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

A custom designed SBFI apparatus (manufactured specifically for the present study; Figure 3) produced the first detailed 3D digital elevation maps (DEMs) of the outer surface texture and morphology of the M. annularis and M. faveolature coral polyps (Figure 4 and SI Videos 1-2). This yielded images of previously undescribed stacked lobes of coral tissue concentrically radiating outward from the center of each polyp (Figures 4B, 4D, and 4E). These lobes are stacked along the uppermost ridge of tissue-covered primary and secondary skeletal septa, with the radially outermost and lowermost elevation lobes eventually synthesizing and merging with coenosarc tissues between polyps. The 3D polyp shape and the associated lobes were well preserved despite the coral tissue sample having gone through several steps of high-temperature infiltration and embedding. The addition of Sudan dye successfully masked the background as opaque, with the black color having helped to maximize the contrast of each sample and increase the signal-to-noise ratio. 3D and orthogonal plane views showed the distribution of fluorescent components along the z-axis (Figure 4B). There were planes with wax flakes that sometimes obscured polyp surface detail, which were removed for clarity when creating projections. The major advantage of this technique is in revealing internal details of any thick sample, thus eliminating the need to take multiple sections and align them at a later time. Further, the images in this technique are already aligned when collected (Figure 4A), providing a z resolution of up to 1 μm from a sample size of up to 10 mm or more (depending on the total drive distance of the block holder in the microtome). It is possible to discriminate different auto-fluorescent components (using multiple filters) as well as individual zooxanthellae at higher magnification, provided the reduced field of view is acceptable.

The ApoTome optical sectioning fluorescence microscope revealed the distribution of coral tissues cells (zooxanthellae and chromatophores) and surface tissue structure at a submillimeter resolution. Hence, the 6-13 μm diameter zooxanthellae could be easily recognized and quantified with respect to number of cells per tissue volume (Figure 5). The dual channel image acquisition using the chlorophyll fluorescence (green) and the mucus labeled with WGB Alexa 647 (shown in red), worked best to quantify the level of mucus and the number of zooxanthellae within a given location of the polyp (major and minor septa of polyp could be calculated independently).

In addition to these microscope approaches, we have also applied TPLSM, a nonlinear technique widely used for thick biological samples. The major advantages of TPLSM over one-photon continuous wave lasers are that: (1) excitation occurs only at the point of focus because the power of the excitation light intensity becomes squared and the chance of a molecule to absorb two consecutive photons to excite one electron from the fluorophore is limited to the depth of field of the objective, thus providing an inherent confocality. and (2) as a consequence there will be no loss of an excitation photon while penetrating deeper into the sample, as opposed to single photon lasers. In addition, most biological tissue samples also absorb visible light. Since the two-photon excitation is inherently long (in near infrared at 780 nm), the absorption is also substantially minimized. On the other hand, the coral skeleton composed of calcium carbonate barely absorbs or scatters the two-photon light. Finally, since we were interested in determining the distribution of objects only on the contoured surface of the coral skeleton, we found deeper imaging using this modality very suitable for coral imaging.

We have attempted to characterize the available spectral signatures in M. annularis (Figures 6-9). Here we show that under the 780 nm two-photon excitation, the chromatophore auto-fluorescence has a peak around 500 nm, while the chlorophyll fluorescence from zooxanthellae is centered at approximately 675 nm (Figure 6). While unmixing of this spectral data (Figures 6-7) is also possible with an unmixing algorithm in the software (Figure 8), since there is a clear spectral distance between the two components, imaging them simultaneously using two detectors at two emission bands proved to be most time saving technique. This is especially true when the sample is tiled and imaged over an entire 1-2 mm depth of tissue. This eliminates the need for physical sectioning, which requires several thousand images per location and sample and polyp. The distribution of auto-fluorescent chromatophores is also distinct (Figure 9) among the two coral species tested. In one case, we observed a significant increase of chromatophores in the shallower-water dwelling coral M. annularis compared to deeper water M. faveolata (Figure 9 and SI Videos 3 and 4).

Figure 1. (A) Map of Curaçao in the southern Caribbean Sea, showing the location of the study site at Playa Kalki on the far northwestern leeward coast of the island. (B) Schematic cross-section of the reef-rimmed shelf at Playa Kalki on Curaçao, showing the water depth distributions of M. annularis and M. faveolata. Please click here to view a larger version of this figure.

