Name: Preparation and Live Imaging of Regenerating Hydra Spheroids (Video) | JoVE Journal
Uploaded: 2026-02-20T14:10:00.000+00:00
Duration: 7 min 4 s
Description: Learn live imaging of regenerating Hydra spheroids for developmental biology using spinning disk confocal and light-sheet microscopy. This protocol captures organizer formation and tissue patterning dynamics in vivo.

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We would like to thank Laure Plantard from the Facility for Advanced Imaging and Microscopy at FMI for her valuable input. This work was supported by the Novartis Research Foundation.

<ol><li>Bode, P. M., Bode, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Formation+of+pattern+in+regenerating+tissue+pieces+of+hydra+attenuata%3A+I.+Head-body+proportion+regulation%5BTitle%5D)+AND+%22Dev+Biol%22%5BJournal%5D)">Formation of pattern in regenerating tissue pieces of hydra attenuata: I. Head-body proportion regulation.</a> Dev Biol. 78 (2), 484-496 (1980).</li>
<li>Fütterer, C., Colombo, C., Jülicher, F., Ott, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Morphogenetic+oscillations+during+symmetry+breaking+of+regenerating+hydra+vulgaris+cells%5BTitle%5D)+AND+%22Europhys+Lett%22%5BJournal%5D)">Morphogenetic oscillations during symmetry breaking of regenerating hydra vulgaris cells.</a> Europhys Lett. 64 (1), 137(2003).</li>
<li>Weevers, S. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mechanochemical+patterning+localizes+the+organizer+of+a+luminal+epithelium%5BTitle%5D)+AND+%22Sci+Adv%22%5BJournal%5D)">Mechanochemical patterning localizes the organizer of a luminal epithelium.</a> Sci Adv. 11 (26), eadu2286(2025).</li>
<li>Oreopoulos, J., Berman, R., Browne, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chapter+9+-+Spinning-disk+confocal+microscopy%3A+present+technology+and+future+trends%5BTitle%5D)+AND+%22Meth+Cell+Biol%22%5BJournal%5D)">Chapter 9 - Spinning-disk confocal microscopy: present technology and future trends.</a> Meth Cell Biol. 123, 153-175 (2014).</li>
<li>Stelzer, E. H. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Light+sheet+fluorescence+microscopy%5BTitle%5D)+AND+%22Nat+Rev+Meth+Primers%22%5BJournal%5D)">Light sheet fluorescence microscopy.</a> Nat Rev Meth Primers. 1 (1), 73(2021).</li>
<li>Moos, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Open-top+multisample+dual-view+light-sheet+microscope+for+live+imaging+of+large+multicellular+systems%5BTitle%5D)+AND+%22Nat+Meth%22%5BJournal%5D)">Open-top multisample dual-view light-sheet microscope for live imaging of large multicellular systems.</a> Nat Meth. 21 (5), 798-803 (2024).</li>
<li>Soriano, J., Colombo, C., Ott, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Hydra+molecular+network+reaches+criticality+at+the+symmetry-breaking+axis-defining+moment%5BTitle%5D)+AND+%22Phys+Rev+Lett%22%5BJournal%5D)">Hydra molecular network reaches criticality at the symmetry-breaking axis-defining moment.</a> Phys Rev Lett. 97 (25), 258102(2006).</li>
<li>Livshits, A., Shani-Zerbib, L., Maroudas-Sacks, Y., Braun, E., Keren, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Structural+inheritance+of+the+actin+cytoskeletal+organization+determines+the+body+axis+in+regenerating+hydra%5BTitle%5D)+AND+%22Cell+Rep%22%5BJournal%5D)">Structural inheritance of the actin cytoskeletal organization determines the body axis in regenerating hydra.</a> Cell Rep. 18 (6), 1410-1421 (2017).</li>
