Name: deep and spatially controlled volume ablations using a two-photon microscope in the zebrafish gastrula
Uploaded: 2021-07-15T15:00:00
Description: Watch this Scientific Journal Video about Deep and Spatially Controlled Volume Ablations using a Two-Photon Microscope in the Zebrafish Gastrula at JoVE.com

We thank Emilie Menant for fish care, the Polytechnique Bioimaging Facility, in particular Pierre Mahou,&#160;for assistance with live imaging on their equipment partly supported by R&#233;gion Ile-de-France (interDIM) and Agence Nationale de la Recherche (ANR-11-EQPX-0029 Morphoscope2, ANR-10-INBS-04 France BioImaging). This work was supported by the ANR grants 15-CE13-0016-1, 18-CE13-0024, 20-CE13-0016, and the European Union&#39;s Horizon 2020 research and innovation programme under the Marie Sk&#322;odowska-Curie grant agreement No 840201, the Minist&#232;re de l&#39;Enseignement Sup&#233;rieur et de la Recherche and the Centre National de la Recherche Scientifique.

All animal work was approved by the Ethical Committee N 59 and the Minist&#232;re de l&#39;Education Nationale, de l&#39;Enseignement Sup&#233;rieur et de la Recherche under the file number APAFIS#15859-2018051710341011v3. Some of the steps described below are specific to our equipment and software but could be easily adapted to different equipment.
1. Injection preparation
<ol>
	<li>Prepare 75 mL of 1% agarose solution in Embryo Medium (EM).</li>
	<li>Place the injecting mold in a 90 mm Pet...

<ol>
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P., Tada, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sphingosine-1-phosphate+receptors+regulate+individual+cell+behaviours+underlying+the+directed+migration+of+prechordal+plate+progenitor+cells+during+zebrafish+gastrulation.">Sphingosine-1-phosphate receptors regulate individual cell behaviours underlying the directed migration of prechordal plate progenitor cells during zebrafish gastrulation.</a> Development. 135 (18), 3043-3051 (2008).</li><li>Smutny, 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=Friction+forces+position+the+neural+anlage.">Friction forces position the neural anlage.</a> Nature Cell Biology. 19 (4), 306-317 (2017).</li><li>Johansson, M., Giger, F. A., Fielding, T., Houart, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dkk1+controls+cell-cell+interaction+through+regulation+of+non-nuclear+&#946;-Catenin+pools.">Dkk1 controls cell-cell interaction through regulation of non-nuclear &#946;-Catenin pools.</a> Developmental Cell. 51 (6), 775-786 (2019).</li><li>Gorelik, R., Gautreau, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+and+unbiased+analysis+of+directional+persistence+in+cell+migration.">Quantitative and unbiased analysis of directional persistence in cell migration.</a> Nature Protocols. 9 (8), 1931-1943 (2014).</li><li>Grill, S. W., Howard, J., Sch&#228;ffer, E., Stelzer, E. H. K., Hyman, A. 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Here, we describe a protocol that uses non-linear optics to perform deep and spatially well-defined volume ablations. The most critical step of the protocol is to find treatment conditions that provide sufficient energy to allow ablations, but not too much energy, to avoid excessive debris or cavitation. The amount of delivered energy at the target site mainly depends on: (1) the laser exit power, (2) the quality of laser alignment, (3) the nature of the tissue through which the light passes to reach the ablation plane, ...