Figure 2. (A) In situ underwater field photograph at 12 m WD of M. faveolata at Playa Kalki, Curaçao. (B) Close up enlargement of the image in (A) showing the individual coral polyps of M. faveolata in daytime lighting conditions (each polyp is approximately 2 mm in diameter, some of which are retracted, denoted by *). Please click here to view a larger version of this figure.

Figure 3. The serial block-face imaging instrumentation. (A and B) The custom designed microscope holder, holds the Stereolumar microscope at 180° facing the block face of the sample placed in the microtome. When each section was removed, the block face image was captured with the objective and the 2D data was digitally stored. This was done until the sample disappeared in the block. The microscope is moved in x and y direction using a motorized lead screw and the z, adjustment is made to focus the sample. It is important to note that when each 1 μm sample is sectioned, the block moves 1 μm forward, so refocus of the scope is not necessary. XYMOT, Custom built xy translational motorized stage; FLS, Fluorescence light source; MT, Microtome; MIC, Stereomicroscope; CAM, Axiocam monochrome camera; BH, Block holder; BF, Block face of the sample; FLL, Fluorescent light excitation spot on the block face; OBJ, Microscope objective; HIP, Human interface panel. One image at the dimension of 1,388 horizontal x 1,040 vertical pixels in monochrome mode was collected at a xy pixel (single) resolution of 1.25 μm. Please click here to view a larger version of this figure.

Figure 4. Serial block face imaging of the corals M. annularis and M. faveolata collected from Playa Kalki, Curaçao. (A) Sample data set gallery showing around 800 images of individual 2D images obtained from a polyp at 1 μm resolution in z. (B) In another coral sample, the sectioning went well over 2,000 μm in z. The image is showing multiple polyps and compilation of all 2D slices in to a 3D volume under shadow projection using the Imaris volume visualization algorithm. (C) Orthogonal projection showing an xz view of the sample in (B), one can see the entire depth of the sample over 2 mm and the arrows indicate the black lines where 2D slices were removed due to poor quality; this image is from M. annularis. 3D volume (D), Isosurface rendering (E) and visualization of the SBFI data. (D) Multiple coral polyps showing the polyp morphology in longitudinal section on the side and the 3D volume (shadow projection) of a single polyp in the middle. Sectioning and visualization at any angle is possible in 3D (see SI video). E. Since voxels are fuzzy in 3D (D), the 3D Isosurface rendered surface shows the contour of the coral polyp surface and their contextual arrangement with adjacent polyps (see SI video). Please click here to view a larger version of this figure.

Figure 5. Fluorescence microscopy of coral polyps. (A) Cross section of a decalcified polyp of M. faveolata exhibiting zooxanthellae with chlorophyll autofluorescence under blue light (shown in green) and mucosal pockets labeled with wheat germ agglutinin (shown in red) as imaged under red light. The image is automatically tiled in two channels and stitched and aligned using the instrument software. (B) Enlargement of the box shown in (A), showing individual zooxanthellae (each green circles, approximately 6-8 μm in diameter in green) and the surface mucus layer (red), and the merge of both channels. Please click here to view a larger version of this figure.

Figure 6. Two-photon spectral characterization of fluorescence from corals. Using the spectral detector of the LSM 710 system, fluorescence signals from the entire visible spectrum (419-722 nm) were scanned over a depth of around 190 μm. The image shows the depth interval at 5 µm in x axis for the z stack and the y axis is wavelength from 419-722 nm acquired at 10 nm interval (a total around 1,154 images showed). One can see the two spectral components, one centered at 500 nm and the second at 650 nm. A two-photon laser excitation wavelength of 780 nm was used to obtain the emission profiles. Please click here to view a larger version of this figure.

Figure 7. Characterization of the two spectral components. (A) The spectral profile of background (blue), autofluorescence from chromatophores (green) and chlorophyll fluorescence (red) excited by the two photon laser excitation wavelength of 780 nm laser. The spectra derived shown at the right side of the multicolor spectral data overlaid and pseudo-colored according to the emission profile. (B) The individual spectral bins where the signals are collected at 10 nm interval to derive the spectra and the image in (A). Note that the two distinct components, one centered at approximately 500 nm (chromatophore autofluorescence) and the other over 675 nm (chlorophyll fluorescence). Please click here to view a larger version of this figure.