<li>Hanson, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Automatic+monitoring+of+neural+activity+with+single-cell+resolution+in+behaving+hydra%5BTitle%5D)+AND+%22Sc+Rep%22%5BJournal%5D)">Automatic monitoring of neural activity with single-cell resolution in behaving hydra.</a> Sc Rep. 14 (1), 5083(2024).</li>
<li>Escobar, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Terminal+differentiation+precedes+functional+circuit+integration+in+the+peduncle+neurons+in+regenerating+hydra+vulgaris%5BTitle%5D)+AND+%22Neural+Dev%22%5BJournal%5D)">Terminal differentiation precedes functional circuit integration in the peduncle neurons in regenerating hydra vulgaris.</a> Neural Dev. 19 (1), 18(2024).</li>
<li>Ferenc, J., Tsiairis, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Studying+Mechanical+Oscillations+During+Whole-Body+Regeneration+in+Hydra%5BTitle%5D)+AND+%22Whole-Body+Regenerat%22%5BJournal%5D)">Studying Mechanical Oscillations During Whole-Body Regeneration in Hydra.</a> Whole-Body Regenerat. 2450, 619-633 (2022).</li>
<li>Bossert, P., Galliot, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((How+to+use+hydra+as+a+model+system+to+teach+biology+in+the+classroom%5BTitle%5D)+AND+%22Int+J+Dev+Biol%22%5BJournal%5D)">How to use hydra as a model system to teach biology in the classroom.</a> Int J Dev Biol. 56 (6-8), 637-652 (2012).</li>
<li>Jemielita, M., Taormina, M. J., Delaurier, A., Kimmel, C. B., Parthasarathy, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Comparing+phototoxicity+during+the+development+of+a+zebrafish+craniofacial+bone+using+confocal+and+light+sheet+fluorescence+microscopy+techniques%5BTitle%5D)+AND+%22J+Biophotonics%22%5BJournal%5D)">Comparing phototoxicity during the development of a zebrafish craniofacial bone using confocal and light sheet fluorescence microscopy techniques.</a> J Biophotonics. 6 (11-12), 920-928 (2013).</li>
<li>Icha, J., Weber, M., Waters, J. C., Norden, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Phototoxicity+in+live+fluorescence+microscopy%2C+and+how+to+avoid+it%5BTitle%5D)+AND+%22Bioessays%22%5BJournal%5D)">Phototoxicity in live fluorescence microscopy, and how to avoid it.</a> Bioessays. 39 (8), 1700003(2017).</li>
<li>Ravichandran, Y., Vogg, M., Kruse, K., Pearce, D. J. G., Roux, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Topology+changes+of+hydra+define+actin+orientation+defects+as+organizers+of+morphogenesis%5BTitle%5D)+AND+%22Sci+Adv%22%5BJournal%5D)">Topology changes of hydra define actin orientation defects as organizers of morphogenesis.</a> Sci Adv. 11 (3), eadr9855(2025).</li>
<li>Maroudas-Sacks, Y., Garion, L., Suganthan, S., Popović, M., Keren, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Confinement+modulates+axial+patterning+in+regenerating+hydra%5BTitle%5D)+AND+%22PRX+Life%22%5BJournal%5D)">Confinement modulates axial patterning in regenerating hydra.</a> PRX Life. 2 (4), 043007(2024).</li>
<li>Bailles, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Anisotropic+stretch+biases+the+self-organization+of+actin+fibers+in+multicellular+hydra+aggregates%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci%22%5BJournal%5D)">Anisotropic stretch biases the self-organization of actin fibers in multicellular hydra aggregates.</a> Proc Natl Acad Sci. 122 (32), e2423437122(2025).</li>
<li>Luu, P., Fraser, S. E., Schneider, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((More+than+double+the+fun+with+two-photon+excitation+microscopy%5BTitle%5D)+AND+%22Comm+Biol%22%5BJournal%5D)">More than double the fun with two-photon excitation microscopy.</a> Comm Biol. 7 (1), 364(2024).</li>
</ol>

