+and+prospects.">Cell mechanics: principles, practices, and prospects.</a> Wiley Interdiscip Rev Syst Biol Med. 6, 371-388 (2014).</li><li>Westerfield, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Zebrafish Book: A Guide for the Laboratory Use of Zebrafish (Danio rerio). , (2000).</li><li>Alcaraz, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microrheology+of+human+lung+epithelial+cells+measured+by+atomic+force+microscopy.">Microrheology of human lung epithelial cells measured by atomic force microscopy.</a> Biophys J. 84, 2071-2079 (2003).</li><li>Jorba, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probing+Micromechanical+Properties+of+the+Extracellular+Matrix+of+Soft+Tissues+by+Atomic+Force+Microscopy.">Probing Micromechanical Properties of the Extracellular Matrix of Soft Tissues by Atomic Force Microscopy.</a> J Cell Physiol. 232, 19-26 (2017).</li><li>Hutter, J. L., Bechhoefer, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calibration+of+atomic-force+microscope+tips.">Calibration of atomic-force microscope tips.</a> Review of Scientific Instruments. 64, 1868-1873 (1993).</li><li>Lomakina, E. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rheological+analysis+and+measurement+of+neutrophil+indentation.">Rheological analysis and measurement of neutrophil indentation.</a> Biophys J. 87, 4246-4258 (2004).</li><li>Alcaraz, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correction+of+Microrheological+Measurements+of+Soft+Samples+with+Atomic+Force+Microscopy+for+the+Hydrodynamic+Drag+on+the+Cantilever.">Correction of Microrheological Measurements of Soft Samples with Atomic Force Microscopy for the Hydrodynamic Drag on the Cantilever.</a> Langmuir. 18, 716-721 (2002).</li><li>Daniels, B. R., Masi, B. C., Wirtz, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probing+single-cell+micromechanics+in+vivo:+the+microrheology+of+C.+elegans+developing+embryos.">Probing single-cell micromechanics in vivo: the microrheology of C. elegans developing embryos.</a> Biophys J. 90, 4712-4719 (2006).</li><li>Kalwarczyk, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+analysis+of+viscosity+of+complex+liquids+and+cytoplasm+of+mammalian+cells+at+the+nanoscale.">Comparative analysis of viscosity of complex liquids and cytoplasm of mammalian cells at the nanoscale.</a> Nano Lett. 11, 2157-2163 (2011).</li><li>Soroldoni, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+oscillations.+A+Doppler+effect+in+embryonic+pattern+formation.">Genetic oscillations. A Doppler effect in embryonic pattern formation.</a> Science. 345, 222-225 (2014).</li><li>He, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apical+constriction+drives+tissue-scale+hydrodynamic+flow+to+mediate+cell+elongation.">Apical constriction drives tissue-scale hydrodynamic flow to mediate cell elongation.</a> Nature. 508, 392-396 (2014).</li><li>Johansen, P. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+micromanipulation+of+nanoparticles+and+cells+inside+living+zebrafish.">Optical micromanipulation of nanoparticles and cells inside living zebrafish.</a> Nat Commun. 7, 10974 (2016).</li></ol>

Here we show that the material properties and some biomechanical parameters of the zebrafish yolk cell during epiboly can be readily estimated by AFM and nanoparticles microrheology.
While AFM has been employed to retrieve rheological features of cells and tissues in physiological conditions4,5,25,28, here we developed a protocol for applying AFM to intact developing...