Figure 8. The same images in Figures 7A and 7B, spectral data unmixed using the software and their corresponding emission peaks/spectra. In both M. annularis and M. faveolata, the spectral components are very similar, as well their emission peaks. The significant difference, however, is due to the location of these spectral components and their abundance (see Figure 9). Please click here to view a larger version of this figure.

Figure 9. Two-photon fluorescence 3D images of coral polyps from M. annularis (A-D) and M. faveolata (E-H). (A and E) were imaged with no spectral separation of components using 780 nm excitation of the two-photon laser, with the optical signal collected from 450-700 nm. (B and F) were collected with the same excitation (780 nm), but two PMT detectors were used simultaneously to collect the data from two components i.e., one from 500-550 nm (green-chromatophore autofluorescence) and the other 650-720 nm (red-Chlorophyll fluorescence). (C and G) are depth coded images of (B and F), red is shallowest and blue is deepest components in 2D. (D and H) are yz images of cut through (B and F) respectively, showing the fluorescence is coming only from the surface of the coral and the skeleton does not have any fluorescence at 780 nm excitation. Note the substantial difference in chromatophore content between the two corals. The M. annularis habitat (0-10 m WD) is shallower than the M. faveolata habitat (10-20 m WD). Please click here to view a larger version of this figure.

Figure 10. The Powers of Ten hierarchical spatial structure of studies of coral reef ecosystems. Please click here to view a larger version of this figure.

SI Figure 1. Screen shots showing steps (A-I) involved in the creation of 3D volume and isosurface rendering from either the SBFI data or the 2Photon microscopy using the image analysis software (see Materials Table). (A) the 2D slices of the raw data in slice mode showing a single plane; (B) 3D volume of all the 2D slices; (C) the isosurface creation wizard with a region of interest (ROI) selected; (D) an algorithm with a background subtraction selected; (E) a threshold value applied to pick up the pixels which represent the data; (F) center seed points of the surface; (G) a volume based filter is applied to eliminate smaller volume of debris and noise; (H) the final 3D rendered isosurface; (I) key frame animation wizard to create the movies as in the SI videos. The final rendered images are shown in both volume and isosurface modalities in the SI Videos 3-7. The protocol steps 3.1.3-3.1.5 covers these procedures. Please click here to view a larger version of this figure.

Table 1. Key materials, equipment, microscopes, and software used in this study.

SI Video 1. In situ underwater field video showing the coral habitat at 12 m WD of M. faveolata at Playa Kalki, Curaçao.

SI Video 2. Individual 2D serial block face images of the 3D rendered image shown in Figure 4E.

SI Video 3. Isosurface 3D rendered serial block face image presented in Figure 4E and SI video 1 showing the topography of the coral polyp.

SI Video 4. 3D volume rendered raw data two-photon microscopy image of the coral polyp M. faveolata. The excitation is 780 nm and the emission captured simultaneously at two band widths for zooxanthellae (pseudo-colored red, 600-700 nm) and chromatophores (pseudo-colored green, 500-550 nm).

SI Video 5. 3D volume rendered in Isosurface-spots two-photon microscopy image of the coral polyp M. faveolata. The excitation is 780 nm and the emission captured simultaneously at two band widths for zooxanthellae (pseudo-colored red, 600-700 nm) and chromatophores (pseudo-colored green, 500-550 nm).

SI Video 6. 3D volume rendered raw data two-photon microscopy image of the coral polyp M. annularis. The excitation is 780 nm and the emission captured simultaneously at two band widths for zooxanthellae (pseudo-colored red, 600-700 nm) and chromatophores (pseudo-colored green, 500-550 nm).

SI Video 7. 3D volume rendered Isosurface-spots two-photon microscopy image of the coral polyp M. annularis. The excitation is 780 nm and the emission captured simultaneously at two band widths for zooxanthellae (pseudo-colored red, 600-700 nm) and chromatophores (pseudo-colored green, 500-550 nm).