The authors declare that they have no conflicts of interest.

Both SDCM and LSFM are well-suited for generating long-term time-lapse datasets of regenerating Hydra spheroids. The optical principles on which each technique is based are distinct and, therefore, they offer unique advantages and limitations (Table 2). SDCM is preferred when a complete, high-resolution view of the sample is not required. Multiple samples can be imaged in a single experiment, making it particularly useful for testing different conditions such as drug treatments. In addition, SDC...

Hydra is a small, freshwater polyp best known for its exceptional regenerative ability. Tissue fragments excised from the middle of its body fold into a hollow spheroid that is composed of Hydra&#39;s two epithelial layers, the ectoderm and endoderm. During regeneration, these Hydra spheroids establish an organizer de novo1. Within approximately 3 days, the body axis elongates, and cells at the head and foot extremities differentiate. Because of this relatively short timeframe, the entire regenerative process can be captured through live imaging. Time-lapse datasets have been instrumental in revealing the characteristic cycles of inflation and deflation that Hydra spheroids go through during regeneration2. The tissue stretching that is generated by these osmotically driven mechanical oscillations contributes to the localization of the organizer3.
Successful long-term live imaging requires rapid image acquisition to avoid movement artefacts, while also minimizing light exposure to prevent photodamaging the sample. Classical single point scanning confocal microscopy does not meet these requirements. However, advanced techniques have been developed. In this protocol, we will focus on two widely popular modalities: spinning disk confocal microscopy (SDCM) and light-sheet fluorescence microscopy (LSFM). SDCM utilizes a rapidly rotating disk with an array of pinholes4. This design enables the simultaneous excitation and detection of multiple points within the sample, while retaining confocality. This parallel acquisition greatly accelerates imaging, thereby allowing for motion-free capture of moving samples. As an additional benefit, the shortened exposure significantly reduces phototoxicity. Still, due to limited imaging depth, live imaging of larger structures is challenging.
LSFM derives its name from the thin sheet of light that is used to excite fluorophores only within this plane5. In contrast to a pinhole, which creates axial resolution by restricting the emitted light that is detected, light-sheet microscopes establish axial resolution by restricting the excited volume instead. The detection objective is positioned orthogonally, collecting fluorescence at a 90&#176; angle to the light sheet. This allows a camera to collect the fluorescence signal from the entire illuminated plane in a single exposure, increasing the speed of acquisition and minimizing phototoxicity. Common LSFM geometries that build around one or two horizontally positioned detection objectives have the additional advantage that the same sample can be imaged from multiple angles. If the microscope is equipped with two detection objectives, samples can simultaneously be imaged from two sides6. Alternatively, a sample can be turned around to acquire views from multiple angles.
Imaging techniques used to study live Hydra have advanced substantially in recent years, enabling observation of regeneration-associated morphological changes down to neuronal activity dynamics7,8,9,10. Here, we expand upon previously published protocols6,11, with a focus on demonstrating the diverse approaches for live imaging of regenerating Hydra spheroids. We describe methods for generating Hydra spheroids from small tissue fragments and provide guidelines for identifying properly folded samples. Step-by-step instructions are outlined for three mounting strategies commonly used for live imaging. For imaging on an inverted microscope, a common geometry for SDCM, spheroids are typically mounted in agarose wells. This allows them to move and rotate freely while remaining within the field of view. Fluoroethylene propylene (FEP) is commonly used for mounting because its refractive index (1.34) is close to water (1.33). FEP foil can be thermoformed into custom imaging chambers, tailored to specific experimental needs. Similarly, embedding spheroids in agarose within glass capillaries is a standard approach for LSFM. Even though this technique limits the number of samples per capillary, it facilitates the rotation required for multiview detection. Finally, we demonstrate how to perform live imaging using both SDCM and LSFM, and we discuss key considerations for selecting the appropriate technique depending on experimental goals. Although this protocol is optimized for Hydra, its underlying principles are broadly applicable to other model systems that rely on long-term imaging to study dynamic morphogenetic processes.

