We acknowledge Dr. Kalidas Kohale and his team for the maintenance of the fish facility and KV Boby for the maintenance of the light sheet microscope. SRN acknowledges financial support from the Department of Atomic Energy (DAE), Govt. of India (Project Identification no. RTI4003, DAE OM no. 1303/2/2019/R\&#38;D-II/DAE/2079 dated 11.02.2020), the Max Planck Society Partner Group program (M.PG.A MOZG0010) and the Science and Engineering Research Board Start-up Research Grant (SRG/2023/001716).

Refined Multiview Light Sheet Microscopy Protocol for Zebrafish Embryos

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The authors declare no competing interest.

Positioning an embryo in the right orientation to image the region of interest is one of the rate-limiting steps that often results in a failed microscopy session for a user. This is more so in a multiview light sheet microscope where manual manipulation of the orientation is difficult as the samples are embedded within a tube. To aid in this process, this study reports the statistics of various positions a zebrafish embryo takes up between 70% epiboly and early somite stages within a chorion when the polymer tube with e...

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.

<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

Light sheet microscopy has become the methodology of choice for live imaging of zebrafish embryos over long time scales with minimal phototoxicity. In particular, a multiview system, which allows sample rotation, enables imaging of entire embryos from different angles. However, in most imaging sessions with a multiview system, sample mounting is a troublesome process as samples are usually prepared in a polymer tube. To aid in this process, this protocol describes basic mounting strategies for imaging early zebrafish development between the 70% epiboly and early somite stages. Specifically, the study provides statistics on the various positions the embryos default to at the 70% epiboly and bud stages within the chorion. Furthermore, it discusses the optimum number of angles and the interval between angles required for imaging whole zebrafish embryos at the early stages of development so that cellular-scale information can be extracted by fusing the different views. Finally, since the embryo covers the entire field of view of the camera, which is required to obtain a cellular-scale resolution, this protocol details the process of using bead information from above or below the embryo for the registration of the different views.

Author Spotlight: Strategies for Mounting Zebrafish Embryos for High-Resolution Multiview Light-Sheet Microscopy &amp;#8212; Techniques for Imaging and Image Reconstruction

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.

Orienting the sample in a precise manner is a vital part of efficiently using a microscopy set-up. However, manually orienting samples is often not possible when using a multiview light sheet system, given the requirement for preparing the samples in a tube. Therefore, to check if there are stereotypical positions that embryos take up within the chorion, zebrafish embryos were imaged at 70% epiboly (about 7 h post-fertilization (hpf)), since time-lapse imaging from gastrulation to early somite stages was the focus of thi...

A sample preparation strategy for imaging early zebrafish embryos within an intact chorion using a light-sheet microscope is described. It analyzes the different orientations that embryos acquire within the chorion at the 70% epiboly and bud stages and details imaging strategies for obtaining cellular-scale resolution throughout the embryo using the light-sheet system.

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

Sample Preparation for Imaging Early Zebrafish Embryos Within an Intact Chorion Using a Light-Sheet Microscope

To begin, remove the prepared agarose tube with added beads from a 37 degree Celsius water bath. Pour the entire 10 milliliters of agarose onto a six centimeter Petri dish. Once the temperature drops below 33 degrees Celsius, transfer 10 to 15 embryos into the agarose solution.Swirl the Petri dish so that the little buffer that would've been transferred gets well dispersed. Next, take a 200 microliter micropipette with an appropriate pipette tip and insert a clean straight tube taken out of the microcentrifuge tube into the pipette tip. Using the micropipette, aspirate a little agarose into the tube, followed by two to three embryos.Without releasing the pressure from the pipette, detach the tube from the pipette tip and place it on the lid of the Petri dish filled with E3 so that the agarose can solidify. Once the agarose has solidified, transfer the tubes to a two milliliter microcentrifuge tube filled with E3.

Analyzing Different Orientations of Zebrafish Embryos Within the Chorion Using Multiview Imaging

Begin by assembling the sample holder to locate the embryo. Insert the tube with embryos directly into a capillary of the right diameter. Now start live scanning and navigate using the specimen navigator to select the first view.Set up the slice interval, followed by the first and last slices of the Z-stack in this view. Change the angle in the specimen navigator to move to the next view. Set up the Z-stack for the second view and add the information in the multi-view dialog box.After setting all the views, save this information in a text file that contains the Z-stack information, coordinates of the tube, and angle specifications for each view. Then clear all positions from the multi-view dialog box. Next, translate to a different Y-coordinate away from the embryo where beads are visible.Add this position to the multi-view dialog box and save this position in a new text file. Then go to the folder where the text files are saved and duplicate the Embryo Positions file to a new file named Beads Positions. Replace the Y-coordinate for all the views in the Beads Positions file with the Y-coordinate from the Beads Y file.Afterwards, return to the imaging software and load the Beads Positions file. If the time series option is activated, select one cycle and start the experiment. Save bead images as beads"to register different views during image processing.Then clear the positions and load the initial Embryo Positions file in the software, set the desirable number of cycles with a suitable time interval and start the experiment.

Multiview Image Analysis to Obtain Cellular-Scale Resolution Throughout the Embryo

To begin, acquire multiview images of early stage zebra fish embryos using a light sheet microscope. Once interest points are detected, select all the views, right click and choose Register using Interest Points. For checking how precisely registration has worked, select two consecutive views.Go to the overlapping region and toggle between the two views to observe if the beads imaged from different angles are superimposed. After successfully registering, click on Save in the multi-view explorer window to save the updated XML file. Now open the bead.XML file in a text editor of choice and copy the entire block under view registrations. Open the embryo. XML file and replace the view registrations block with the block copied from the beads file.Then open the embryo. XML file in Big Stitcher and carefully check if the registration has worked successfully for the embryo. Repeat the same for the beads by checking the overlap of structures of interest in every two consecutive views.To perform multi-view deconvolution, select all the views in the bead file, right click, and choose point spread functions and extract options. Proceed with the default point spread function sizes and click on OK.Then re-save the XML file. Next, open the embryo.XML file and assign the point spread function for each view separately. Right click a view, click on Point Spread Functions, choose Assign, and select Advanced, followed by Assign new PSF to all selected views. Click on Browse, go to the XML file path and open the PSF folder.In the selected view, choose the matching point spread function with the corresponding ID, and click on OK.After the point spread function has been assigned for all the views, right click and choose multi-view deconvolution. Select the bounding box as currently selected views. At 70%epiboly, embryos assumed horizontal, vertical or oblique orientations within the polymer tube with horizontal being the least represented, while the vertical and oblique positions were equally observed.When samples were prepared before gastrulation, about 25%of the embryos exhibited horizontal orientation. The rest of the embryos exhibited various other orientations at the bud stage. However, many of them reoriented to the horizontal position during early somite formation.A comparison of cellular scale information infused images showed no significant difference in nuclei information, but cell boundaries were better resolved in the 30 degree set. Compared to the original input images, the segmented images contained errors. The software either failed to detect a cell boundary or drew non-existent cell boundaries.The number of errors drastically increased infused images obtained from imaging at 45 and 60 degree angular intervals compared to 30 degrees.

363 Views.  Tata Institute of Fundamental Research.  A sample preparation strategy for imaging early zebrafish embryos within an intact chorion using a light-sheet microscope is described. It analyzes the different orientations that embryos acquire within the chorion at the 70% epiboly and bud stages and details imaging strategies for obtaining cellular-scale resolution throughout the embryo using the light-sheet system.

Refined Multiview Light Sheet Microscopy Protocol for Zebrafish Embryos

Summary