Multimodal Optical Microscopy Methods Reveal Polyp Tissue Morphology and Structure in Caribbean Reef Building Corals

The overall goal of the following experiment is to image one to two millimeter thick coral polyps in 3D to understand the fine structure as well as the coral pigments and their localization. This is achieved by first embedding the sample in wax, then cut the sample into one micron sections using a microtome. Next, a custom designed serial block face technique is used to image the coral polyps and reconstruct the 3D image.

Using multiple 2D images, results are obtained that show the ability of the technique to resolve fine structural details of the coral tissue without compromising its structural integrity. Demonstrating the procedure. Today will be Dr.Maunde Siva guru, a collaborator with my research group.

To prepare the pre-filtration wax melt 3.6 grams of steer in flakes in a glass beaker on a hot plate. Next, add 400 milligrams of Sudan. Four to minimize the wax background fluorescence, mix well and wait until a red translucent solution is achieved.

Then add 81 grams of paraffin and mix well. Subsequently, add 15 grams of white granular VI bar and melt completely in the same beaker. Once melted, mix it again to get a completely mixed wax solution.

Now add the mixture to a glass bottle and close loosely with a lid. Place the glass bottle in a 60 degree Celsius convection oven to keep the ingredients in a liquid state and for all the infiltrations to take place. This solution can be made to 200 milliliters and split into two 100 milliliter bottles.

One for the infiltration and one for final embedding Caribbean reef building coral samples were collected from AU and fixed in para formaldehyde in the field. To embed the coral tissues for SBFI first wash the coral polyps collected in the field and store them at four degrees Celsius in para formaldehyde. Afterward, wash them in PB S3 times for five minutes each time when ready to be imaged.

Decalcify the polyps in ex Cal two solution for 24 hours or until the polyps are totally devoid of calcium carbonate. Incubate several decalcified coral polyps as a single block in a 25, 50, 75, and 100%ethanol series followed by one x xylene. Substitute for 30 minutes each session to dehydrate the samples.

Next place the processed polyps in 100%xylene. Substitute in a 65 degree Celsius oven for 30 minutes. Afterward, replace with fresh solution and incubate for another 30 minutes.

Orient the polyps with the tops facing down and the surface as flat as possible. Make solutions of two to one, one to one, and one to two. Xylene substitute and pre-filtration wax.

In 50 milliliter Falcon tubes incubate the coral polyps with these three increasing concentrations of pre-filtration wax, followed by three incubations in 100%Pre-filtration wax for 30 minutes to one hour each time. Now transfer the sample to the embedding wax. Remove the embedding wax after 30 minutes, then replace with fresh embedding wax and continue the incubation for a minimum of four hours at 65 degrees Celsius.

Afterward, photograph the block. This is necessary because once embedded in the wax, the location of the sample will become invisible as the embedding red wax is opaque. Next place small drops of high melting point wax around the new preheated stainless steel embedding mold and allow it to cool.

Pour a small volume of freshly melted and bedding wax from the second aliquot and position the coral polyp facing down over the white wax dot quickly. Then place a plastic sample holder on the stainless steel tray and pour more embedding wax so that the wax comes up to the surface of the plastic mold. Now take the entire setup out of the 65 degrees Celsius oven and allow it to cool on a bench or a cool surface until the wax completely hardens.

After that, place the desiccated block in a refrigerator at four degrees Celsius, protected from light for long-term storage to section the block. First, trim it and cut one micron sections. Using a microtome capture the image of the smooth block face, which contains the sample.

Every time. When a section is removed, the sample appears when the white wax begins to disappear. Then capture the images with a monochrome camera using a fitzy fluorescent filter to pick up the autofluorescence of the chromatophores or coral polyp image, three to four decalcified cores from each sample.

To perform two photon fluorescence imaging, place the washed coral polyp cores in 4%para formaldehyde upside down in a covered glass bottom dish using a two photon laser at 780 nanometers excitation image three to four polyps at two different magnifications using a 10 x objective. Next, use the tile scan mode to collect approximately 25 to 100 images per focal plane in XY at 10 or 20 micron intervals. Then collect 50 to 100 images through the Z axis at 10 or 20 micron intervals, which will be around 5, 000 to 10, 000 images total per coral polyp.

After that image, three to four polyp areas to represent a core and coral species. Then store all images in raw data format on the system's hard disc. In this procedure, crop the 2D data to reduce the file size by focusing on a single polyp using the square cropping tool and compile it as a single TIF file.

In the acquisition software. Open the assembled files of the SBFI data or the tiled multiple Z stacks of two photon optical sections in the EMERIS surpass program module under volume algorithm project, the SBFI data rendered in 3D. Using a shadow projection, create an ISOSURFACE mode where the voxels are threshold to create a solid surface pattern.

Visualize the 3D projections using a clipping plain algorithm at XY xz and YZ orthogonal modes to reveal the 3D structure and shape of the corals. Then animate the projections using a key frame animation module. In the same erris program, generate video files at 5%compression and generate movie clips in a VI format using volume 3D and isosurface modalities.

Here are the two photon fluorescence 3D images of coral polyps from M Anis and M fa Lata. These two images were captured with no spectral separation of components using 780 nanometer excitation of the two photon laser with the optical signal collected from 450 to 700 nanometers, and these two images were collected with the same excitation, but two photo multiplier tube detectors were used simultaneously to collect the data from two components, one from 500 to 550 nanometers and the other from 650 to 720 nanometers. These are depth coated images.

Red are the shallowest and blue are the deepest components in 2D.Yeah. While attempting this procedure, it is important to remember to prepare the red wax embedded sample properly. Combination of serial block face imaging and two foot on fluorescent microscopy allows analysis of coral pigmentation and cellular structure on a variety of lens scales.

After watching this video, you should have a good understanding of how to handle and image reef building corals and their spectral properties in three dimensions.