<table><tbody><tr><td>20&amp;times;/0.8 Plan-Apochromat objective</td><td>Zeiss</td><td>440640-9903-000</td><td>For spinning disk confocal microscopy</td></tr><tr><td>20x/1.0 W Plan-Apochromat water dipping objective</td><td>Zeiss</td><td>421452-9700-000</td><td>Used as detection objective in the Zeiss Lightsheet 7</td></tr><tr><td>3D-printed holder for FEP chamber mounting</td><td></td><td></td><td>custom-made</td></tr><tr><td>4-well glass bottom imaging plate (#1.5H)</td><td>Ibidi</td><td>80427</td><td></td></tr><tr><td>Agarose</td><td>Carl Roth</td><td>3810.4</td><td></td></tr><tr><td>Aluminium mold for FEP chamber</td><td></td><td></td><td>custom-made</td></tr><tr><td>Calcium chloride</td><td>Sigma-Aldrich</td><td>C4901</td><td></td></tr><tr><td>Canned air</td><td>Durable</td><td>POWERCLEAN STANDARD 400</td><td></td></tr><tr><td>Cell culture plate (6 cm)</td><td>Thermo Fisher Scientific</td><td>150288</td><td></td></tr><tr><td>D-(+)-glucose</td><td>Sigma-Aldrich</td><td>G8769</td><td></td></tr><tr><td>FEP foil (127 &amp;mu;m thickness)</td><td>Adtech Polymer Engineering</td><td></td><td></td></tr><tr><td>FluoSpheres carboxylate-modified (0.2 &amp;mu;m, dark red)</td><td>Thermo Fisher Scientific</td><td>F8807</td><td></td></tr><tr><td>Glass capillary</td><td>Brand</td><td>701904</td><td></td></tr><tr><td>Glass pasteur pipette (150 mm)</td><td>VWR</td><td>612-1701</td><td></td></tr><tr><td>Hydra vulgaris</td><td></td><td></td><td></td></tr><tr><td>Imaging software</td><td>Visitron</td><td>VisiView</td><td>For spinning disk confocal microscopy</td></tr><tr><td>Inverted light microscope</td><td>Zeiss</td><td>Axio observer 7</td><td>For spinning disk confocal microscopy</td></tr><tr><td>Light sheet microscope</td><td>Leica</td><td>Viventis Deep LS2</td><td></td></tr><tr><td>Light sheet microscope</td><td>Zeiss</td><td>Lightsheet 7</td><td></td></tr><tr><td>Low melting point Agarose</td><td>Thermo Fisher Scientific</td><td>R0801</td><td></td></tr><tr><td>Magnesium chloride</td><td>Sigma-Aldrich</td><td>M8266</td><td></td></tr><tr><td>Magnesium sulfate</td><td>Sigma-Aldrich</td><td>M2643</td><td></td></tr><tr><td>Microscalpel</td><td>pfm medical</td><td>200300745</td><td></td></tr><tr><td>Pipette bulb</td><td></td><td></td><td></td></tr><tr><td>Piston rod transferpettor</td><td>Brand</td><td>701932</td><td></td></tr><tr><td>Potassium chloride</td><td>Sigma-Aldrich</td><td>P9333</td><td></td></tr><tr><td>Safety Bunsen burner</td><td>INTEGRA Biosciences</td><td>FIREBOY plus</td><td></td></tr><tr><td>sCMOS cameras</td><td>Photometrics</td><td>Prime 95B</td><td>For spinning disk confocal microscopy</td></tr><tr><td>Sodium bicarbonate</td><td>Fluka Analytical, Sigma-Aldrich</td><td>71630</td><td></td></tr><tr><td>Sodium citrate dihydrate</td><td>Sigma-Aldrich</td><td>W302600</td><td></td></tr><tr><td>Sodium pyruvate</td><td>Sigma-Aldrich</td><td>P2256</td><td></td></tr><tr><td>Spinning disk confocal scanning unit (50 &amp;mu;m pinholes)</td><td>Yokogawa</td><td>CSU-W1</td><td>For spinning disk confocal microscopy</td></tr><tr><td>Stereoscope</td><td>Leica</td><td>M80</td><td></td></tr><tr><td>TES</td><td>Sigma-Aldrich</td><td>T1375</td><td></td></tr><tr><td>Thermoformer</td><td>Yuyao Jintai Machine Factory</td><td>Jintai JT-18</td><td></td></tr><tr><td>Tissue culture dish (10 cm)</td><td>VWR</td><td>734-2321</td><td></td></tr><tr><td>Tris-HCl buffer</td><td>Roche Diagnostics</td><td>10812846001</td><td></td></tr></tbody></table>

preparation and live imaging of regenerating hydra spheroids

Preparation and Live Imaging of Regenerating Hydra Spheroids

Hydra is renowned for its exceptional regenerative capacity. Remarkably, small regenerating fragments of Hydra tissue, called Hydra spheroids, can establish an organizer de novo. Once established, this organizer directs the surrounding tissue to differentiate into the appropriate oral structures. Within days, the spheroid can develop into a small yet fully patterned polyp. The relatively short timescale of this process enables continuous time-lapse imaging of the entire process, offering a unique opportunity to study the spatiotemporal dynamics of regeneration. Here, we present a detailed workflow and show representative results for generating time-lapse datasets of regenerating Hydra spheroids. The protocol includes a detailed procedure for spheroid generation and guidelines for selecting optimal samples for imaging. We discuss mounting strategies suitable for the long-term imaging of live samples and demonstrate imaging approaches using both spinning disk confocal and light-sheet microscopes. The principles described here are broadly applicable to other model systems in which dynamic morphogenetic processes can be studied using long-term imaging.

We successfully generated long-term time-lapse datasets for regenerating Hydra spheroids using both spinning disk confocal microscopy (SDCM) and light-sheet fluorescence microscopy (LSFM). Regeneration occurred normally in both imaging setups: spheroids broke symmetry, elongated, and developed tentacles and a foot within 3 days (Figure 5A, Figure 6A, Supplementary Video 1, and Supplementary Video 2). After prolonged imaging, the regener...

Using time-lapse imaging, the complete regeneration process of Hydra spheroids can be followed. This protocol describes the generation of Hydra spheroids, discusses a variety of mounting strategies, and details how to perform long-term, live imaging using spinning disk confocal microscopy as well as light-sheet fluorescence microscopy.

Hydra is renowned for its exceptional regenerative capacity. Remarkably, small regenerating fragments of Hydra tissue, called Hydra spheroids, can ...

