TITLE: Measuring Coral Skeletal Architecture Using Image Analysis
AUTHORS: Rowan Mclachlan, Ashruti Patel, Andrea Grottoli

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Coral morphology is influenced by genetics, the environment, or the interaction of both, and thus is highly variable. This protocol outlines a non-destructive and relatively simple method for measuring Scleractinian coral sub-corallite skeletal structures (such as the septa length, theca thickness, and corallite diameter, etc.) using digital images produced as a result of digital microscopy or from scanning electron microscopy. This method uses X and Y coordinates of points placed onto photomicrographs to automatically calculate the length and/or diameter of a variety of sub-corallite skeletal structures in the Scleractinian coral </span><span style = "font-style:italic;">Porites lobata. </span><span>However, this protocol can be easily adapted for other coral species - the only difference may be the specific skeletal structures that are measured (for example, not all coral species have a pronounced columella or pali, or even circular corallites). This protocol is adapted from the methods described in Forsman et al. (2015) & Tisthammer et al. (2018).</span></div><br/><div class = "text-block">There are 4 steps to this protocol:</div><div class = "text-block">1) Removal of Organic Tissue from Coral Skeletons </div><div class = "text-block">2) Imaging of Coral Skeletons</div><div class = "text-block">3) Photomicrograph Image Analysis</div><div class = "text-block">4) Calculation of Corallite Microstructure Size</div><br/><div class = "text-block">This protocol was written by Dr. Rowan McLachlan and was reviewed by Ashruti Patel and Dr. Andréa Grottoli.</div><br/><div class = "text-block"><span style = "font-weight:bold;">Acknowledgments</span></div><div class = "text-block">Leica DMS 1000 and Scanning Electron Microscopy photomicrographs used in this protocol were acquired at the Subsurface Energy Materials Characterization and Analysis Laboratory (SEMCAL), School of Earth Sciences at The Ohio State University, Ohio, USA. I would like to thank Dr. Julie Sheets, Dr. Sue Welch, and Dr. David Cole for training me on the use of these instruments.</div><br/><br/><br/><br/><br/></div>

[STEPS]
?. [Removal of Organic Tissue from Coral Skeletons ]
Remove coral fragments from the freezer (if applicable) and place them in individual 500 ml plastic beakers filled with deionized water. Soak coral fragments for 30 minutes at room temperature to remove excess salt and prevent NaCl precipitation.
?. [Imaging of Coral Skeletons]
The photomicrographs used in this protocol were produced using a Leica DMS1000 Microscope. For detailed instructions on how to use this instrument, please refer to the User Manual.
?. [Imaging of Coral Skeletons]
Multiple photomicrographs should be taken per individual coral skeleton at a variety of angles and magnifications (Fig. 2). First, take lower magnification images in order to visualize the gross morphology of the skeleton (Fig. 2 A).  Next, take medium-magnification images from above (i.e., dorsal view) in order to visualize individual corallites (Fig. 2 B). Finally, take multiple high-magnification images to visualize sub-corallite structures (Fig. 2 C, D). Specific magnification levels vary and will depend on the size of coral fragments and the size of corallites. The angle of the skeleton relative to the microscope should be adjusted between high-magnification photomicrographs as later image-analysis measurements can only be conducted on corallites that were photographed perfectly perpendicular to the field-of-view rather than at an angle.
?. [Removal of Organic Tissue from Coral Skeletons ]
Using plastic forceps, transfer coral fragments to new 500 ml beakers filled with ~ 200 ml of 3% sodium hypochlorite bleach (Fig 1A). Take care not to damage fragile corallite micro-skeleton. Soak for 1 hour at room temperature.
?. [Removal of Organic Tissue from Coral Skeletons ]
Replace liquid contents of the beakers with fresh bleach solution and arrange beakers in a sonicator bath (Fig. 1B). Sonicate for approximately 30 minutes at room temperature.
?. [Removal of Organic Tissue from Coral Skeletons ]
After sonication, visually inspect skeletons. If the coral skeletons are completely white and devoid of tissue, proceed to the next step (Step 5). However, if tissue is still visible (i.e., the coral surface is light brown or green) then sonicate skeletons in fresh bleach solution for an additional 30 minutes (or longer) until all tissue disappears.
?. [Removal of Organic Tissue from Coral Skeletons ]
Drain and dispose of bleach, transfer coral skeletons into individual pre-labeled aluminum weighing pans, and dry overnight at 60 °C in a drying oven (Fig. 1C).
?. [Photomicrograph Image Analysis]
Download ImageJ. ImageJ can be downloaded for free from the NIH website: https://imagej.nih.gov/ij/download.html.
