<meta charset="utf8"></head><body>제브라피시 배아를 위한 개선된 Multiview Light Sheet Microscopy 프로토콜

<ol>
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K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiview+image+fusion+improves+resolution+in+three-dimensional+microscopy.">Multiview image fusion improves resolution in three-dimensional microscopy.</a> Opt Express. 15 (13), 8029-8042 (2007).</li><li>Swoger, J., Huisken, J., Stelzer, E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+imaging+axis+microscopy+improves+resolution+for+thick-sample+applications.">Multiple imaging axis microscopy improves resolution for thick-sample applications.</a> Opt Lett. 28 (18), 1654-1656 (2003).</li><li>Kaufmann, A., Mickoleit, M., Weber, M., Huisken, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multilayer+mounting+enables+long-term+imaging+of+zebrafish+development+in+a+light+sheet+microscope.">Multilayer mounting enables long-term imaging of zebrafish development in a light sheet microscope.</a> Development. 139 (17), 3242-3247 (2012).</li><li>Weber, M., Mickoleit, M., Huisken, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multilayer+mounting+for+long-term+light+sheet+microscopy+of+zebrafish.">Multilayer mounting for long-term light sheet microscopy of zebrafish.</a> J Vis Exp. 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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Left-right+symmetry+of+zebrafish+embryos+requires+somite+surface+tension.">Left-right symmetry of zebrafish embryos requires somite surface tension.</a> Nature. 605 (7910), 516-521 (2022).</li><li>Shah, G., 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=Multi-scale+imaging+and+analysis+identify+pan-embryo+cell+dynamics+of+germlayer+formation+in+zebrafish.">Multi-scale imaging and analysis identify pan-embryo cell dynamics of germlayer formation in zebrafish.</a> Nat Commun. 10 (1), 5753 (2019).</li><li>Schindelin, 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=Fiji:+An+open-source+platform+for+biological-image+analysis.">Fiji: An open-source platform for biological-image analysis.</a> Nat Methods. 9 (7), 676-682 (2012).</li><li>H&#246;rl, 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=BigStitcher:+Reconstructing+high-resolution+image+datasets+of+cleared+and+expanded+samples.">BigStitcher: Reconstructing high-resolution image datasets of cleared and expanded samples.</a> Nat Methods. 16 (9), 870-874 (2019).</li><li>Behrndt, 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=Forces+driving+epithelial+spreading+in+zebrafish+gastrulation.">Forces driving epithelial spreading in zebrafish gastrulation.</a> Science. 338 (6104), 257-260 (2012).</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>Icha, 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=Using+light+sheet+fluorescence+microscopy+to+image+zebrafish+eye+development.">Using light sheet fluorescence microscopy to image zebrafish eye development.</a> J Vis Exp. 110, e53966 (2016).</li><li>Tomer, R., Khairy, K., Amat, F., Keller, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+high-speed+imaging+of+entire+developing+embryos+with+simultaneous+multiview+Light+sheet+microscopy.">Quantitative high-speed imaging of entire developing embryos with simultaneous multiview Light sheet microscopy.</a> Nat Methods. 9 (7), 755-763 (2012).</li><li>Aigouy, B., Prud'homme, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Segmentation+and+quantitative+analysis+of+epithelial+tissues.">Segmentation and quantitative analysis of epithelial tissues.