My research involves investigating the mechanisms of symmetry breaking in hydra spheroards. This protocol is broadly applicable to other model systems that also rely on long-term life imaging to study dynamic morphogenetic processes. To begin, using a glass pasteur pipette, move a hydra to a lid of a 10-centimeter culture dish containing hydra medium.Position it under a stereoscope. Bisect the hydra at body regions B2 and B3 by creating a single straight cut between them. Wait until the hydra is completely relaxed after the cut.The bisected tissue will relax while the cut-end will remain swollen. Make a second cut where the swelling ends to generate a ring of tissue. Generate tissue rings from each of the hydra halves.Remove the remaining head and foot-ends from the lid. Then cut the tissue rings open and cut the opened-rings into multiple rectangular tissue pieces of approximately 100 to 150 micrometers by 300 to 500 micrometers. Incubate the tissue pieces in room temperature dissociation medium for three to four hours.To mount the spheroids in agarose, first completely coat the bottom of a four-well glass bottom imaging plate with a two millimeter thick layer of 1%agarose. When the agarose has solidified, push the tip of a P1000 pipette into the agarose until it touches the bottom of the imaging plate. Hold the pipette straight and flush with the bottom.Then wiggle and rotate the tip to cut the agarose. Release the plunger and slowly remove the pipette tip straight up. Repeat the gel-cutting process until enough wells have been created.Then cover the agarose with a thin layer of hydra medium. Release any air bubbles in the agarose'wells by touching them with a P20 pipette tip. After incubation, assess whether the spheroids have properly folded by checking for a rounded shape and complete ectoderm coverage.Select folded spheroids with a diameter of 250 to 350 micrometers and move the selected spheroids to hydromedia before mounting. Next, move the hydra spheroids one-by-one into the imaging plate. If a spheroid does not immediately drop into a well, gently nudge it toward a well using the pipette.To mount in FEP wells, first fill the chamber with hydra medium. Release air bubbles from the wells using prepared glass pipettes with a scooping motion. Once all wells are free of bubbles, move the hydra spheroids one-by-one into the FEP chamber.Nudge spheroids into wells with a pipette if needed. Image the samples on an inverted microscope outfitted with a spinning disc unit. Set the microscope chamber temperature to 18 to 21 degrees Celsius.Choose an objective with a field of view that covers the complete sample. Then place the sample holder into the microscope stage. Turn on transmitted light and set the exposure time.Select the laser wavelength, laser power and exposure time for the fluorophores present. Find all samples and define them as multiple imaging positions. For each sample, create a Z-stack from the plane closest to the objective to the first out of focus plane.Set time lapse parameters with an interval of 10 minutes and start imaging. Alternatively, image the samples on a light sheet microscope with horizontally-positioned detection objectives. Set the microscope chamber temperature to 18 to 21 degrees Celsius.If that is not possible, cool the room instead. Place the sample holder into the microscope. Set all the required parameters as demonstrated previously and start imaging.Long-term time-lapse data sets of regenerating hydra spheroids were successfully generated using spinning disc confocal and light-sheet fluorescence microscopies. In both imaging setups, spheroids broke symmetry, elongated and developed tentacles and a foot within three days. After prolonged-imaging, regenerated animals became highly-modal and moved partially or completely out of the field of view.Using fluorescent markers, rapid reorganization of actin fibers was observed during hydra spheroid regeneration. Although only half of the spheroid was imaged, the region of interest containing the actin reorganization was successfully captured. Organizer formation was visualized by imaging beta-catenin green fluorescent protein expressing spheroids with light-sheet fluorescence microscopy.Three stages of regeneration were resolved, including elevated-nuclear beta-catenin across the spheroid, localized-nuclear beta-catenin enrichment and subsequent axis establishment with tentacle outgrowth. Fusion of the two opposing views acquired using light-sheet fluorescence microscopy produced a data set spanning the entire spheroid volume without apparent loss of image quality. These microscopy techniques allow researchers to create long-term life imaging data sets of their samples with minimal photo damage.It's important to choose the best suit in mounting and microscopy techniques for the intended results. Once image the data should be analyzed properly, there is many image analysis softwares or other methods that you can use.

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JoVE Journal

Developmental Biology

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Generation of Transgenic Hydra by Embryo Microinjection

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Learn live imaging of regenerating Hydra spheroids for developmental biology using spinning disk confocal and light-sheet microscopy. This protocol captures organizer formation and tissue patterning dynamics in vivo.

Preparation and Live Imaging of Regenerating Hydra Spheroids

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