Cell-cell interactions play vital roles in development. Cells provide signals that their direct neighbors, or cells further away, can perceive, thereby influencing their fate and/or behavior. Many of these signals are chemical in nature. For instance, in the well-characterized induction events, one cell group produces diffusible molecules affecting the fate of another cell population1. Other signals, however, are mechanical; cells exert forces and constraints on their neighbors, which the neighbors perceive and respond to2.
One way of studying the importance of these cell-cell interactions in vivo is to eliminate some cells and observe subsequent development. Unfortunately, available techniques to remove or destroy cells are limited. Cells can be removed surgically3,4, using needles or small wires, but such treatments are invasive, not very precise, and usually performed under a stereomicroscope, preventing immediate imaging under a microscope. Furthermore, targeting deep cells implies piercing a hole in overlying tissues, creating unwanted perturbations. Genetically encoded photosensitizers, such as KillerRed, have been used to induce cell death via light illumination5. Photosensitizers are chromophores that generate reactive oxygen species upon light irradiation. Their main limitation is that they require long light illuminations (around 15 min), which may be difficult to achieve if cells are moving, and that they induce cell death through apoptosis, which is not immediate.
Finally, laser ablations have been developed and widely used in the past 15 years6,7,8,9,10,11,12. A laser beam is focused on the targeted cell/tissue. It induces its ablation through heating, photoablation, or plasma-induced ablation; the involved process depends on the power density and exposure time13. Most ablation protocols use UV lasers for their high energy. However, UV light is both absorbed and scattered by biological tissues. Thus, targeting deep cells requires a high laser power, which then induces damages in more superficial, out-of-plane tissues. This limits the use of UV lasers to superficial structures and explains their relatively low axial resolution. Non-linear optics (so-called two-photon microscopy) uses non-linear properties of light to excite a fluorophore with two photons of approximately half-energy in the infrared domain. When applied to ablations, this has three main advantages. First, the infrared light is less scattered and less absorbed than UV light by biological tissues14, allowing to reach deeper structures without increasing the required laser power. Second, the use of a femtosecond pulsed laser provides very high power densities, creating an ablation through plasma induction, which, contrary to heating, does not diffuse spatially15. Third, the power density inducing plasma formation is reached at the focal point only. Thanks to these properties, two-photon laser ablations can be used to precisely target deep cells without affecting the surrounding tissue environment.
Collective migrations are an excellent example of developmental processes in which cell-cell interactions are fundamental. Collective migrations are defined as cell migrations in which neighboring cells&#160;influence the behavior of one cell16. The nature of these interactions (chemical or mechanical) and how they affect cell migration can vary greatly and is often not entirely understood. The ability to remove cells and observe how this affects the others is critical in further unraveling these collective processes. A few years ago, we established &#8212; using surgical approaches &#8212; that the migration of the polster during zebrafish gastrulation is a collective migration17. The polster is a group of cells that constitutes the first internalizing cells on the dorsal side of the embryo18. These cells, labeled in green in the Tg(gsc:GFP) transgenic line, are located deep in the embryo, below several layers of epiblast cells. During gastrulation, this group leads the extension of the axial mesoderm, migrating from the embryonic organizer to the animal pole19,20,21,22,23 (Figure 1A). We established that cells require contact with their neighbors to orient their migration in the direction of the animal pole. However, better understanding the cellular and molecular bases of this collective migration involves removing some cells to see how this influences the remaining ones. We, therefore, developed ablations of large and deep volumes using a two-photon microscopy setup. Here, we demonstrate the use of this protocol to sever the polster in its middle and observe the consequences on cell migration by tracking nuclei labeled with Histone2B-mCherry.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>25x water immersion objective</td>
			<td>Olympus</td>
			<td>XLPLN25XWMP2</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>PanReac AppliChem</td>
			<td>A8963,0500</td>
			<td></td>
		</tr>
		<tr>
			<td>Data analysis software : Matlab</td>
			<td>Math Works</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Electro-optic modulator (EOM)</td>
			<td>ConOptics</td>
			<td>350-80LA</td>
			<td></td>
		</tr>
		<tr>
			<td>Embryo Medium (EM) solution</td>
			<td></td>
			<td></td>
			<td>Westerfield, M. The Zebrafish Book. A Guide for the Laboratory Use of Zebrafish (Danio rerio), 5th Edition. University of Oregon Press, Eugene (Book). (2000).</td>
		</tr>
		<tr>
			<td>Environmental chamber chamber</td>
			<td>Okolab</td>
			<td>H201-T-UNIT-BL</td>
			<td></td>
		</tr>
		<tr>
			<td>EOM driver</td>
			<td>ConOptics</td>
			<td>302RM</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescence source</td>
			<td>Lumencor</td>
			<td>SOLA</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass bottom dishes</td>
			<td>MatTek</td>
			<td>P35G-0-10-C</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass capillaries</td>
			<td>Harvard Apparatus</td>
			<td>300085</td>
			<td>Outside diameter 1.0 mm, inside diameter 0.58&#160;mm</td>
		</tr>
		<tr>
			<td>Glass pipettes</td>
			<td>Volac</td>
			<td>D810</td>
			<td>Tip should be fire polished</td>
		</tr>
		<tr>
			<td>Green/ablation laser</td>
			<td>Spectra Physics</td>
			<td>Mai Tai HP DeepSee</td>
			<td></td>
		</tr>
		<tr>
			<td>Histone2B-mCherry mRNA</td>
			<td></td>
			<td></td>
			<td>Synthesized from pCS2-H2B-mCherry plasmid (Dumortier&#38; al. 2012)</td>
		</tr>
		<tr>
			<td>Image analysis software: IMARIS</td>
			<td>Bitplane</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ImSpector software</td>
			<td>Abberior Instruments Development Team</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Injection mold</td>
			<td>Adapative Science Tools</td>
			<td>I-34</td>
			<td></td>
		</tr>
		<tr>
			<td>Microloader tips</td>
			<td>Eppendorf</td>
			<td>5242956003</td>
			<td></td>
		</tr>
		<tr>
			<td>Micromanipulator</td>
			<td>Narishige</td>
			<td>MN-151</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette puller</td>
			<td>Sutter</td>
			<td>P-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>mMESSAGE mMACHINE SP6 Transcription Kit</td>
			<td>Invitrogen</td>
			<td>AM1340</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Thermofisher</td>
			<td>15140-122</td>
			<td>10 000 units penicillin and 10 mgstreptomycin per ml</td>
		</tr>
		<tr>
			<td>Photomultiplier tube (PMT)</td>
			<td>Hammamatsu</td>
			<td>H7422-40</td>
			<td></td>
		</tr>
		<tr>
			<td>PicoPump (Air injector)</td>
			<td>World Precision Instrument</td>
			<td>PV820</td>
			<td></td>
		</tr>
		<tr>
			<td>Red laser</td>
			<td>Spectra Physics</td>
			<td>OPO/Insight DeepSee</td>
			<td></td>
		</tr>
		<tr>
			<td>RNAse free water for injection</td>
			<td>Sigma</td>
			<td>W3500</td>
			<td></td>
		</tr>
		<tr>
			<td>Spreadsheet software: Excel</td>
			<td>Microsoft</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Stereomicroscope</td>
			<td>Nikon</td>
			<td>SMZ18</td>
			<td></td>
		</tr>
		<tr>
			<td>Tg(gsc:GFP) zebrafish line</td>
			<td></td>
			<td></td>
			<td>Doitsidou, M. et al. Guidance of primordial germ cell migration by the chemokine SDF-1. Cell. 111 (5), 647&#8211;59, doi: doi.org/10.1016/S0092-8674(02)01135-2 (2002).</td>
		</tr>
		<tr>
			<td>TriM Scope II microscope</td>
			<td>La Vision Biotech</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