</a> Methods Mol Biol. 2540, 387-399 (2022).</li><li>Mancini, 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=Apical+size+and+deltaA+expression+predict+adult+neural+stem+cell+decisions+along+lineage+progression.">Apical size and deltaA expression predict adult neural stem cell decisions along lineage progression.</a> Science Adv. 9, eadg7519 (2023).</li><li>Piscitello-Gomez, R., Mahmoud, A., Dye, N., Eaton, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensitivity+of+the+timing+of+Drosophila+pupal+wing+morphogenesis+to+external+perturbations.">Sensitivity of the timing of Drosophila pupal wing morphogenesis to external perturbations.</a> bioRxiv. , (2023).</li><li>Tsuboi, A., Fujimoto, K., Kondo, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatiotemporal+remodeling+of+extracellular+matrix+orients+epithelial+sheet+folding.">Spatiotemporal remodeling of extracellular matrix orients epithelial sheet folding.</a> Science Adv. 9, eadh2154 (2023).</li><li>Fu, Q., Martin, B. L., Matus, D. Q., Gao, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+multicellular+specimens+with+real-time+optimized+tiling+Light+sheet+selective+plane+illumination+microscopy.">Imaging multicellular specimens with real-time optimized tiling Light sheet selective plane illumination microscopy.</a> Nat Commun. 7 (1), 11088 (2016).</li><li>Chen, B. C., 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=Lattice+Light+sheet+microscopy:+imaging+molecules+to+embryos+at+high+spatiotemporal+resolution.">Lattice Light sheet microscopy: imaging molecules to embryos at high spatiotemporal resolution.</a> Science. 346 (6208), 1257998 (2014).</li><li>York, H. 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<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Agarose, low gelling temperature</td>
			<td>Sigma-Aldrich</td>
			<td>A9414</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium Chloride dihydrate</td>
			<td>Sigma-Aldrich</td>
			<td>12022</td>
			<td></td>
		</tr>
		<tr>
			<td>FIJI</td>
			<td></td>
			<td></td>
			<td>Version: ImageJ 1.54f</td>
		</tr>
		<tr>
			<td>Latex beads, carboxylate-modified polystyrene, fluorescent red, 0.5 &#956;m mean particle size, aqueous suspension</td>
			<td>Sigma-Aldrich</td>
			<td>L3280</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium sulfate heptahydrate</td>
			<td>Sigma-Aldrich</td>
			<td>M2773</td>
			<td></td>
		</tr>
		<tr>
			<td>mMESSAGE mMACHINE SP6 Transcription kit</td>
			<td>ThermoFischer Scientific</td>
			<td>AM1340</td>
			<td>For in vitro transccription of H2A-mCherry plasmid</td>
		</tr>
		<tr>
			<td>Potassium Chhloride</td>
			<td>Sigma-Aldrich</td>
			<td>P9541</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium phosphate monobasic</td>
			<td>Sigma-Aldrich</td>
			<td>P0662</td>
			<td></td>
		</tr>
		<tr>
			<td>PTFE Sleeving AWG 15L - 1.58 mm ID x 0.15 mm Wall +/-0.05&#160;</td>
			<td>Adtech Innovations in Fluoroplastics</td>
			<td>STW15</td>
			<td>PTFE tubes</td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Sigma-Aldrich</td>
			<td>S3014</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium phosphate dibasic</td>
			<td>Sigma-Aldrich</td>
			<td>71640</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrasonic Cleaner</td>
			<td>Labman</td>
			<td>LMUC3</td>
			<td>Ultrasonicator</td>
		</tr>
		<tr>
			<td>Zeiss LightSheet 7 System</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