?. [Photomicrograph Image Analysis]
After setting the scale, zoom out again (Fig. 5A), select an appropriately angled corallite, and then zoom in such that a single corallite fills the entire ImageJ window (Fig. 5B).
?. [Photomicrograph Image Analysis]
After selecting an appropriately-angled corallite, next, identify the dorsal directive septa (ubiquitous among Porites corals) (Fig. 7). Rotating clockwise from the dorsal directive are lateral pair 1, lateral pair 2, the ventral triplet, lateral pair 3, and finally, lateral pair 4.
?. [Photomicrograph Image Analysis]
Make sure to only select corallites which are perpendicular to the field-of-view (Fig. 6). If the cupped-base of the corallite is at an angle relative to the field-of-view, then measurements made from images will not be accurate. You will notcie that in Fig. 6A, only the central corallite is appropriately angled, whereas those around it are all tilted.
?. [Photomicrograph Image Analysis]
Open one high-magnification photomicrograph in ImageJ (Fig. 3).
?. [Photomicrograph Image Analysis]
Set the scale. Using the hand tool and the zoom function (zoom shortcuts: Ctrl and + on PC keyboard, or left-click and scroll on PC mouse), zoom in to the upper left corner of the photomicrograph where the scale bar is located (Fig. 4A). Using the "Straight" tool (fifth button from the left), carefully select the entire 1 mm scale bar (Fig. 4A). Once the scalebar is selected, go to "Analyze" followed by "Set Scale" (Fig. 4A). In the Set Scale window, type "1" into the "Known Distance" field, and type "mm" into the "Unit of length" field (Fig. 4B).
?. [Photomicrograph Image Analysis]
Select the "Multi-point" tool (seventh button from the left) (Fig. 8A). Place two points on top of the image. The first point should be placed at the outer end of the dorsal directive septa, and the second point on the inner end of the dorsal directive septa (Fig. 8B). These points will be used to calculate septal-length. Notice in Fig. 8 the small blue numbers next to each point (you may need to enlarge the image in order to see these numbers).Take care when placing these septa end-point and try to be consistent within corallites, among corallites, and among different skeleton samples. One method we used to improve consistency was to use the negative space (black holes) as a guide (Fig. 9). Notice in Fig. 9 how the blue point #1 is placed between the yellow arrows which align with the skeleton holes between septa. If the blue point had instead been placed where the red cross symbol is, it would have been too far out towards the theca wall. Conversely, if the blue point had instead been placed where the orange cross symbol is, it would have been too far inside the calyx area. Likewise, for the placement of point #2, we used the ring of the fossa (purple circle in Fig. 9) as a guide and tried to place point 2 on, or as close to this ring as possible.
?. [Photomicrograph Image Analysis]
Repeat this process for all 12 septa (proceeding clockwise from the dorsal directive) until points #1 through #24 have been placed onto the photomicrograph (Fig. 10).
?. [Photomicrograph Image Analysis]
Next, place an additional 24 points on the photomicrograph on either side of each septa, located at the midpoint (i.e., halfway between the endpoints) (Fig. 11A). These points will be used to calculate septal-width. Proceed clockwise from the dorsal directive septa until points #25 through #48 have been placed (Fig. 11B).
?. [Photomicrograph Image Analysis]
Next, place an additional 12 points on the photomicrograph opposite from the septa outer-edge points (Fig. 12A). These points should be placed on the inner edge of the theca walls of neighboring corallites (Fig. 12A). The distance between septa outer-edge points and these points will be used to calculate the thickness of the theca wall. Proceed clockwise from the dorsal directive septa until points #49 through #60 have been placed (Fig. 12B).
?. [Photomicrograph Image Analysis]
Next place point #61 at the center of the primary corallite (Fig. 13 green text). Following this, place one point in the center of all the neighboring corallites (only those which share a thecal wall with the primary corallite (Fig. 13 yellow text). The number of neighboring corallites will vary depending upon the individual (anywhere from 4–8 in my experience).
?. [Photomicrograph Image Analysis]
Next, navigate to the toolbar, press "Analyze" followed by "Measure" [keyboard shortcut is Ctrl + M] (Fig. 14A). This will bring up a Results window of the X and Y coordinates for all 67 points on the photomicrograph (Fig. 14B). Copy and paste these values into an excel spreadsheet.