The physical principles underlying the biomechanical control of morphogenetic processes are largely undefined. While biomechanical studies at the molecular and cellular level are gathering considerable momentum, the exploration of biomechanical parameters at the tissue/organism level is at its infancy. Hydrodynamic Regression1 or Video Force Microscopy2 allow researchers to distinguish active and passive forces, while laser microsurgery3, or the less intrusive atomic force microscopy (AFM) of dissociated cells4 or in vivo5 or nanoparticle microrheology6 allow the anticipation of a tissue&#39;s mechanical properties and responses.
AFM is a three-dimensional topographic technique with high atomic resolution resolving surface roughness and compliance from the deflection of cantilever tips upon contact with a probed surface7. Different methodologies employing AFM have been developed to investigate surface properties, including measuring friction, adhesion forces, and viscoelastic properties of diverse materials. Recently, AFM has emerged as a powerful tool to retrieve information on the mechanical properties of biological samples. In particular, AFM can non-invasively extract the complex shear modulus from single cells by applying oscillatory indentations with a micro tip attached to a cantilever of known bending constant8. This lets us infer resulting forces. Yet, AFM is not unique and different methodologies allow extracting rheological data and mechanical properties from individual cells (e.g., Micropipette aspiration9, magnetic twisting cytometry (MTC)10, or uniaxial tensile testing11).
Yet, the application of these novel methodologies to complex morphogenetic processes is not straightforward. The main challenges faced when determining the mechanical properties of embryonic tissues are the small (&#181;m to mm), soft (in the 102&#160;- 104 Pa range) and visco-elastic (giving rise to time-dependent phenomena) nature of the material12. It is therefore important to adapt the methods employed for the determination of mechanical properties (stiffness, viscosity, adhesion) to the specific case of embryos and developing organisms. Two important issues need to be taken into consideration when analyzing rheological approaches to study development: to avoid intrusive interference and to provide easy accessibility. In this scenario, micropipette aspiration, MTC, and tensile testing exhibit limitations. In the first case, the high deformation that a cell suffers might alter its physiological and mechanical properties9. In the second case, the need to firmly adhere the experimental tissue to a substrate to ensure that the stresses applied deform the cytoskeleton locally could also introduce effects on the cells&#39; activation state and, hence, in their mechanical features10. Last, uniaxial tensile approaches are limited by the geometry of developing organisms and accessibility11.
AFM seems to be better suited for the study of developmental processes as it allows the study of biological samples directly in their natural environment without any cumbersome sample preparation. Moreover, as dissociation of embryonic tissues is generally difficult, the reduced size of AFM probes provides a high degree of versatility with no limitation in the choice of the type of medium (either aqueous or non-aqueous), sample temperature, or chemical composition of the sample. AFM is versatile enough to be applied to large domains and time scales and has been employed to retrieve material properties of tissues in distinct developmental stages or physiological conditions. Whole native tissues such as arteries13 or bones14 have been studied from a topographical point of view and in some cases, like the sclera, mechanical properties have also been retrieved15. Topography has also been explored in live embryos allowing the visualization of, for instance, cell rearrangement during morphogenesis in Xenopus16. Last, comprehensive sample preparation protocols have been developed to determine mechanical parameters during different developmental processes. Nanoindentation maps have been generated on native unfixed tissue sections for the chick embryonic digestive tract11; adhesive and mechanical properties retrieved for individual ectoderm, mesoderm, and endoderm progenitor cells isolated from gastrulating zebrafish embryos4; stiffness measurements performed for the epiblast and primitive streak on explants from avian embryos17; and a distinct pattern of stiffness gradients directly defined in the embryonic brain of Xenopus5.
A significant qualitative leap for applying AFM for exploring embryonic development may come from approaching simple accessible morphogenetic processes. Zebrafish epiboly is an essential and conserved event, in which different tissues coordinate in a restricted spherical space to direct the expansion of the blastoderm in a few hours. At the onset of zebrafish epiboly (spherical stage), a superficial layer of cells, the enveloping layer (EVL), covers a semi-spherical cap of blastomeres centered on the animal pole of the embryo sitting on a massive yolk syncytial cell. Epiboly consists of the cortical vegetal ward expansion of the EVL, the deep cells (DCs) of the blastoderm, and the external layer of the syncytial yolk cell (E-YSL) around the yolk. Epiboly ends with the closure of the EVL and the DCs at the vegetal pole18,19,20. How and where forces are generated during epiboly and how they are globally coupled is not yet clear1,21.
Here we describe in detail how to apply AFM to infer passive mechanical tissue properties during epiboly progression in the zebrafish yolk cell in vivo. To do so, we employed small microspheres attached to AFM cantilevers as probes. This allows the retrieval of precise local information within the non-uniform yolk cell surface and to study tensional gradients and their dynamics over time. Alternative AFM approaches such as those using wedge cantilevers22, will not render local data that is precise enough. The wedge technique, which requires very careful manipulation skills to glue a wedge larger than the sample size at the end of the cantilever, is not well suited for probing embryo (~2-fold larger than the length of the cantilever) mechanics.
Modeling and characterizing the viscoelastic properties of cells in qualitative and quantitative ways allows for an improved understanding of their biomechanics. From a biomechanical point of view, it is essential to understand epiboly, and not just demand knowledge on cortical tension measurements and dynamics, which can be extracted by AFM; thus there is need for information on the biophysical properties of the tissue. To extract this information, a variety of cell rheology techniques have been developed over the years in order to characterize different cell types under different physiological conditions23. They include AFM, MTC, and Optical Tweezers (OT). These methods, however, have been proven inadequate for mesoscopic analyses at the scale of embryos or organs. As an alternative, we have successfully adapted nanoparticle-tracking microrheology6 for in vivo rheological measurements. This technique analyzes the Brownian displacements of individual particles and infers local micromechanical properties.
Together AFM and nanoparticle microrheology allow the definition of cortical tensional dynamics and internal mechanical properties of the yolk during epiboly. This information fully endorses a model in which anisotropic stress develops as a consequence of the differences in the deformation response of the EVL and the Yolk Cytoplasmic Layer (YCL) to the isotropic actomyosin contraction of the E-YSL cortex is essential for the directional net movement of the EVL and epiboly progression1.

