Preparation and Live Imaging of Regenerating Hydra Spheroids

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.