?. [Calculation of Corallite Microstructure Size]
There are a variety of different measurements that can be made using this data set of X and Y coordinates. Table 1 summarizes which points can be used to calculate the length and width of various corallite microstructures. This is not an exhaustive list, however, and other measurements are possible with the placement of additional points (e.g., pali area, columella diameter, etc.) or from observations (e.g., number of radii, the shape of the ventral triplet, etc.). Please see Forsman et al. (2015) or Tisthammer et al. (2018) for such examples. DescriptionPointsDescriptionPointsDescriptionPointsSepta length 11:2Septa width 125:26Septa spacing 11:3Septa length 23:4Septa width 227:28Septa spacing 23:5Septa length 35:6Septa width 329:30Septa spacing 35:7Septa length 47:8Septa width 431:32Septa spacing 47:9Septa length 59:10Septa width 533:34Septa spacing 59:11Septa length 611:12Septa width 635:36Septa spacing 611:13Septa length 713:14Septa width 737:38Septa spacing 713:15Septa length 815:16Septa width 839:40Septa spacing 815:17Septa length 917:18Septa width 941:42Septa spacing 917:19Septa length 1019:20Septa width 1043:44Septa spacing 1019:21Septa length 1121:22Septa width 1145:46Septa spacing 1121:23Septa length 1223:24Septa width 1247:48Septa spacing 1223:1Theca thickness 11:49Corallite width 11:13Corallite spacing 161:62Theca thickness 23:50Corallite width 23:15Corallite spacing 261:63Theca thickness 35:51Corallite width 35:17Corallite spacing 361:64Theca thickness 47:52Corallite width 47:19Corallite spacing 461:65Theca thickness 59:53Corallite width 59:21Corallite spacing 561:66Theca thickness 611:54Corallite width 611:23Corallite spacing 661:67Theca thickness 713:55Fossa width 12:14Corallite spacing 761:68Theca thickness 815:56Fossa width 24:16Corallite spacing 861:69Theca thickness 917:57Fossa width 36:18Theca thickness 1019:58Fossa width 48:20Corallite Area1:3:5:7:9:11:13:15:17:19:21:23Theca thickness 1121:59Fossa width 510:22Fossa Area2:4:6:8:10:12:14:16:18:20:22:24Theca thickness 1223:60Fossa width 612:24
DescriptionPointsDescriptionPointsDescriptionPointsSepta length 11:2Septa width 125:26Septa spacing 11:3Septa length 23:4Septa width 227:28Septa spacing 23:5Septa length 35:6Septa width 329:30Septa spacing 35:7Septa length 47:8Septa width 431:32Septa spacing 47:9Septa length 59:10Septa width 533:34Septa spacing 59:11Septa length 611:12Septa width 635:36Septa spacing 611:13Septa length 713:14Septa width 737:38Septa spacing 713:15Septa length 815:16Septa width 839:40Septa spacing 815:17Septa length 917:18Septa width 941:42Septa spacing 917:19Septa length 1019:20Septa width 1043:44Septa spacing 1019:21Septa length 1121:22Septa width 1145:46Septa spacing 1121:23Septa length 1223:24Septa width 1247:48Septa spacing 1223:1Theca thickness 11:49Corallite width 11:13Corallite spacing 161:62Theca thickness 23:50Corallite width 23:15Corallite spacing 261:63Theca thickness 35:51Corallite width 35:17Corallite spacing 361:64Theca thickness 47:52Corallite width 47:19Corallite spacing 461:65Theca thickness 59:53Corallite width 59:21Corallite spacing 561:66Theca thickness 611:54Corallite width 611:23Corallite spacing 661:67Theca thickness 713:55Fossa width 12:14Corallite spacing 761:68Theca thickness 815:56Fossa width 24:16Corallite spacing 861:69Theca thickness 917:57Fossa width 36:18Theca thickness 1019:58Fossa width 48:20Corallite Area1:3:5:7:9:11:13:15:17:19:21:23Theca thickness 1121:59Fossa width 510:22Fossa Area2:4:6:8:10:12:14:16:18:20:22:24Theca thickness 1223:60Fossa width 612:24
?. [Calculation of Corallite Microstructure Size]
In addition to distances between points, the area of polygons constructed from multiple points can also be calculated. For instance, Corallite Area or Fossa Area (cm^2) can be calculated using the following equation:
?. [Calculation of Corallite Microstructure Size]
To calculate the distance between two points, use the following formula:
?. [Calculation of Corallite Microstructure Size]
EXAMPLE: To calculate the length of the dorsal septa (aka "Septa Length 1") we will use the X and Y coordinates of Point #1 and Point #2 (recall Table 1) which were measured and are shown in Figure 14.Point 1 (X = 2.869, Y = 2.914)Point 2 (X = 3.248, Y = 2.991)Next, using the equation shown in Step 21:Septa Lenth 1 =  √ [ (3.248 – 2.869)2 + (2.991 – 2.914)2 ]Next, using the equation shown in Step 21:Therefore, Septa Length 1 = 0.387 cm