light sheet microscopy imaging and mounting strategies for early zebrafish embryos

Ensuring minimal phototoxicity is a major requirement for imaging live embryos with high spatiotemporal resolution for long periods. Over the last decade, light sheet microscopy has become the methodology of choice to meet this requirement1,2,3,4,5,6,7. Briefly, in this technique that was first used in 2004 to capture developmental processes8, two aligned thin sheets of laser pass through the embryo from opposing ends, illuminating only the plane of interest. A detection objective is placed orthogonally, and then the emitted fluorescent light from all illuminated points in the sample is collected simultaneously. A 3D image is then obtained by sequentially moving the embryo through the static light sheet.
In addition, in a specific form of this methodology, termed multiview light sheet microscopy, the samples can be suspended in a polymer tube that can be rotated using a rotor, enabling imaging of the same embryo from multiple angles9,10,11. Following imaging, the images from multiple angles are fused based on registration markers, which are typically globular fluorescent markers within the embryo (e.g., nuclei) or in the tube (e.g., fluorescent beads). Multiview imaging and fusion significantly improve the axial resolution, providing isotropic resolution across all three dimensions12. While this is a big advantage, a major challenge of multiview methodology is sample mounting, where embryos have to be mounted and kept in place in the tubes during the entire time course of imaging.
For performing multiview imaging, to keep the embryos in place and prevent movement while imaging, embryos can be embedded in agarose. However, this often leads to detrimental growth and development, particularly for early-stage zebrafish embryos13, the model system that is discussed here. A second mounting strategy is to use a thin tube that is only slightly bigger than the diameter of the embryo, where the embryo can be pulled into the tube along with the embryo medium, followed by closing the bottom of the tube with an agarose plug14. In this method, because the tube is filled with embryo medium, registration markers such as fluorescent beads cannot be used for the fusion of the different views, and registration is therefore reliant on markers within the embryo. In general, beads act as better registration markers as the signal of the markers within the embryo degrades upon moving deeper into the sample owing to both illumination and detection limitations of any microscope.
Thus, a third approach, which will be detailed here and used previously5,13,14,15,16, is imaging early zebrafish embryos with an intact chorion and filling the tube with a minimal percentage of agarose, which contains beads as registration markers. In this scenario, because manual intervention for positioning embryos within a chorion is not possible, this study provides statistics on the default orientation early zebrafish embryos fall into, particularly focusing on 70% epiboly and bud stages. It then discusses the optimal number of views required for imaging early-stage embryos at cellular-scale resolution and details the process of fusion using BigStitcher, a FIJI-based plugin10,17,18. Together, this protocol, which uses a 20x/1 NA objective, aims to facilitate zebrafish embryologists in using multiview light sheet systems for imaging embryos with nuclei and membrane markers from gastrulation to early somite stages.

<meta charset="utf8"></head><body>저자 스포트라이트: 고해상도 멀티뷰 광시트 현미경을 위한 제브라피시 배아 장착 전략 — 이미징 및 이미지 재구성 기법

Light Sheet Microscopy Imaging and Mounting Strategies for Early Zebrafish Embryos

Despite internal asymmetries along the left-right axis, in most higher organisms, external structures such as skeleton and muscle develop in a symmetric manner. Using zebrafish embryos, we combine high-resolution live imaging, quantitative analysis, and theoretical modeling to investigate the principles of symmetry establishment. The field heavily depends on various microscopy techniques for imaging developing embryos with high spatiotemporal resolution, followed by quantitative analysis of the acquired images.Different forms of microscopy provide unique advantages. However, of late, light-sheet and live super-resolution microscopy have significantly advanced our view of dynamic processes in embryonic development. Traditional confocal and multiphoton microscopy often lead to photobleaching and phototoxicity, in addition to a limited field of view when imaging live embryos.A multiview light-sheet microscope addresses these issues, allowing for longer observation times and sample rotation. However, challenges remain in sample preparation and mounting for imaging using this technique. A major limitation of a successful imaging session is mounting and orienting the sample appropriately.Here we describe strategies to mount early zebrafish embryos for imaging in a multiview light-sheet microscope and the general procedure for acquiring and reconstructing multiview images.

Light sheet microscopy has become the methodology of choice for live imaging of zebrafish embryos over long time scales with minimal phototoxicity. In ...

light-sheet-microscopy-imaging-mounting-strategies-for-early

Research

JoVE Journal

Developmental Biology

369 조회수.  Tata Institute of Fundamental Research. 좌우 축을 따라 내부 비대칭이 있음에도 불구하고 대부분의 고등 유기체에서 골격 및 근육과 같은 외부 구조는 대칭적인 방식으로 발달합니다. 제브라피시 배아를 사용하여 고해상도 실시간 이미징, 정량 분석 및 이론적 모델링을 결합하여 대칭 확립의 원리를 조사합니다. 이 분야는 높은 시공간 해상도로 발달 중인 배아를 이미징한 후 획득한 이미...