The Institute for Genomic Biology

December 2008

Serial Block Face Imaging (SBFI) of corals using autofluorescence of zooxanthellae to create a 1micron-resolution digital elevation map of the tissue surface and polyp morphology

During the last year in the IGB core microscopy facility, a graduate student named Alan Piggot from the Department of Geology, who is working with his advisor Professor Bruce W. Fouke, has determined the three-dimensional distribution of zooxanthellae (zoox) and mucocytes in coral tissues. Zoox (the green spots, click on image for a high resolution image) are symbiotic dinoflagellates closely related to algae that began living in coral tissues 250 million years before present. Piggot studied the distribution and tissue density of zooxanthellae and mucocytes in corals as they respond to several different types of environmental and climatic change. The red cells are mucocytes loaded with mucin, which is expelled when the coral is stressed to create a protective covering from the environment. For the first time, Piggot has been able to reveal these details and quantify the zoox/mucocyte tissue density relationships using tiled images of two-dimensional cross sections at different depths within a single coral polyp (shown below). The results will be published next year in the Marine Biology Journal. However, this ground- breaking technique is limited in that some of the fine-scale three-dimensional details of the complex coral structure and zoox/mucocyte distributions are lost due to lack of coverage of the tissue in regions between individual histology sections. Coral polyps range from 1-3 mm in diameter and thickness, and thus it is impossible to process enough histology sections for complete coverage.

2D image of a single coral polyp, tiled image using Mosaic module in Zeiss Axiovision software in two channels (GFP and Cy5). The GFP channel showing the autofluorescence of zooxanthellae and the CY5 channel showing the mucus labeled with wheat germ agglutinin.

A different approach is to use all of the sections from the 1 mm to 3mm thick polyp.   This would be prohibitively time consuming if each section were mounted on a slide, labeled, imaged at high resolution, and aligned before reconstructing the complete polyp.  To shorten this process the autofluorescence from each section was imaged before the section was cut from the sample block, eliminating the mounting, labeling and aligning steps.  Using a Microtome, and Dissecting microscope combination in the Core Facility, 1500 2 um thick sections were imaged and cut in less than a day.  The samples (autofluorescing or fluorescently labeled) are mounted in a red wax formulation making the background opaque (Rosenthal et al., 2004). We have slightly modified the original technique to suit the current samples. Briefly, the paraffin wax in combination with 10% Vybar and 3% Stearin and the dye Sudan IV have been used to make a high melting point wax used for embedding the decalcified coral samples which were first processed in a tissue processor for routine histology.

Coral serial block face images at 1 micron resolution in Z
(Click on the images for high resolution images or a movie of volume rendered images in 3D).

3D Volume of multiple coral polyps side view showing XZ and YZ dimensions with intensity differences.

Click on the image above for a movie.

Gallery of individual sections of a sample polyp.

3D Volume of multiple coral polyps around 2500 images at 1 micron Z resolution.

Instrumental set up showing Serial Block Face Imaging with a Leica Microtome and Zeiss Fluorescence Stereoscope.  A home built XYZ stage was built to align the microscope with sample block at the top of the cutting cycle. The sample is exposed with a fluorescent light source and a band pass GFP filter with appropriate excitation and emission wavelength was used to collect the autofluorescence image of the zooxanthellae distribution.  The single images obtained at 1388x1040 dimensions at a resolution of around 2 microns in XY and 1 micron in the Z. The images were pooled in the program Zeiss Axiovision and the sections with debris were removed and then sequentially arranged, aligned and then 3D volume rendered in the program Bitplane Imaris version 6.2. To reveal the surface details and contours the images were surface rendered using the Isosurface mode in the Surpass module as well as combined with volume projection of all slices from single images in the range of 700-2500 microns in Z.

Literature cited:

Rosenthal J, Mangal V, Walker D, Bennett M, Mohun TJ, Lo CW. Rapid high resolution three dimensional reconstruction of embryos with episcopic fluorescence image capture. Birth Defects Research 72: 213-223 (2004).

Image courtesy and copyright- Alan Piggot, Bruce Fouke’s lab.