<table>
	<tbody>
		<tr>
			<td>Inverted optical microscope</td>
			<td>Nikon</td>
			<td>TE2000</td>
			<td>Visualization of the sample and AFM cantilever</td>
		</tr>
		<tr>
			<td>Atomic force microscope (AFM)</td>
			<td>Custom made</td>
			<td>-</td>
			<td>Any commercialized AFM could be employed</td>
		</tr>
		<tr>
			<td>Inverted Confocal microscope</td>
			<td>Zeiss</td>
			<td>LSM780</td>
			<td>Nanorheology data collection</td>
		</tr>
		<tr>
			<td>Agarose D1 Low EEO</td>
			<td>Conda</td>
			<td>8016.00</td>
			<td>Casting embryos</td>
		</tr>
		<tr>
			<td>Ultrapure LMP Agarose (low melting)</td>
			<td>Invitrogen</td>
			<td>16520-100</td>
			<td>Securing embryos</td>
		</tr>
		<tr>
			<td>Zebrafish Microinjection and Transplantation Molds</td>
			<td>World Precision Instruments</td>
			<td>Z-MOLDS</td>
			<td>Casting embryos. Custom made with the original dimensions can also be employed</td>
		</tr>
		<tr>
			<td>Dumont 5 Forceps</td>
			<td>FST</td>
			<td>11251-20</td>
			<td>1.5 mm diameter</td>
		</tr>
		<tr>
			<td>Si3N4 cantilever</td>
			<td>Novascan</td>
			<td>PT.GS</td>
			<td>Measuring the embryo by AFM. Spherical tip: 4.5 &#181;m diameter and k=0.01 N/m</td>
		</tr>
		<tr>
			<td>Fluorescent nanoparticles</td>
			<td>Life Technologies</td>
			<td>FluoSpheres F8811,</td>
			<td>For tracking nanorheology</td>
		</tr>
		<tr>
			<td>Micropipette puller</td>
			<td>Sutter Instruments</td>
			<td>P-2000</td>
			<td>Fabricating tailored microneedles</td>
		</tr>
		<tr>
			<td>Borosilicate capillary glass</td>
			<td>Warner Instruments</td>
			<td>G100TF-4</td>
			<td>Fabricating tailored microneedles</td>
		</tr>
		<tr>
			<td>Micromanipulator</td>
			<td>Narishige</td>
			<td>MN-153</td>
			<td>Manipulating the micropipette</td>
		</tr>
		<tr>
			<td>Microinjector</td>
			<td>Eppendorf</td>
			<td>FemtoJet Express</td>
			<td>Controlling injection time and pressure</td>
		</tr>
		<tr>
			<td>Stereomicroscope</td>
			<td>Leica</td>
			<td>DFC365FX</td>
			<td>Visualization of the embryos during injection</td>
		</tr>
		<tr>
			<td>Analysis Software</td>
			<td>MathWorks</td>
			<td>Matlab</td>
			<td>Analyzing AFM and particle tracking data</td>
		</tr>
		<tr>
			<td>Zebrafish</td>
			<td>AB and TL wild type</td>
			<td>-</td>
			<td>Strains employed for embryo collection</td>
		</tr>
		<tr>
			<td>Glass Bottom Plates</td>
			<td>Mat Tek</td>
			<td>P35G-0.170-14-C</td>
			<td>Mounting embryos for nanorheology data collection</td>
		</tr>
		<tr>
			<td>Stage micrometer</td>
			<td>FST</td>
			<td>29025-02</td>
			<td></td>
		</tr>
	</tbody>
</table>