deep and spatially controlled volume ablations using a two-photon microscope in the zebrafish gastrula

Morphogenesis involves many cell movements to organize cells into tissues and organs. For proper development, all these movements need to be tightly coordinated, and accumulating evidence suggests this is achieved, at least in part, through mechanical interactions. Testing this in the embryo requires direct physical perturbations. Laser ablations are an increasingly used option that allows relieving mechanical constraints or physically isolating two cell populations from each other. However, many ablations are performed with an ultraviolet (UV) laser, which offers limited axial resolution and tissue penetration. A method is described here to ablate deep, significant, and spatially well-defined volumes using a two-photon microscope. Ablations are demonstrated in a transgenic zebrafish line expressing the green fluorescent protein in the axial mesendoderm and used to sever the axial mesendoderm without affecting the overlying ectoderm or the underlying yolk cell. Cell behavior is monitored by live imaging before and after the ablation. The ablation protocol can be used at different developmental stages, on any cell type or tissue, at scales ranging from a few microns to more than a hundred microns.

To sever the polster in its middle, a Tg(gsc:GFP) embryo, injected with Histone2B-mCherry mRNAs was mounted at the 70% epiboly stage, as described in step 4. The polster was identified by GFP expression, and the embryo was mounted so that the plane of the polster is perpendicular to the optical axis (Figure 1B). Tilting the embryo away from this position will complicate the procedure. The light will have to go through more tissues to reach the ablation planes, and ablation planes wi...

Watch this Scientific Journal Video about Deep and Spatially Controlled Volume Ablations using a Two-Photon Microscope in the Zebrafish Gastrula at JoVE.com

Deep and Spatially Controlled Volume Ablations using a Two-Photon Microscope in the Zebrafish Gastrula

Embryonic development requires large-scale coordination of cell motion. Two-photon excitation mediated laser ablation allows the spatially controlled 3-dimensional ablation of large groups of deep cells. In addition, this technique can probe the reaction of collectively migrating cells in vivo to perturbations in their mechanical environment.

Morphogenesis involves many cell movements to organize cells into tissues and organs. For proper development, all these movements need to be tightly ...

Research

JoVE Journal

Developmental Biology

Measuring Protein Stability in Living Zebrafish Embryos Using Fluorescence Decay After Photoconversion (FDAP)

Measuring Protein Stability in Living Zebrafish Embryos Using Fluorescence Decay After Photoconversion FDAP

Protein stability influences many aspects of biology, and measuring the clearance kinetics of proteins can provide important insights into biological ...

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 ...

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 ...

Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos

Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos

Cell migration is key to many physiological and pathological conditions, including cancer metastasis. The cellular and molecular bases of cell migration ...

Analysis of Zebrafish Kidney Development with Time-lapse Imaging Using a Dissecting Microscope Equipped for Optical Sectioning

In order to understand organogenesis, the spatial and temporal alterations that occur during development of tissues need to be recorded. The method ...

Using Light Sheet Fluorescence Microscopy to Image Zebrafish Eye Development

Light sheet fluorescence microscopy (LSFM) is gaining more and more popularity as a method to image embryonic development. The main advantages of LSFM ...

Using Touch-evoked Response and Locomotion Assays to Assess Muscle Performance and Function in Zebrafish

Zebrafish muscle development is highly conserved with mammalian systems making them an excellent model to study muscle function and disease. Many ...

Induction of Hypoxia in Living Frog and Zebrafish Embryos

Here, we introduce a novel system for hypoxia induction, which we developed to study the effects of hypoxia in aquatic organisms such as frog and ...

Visualizing the Developing Brain in Living Zebrafish using Brainbow and Time-lapse Confocal Imaging

Development of the vertebrate nervous system requires a precise coordination of complex cellular behaviors and interactions. The use of high resolution in ...