afm and microrheology in the zebrafish embryo yolk cell

Elucidating the factors that direct the spatio-temporal organization of evolving tissues is one of the primary purposes in the study of development. Various propositions claim to have been important contributions to the understanding of the mechanical properties of cells and tissues in their spatiotemporal organization in different developmental and morphogenetic processes. However, due to the lack of reliable and accessible tools to measure material properties and tensional parameters in vivo, validating these hypotheses has been difficult. Here we present methods employing atomic force microscopy (AFM) and particle tracking with the aim of quantifying the mechanical properties of the intact zebrafish embryo yolk cell during epiboly. Epiboly is an early conserved developmental process whose study is facilitated by the transparency of the embryo. These methods are simple to implement, reliable, and widely applicable since they overcome intrusive interventions that could affect tissue mechanics. A simple strategy was applied for the mounting of specimens, AFM recording, and nanoparticle injections and tracking. This approach makes these methods easily adaptable to other developmental times or organisms.

Cortical tension measurements 
For each measurement point, five force-displacement (F-z) curves were acquired by AFM by ramping the cantilever at 1 Hz with a peak-to-peak amplitude of 5 &#181;m (velocity = 10 &#181;m/s) up to a maximum indentation of ~2 &#181;m. This procedure was taking less than 20 min and was not affected by epiboly progression. Each experimental condition was tested in at least 5 embryos.
<p class="jove_content" fo:keep-together.within-page="...

Watch this Scientific Journal Video about AFM and Microrheology in the Zebrafish Embryo Yolk Cell at JoVE.com

AFM and Microrheology in the Zebrafish Embryo Yolk Cell

The lack of tools to measure material properties and tensional parameters in vivo prevents validating their roles during development. We employed atomic force microscopy (AFM) and nanoparticle-tracking to quantify mechanical features on the intact zebrafish embryo yolk cell during epiboly. These methods are reliable and widely applicable avoiding intrusive interventions.

Elucidating the factors that direct the spatio-temporal organization of evolving tissues is one of the primary purposes in the study of development. ...

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.

Research

JoVE Journal

Developmental Biology

Microbead Implantation in the Zebrafish Embryo

The zebrafish has emerged as a valuable genetic model system for the study of developmental biology and disease. Zebrafish share a high degree of genomic ...

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

Imaging Subcellular Structures in the Living Zebrafish Embryo

In vivo imaging provides unprecedented access to the dynamic behavior of cellular and subcellular structures in their natural context. Performing such ...

Tracking Cells in GFP-transgenic Zebrafish Using the Photoconvertible PSmOrange System

The rapid development of transparent zebrafish embryos (Danio rerio) in combination with fluorescent labelings of cells and tissues allows visualizing ...

Primary Endodermal Epithelial Cell Culture from the Yolk Sac Membrane of Japanese Quail Embryos

We established an endodermal epithelial cell culture model (EEC) for studying the function of certain enzymes and proteins in mediating nutrient ...

Observing Mitotic Division and Dynamics in a Live Zebrafish Embryo

Mitosis is critical for organismal growth and differentiation. The process is highly dynamic and requires ordered events to accomplish proper chromatin ...

Identification Of Erythromyeloid Progenitors And Their Progeny In The Mouse Embryo By Flow Cytometry

Macrophages are professional phagocytes from the innate arm of the immune system. In steady-state, sessile macrophages are found in adult tissues where ...

Use of Immunolabeling to Analyze Stable, Dynamic, and Nascent Microtubules in the Zebrafish Embryo

Microtubules (MTs) are dynamic and fragile structures that are challenging to image in vivo, particularly in vertebrate embryos. Immunolabeling methods ...

Following Endocardial Tissue Movements via Cell Photoconversion in the Zebrafish Embryo

During embryogenesis, cells undergo dynamic changes in cell behavior, and deciphering the cellular logic behind these changes is a fundamental goal in the ...

Minimally Invasive Embryo Transfer and Embryo Vitrification at the Optimal Embryo Stage in Rabbit Model

Assisted reproductive techniques (ARTs), such as in vitro embryo culture or embryo cryopreservation, affect natural development patterns with perinatal ...