Aug 21, 2025
Quantifying RGB-Based Color Score in Corals
- Serena Hackerott1,2,
- Lauren Gregory2,
- John Howard2,
- Jose Eirin-Lopez2
- 1University of Delaware;
- 2Florida International University
Protocol Citation: Serena Hackerott, Lauren Gregory, John Howard, Jose Eirin-Lopez 2025. Quantifying RGB-Based Color Score in Corals. protocols.io https://dx.doi.org/10.17504/protocols.io.14egnr3wpl5d/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
This protocol has been evaluated in Hackerott et al (2025), published in Coral Reefs.
Created: August 18, 2025
Last Modified: August 21, 2025
Protocol Integer ID: 224903
Keywords: color score in coral, quantitative color score protocol, based color score, color score, quantifying rgb, coral, coral reef, bleaching severity, blue intensity
Abstract
This protocol describes the steps required for photo capture, image processing, and data analysis to quantify color scores based on standardized red, green, and blue intensities, which can be used as metrics of coral bleaching severity. This is the quantitative color score protocol evaluated in Hackerott et al. (2025), published in Coral Reefs.
Guidelines
To maximize comparability, use the same color standards (e.g., same rolls of red, green, and blue electrical tape) across measurement sessions (e.g., sites, timepoints, species, etc.).
Include comparison groups (e.g., control vs treatment, pre- vs post-heating) to evaluate color changes as a metric of bleaching severity.
While color scores are quantitative, they are also relative. Therefore, it is important to conduct the PCA-LDA approach on the entire dataset in which comparisons will be made (e.g., comparing bleaching severity across species or sites).
This specific method was validated using Acropora cervicornis, staghorn corals, under artificial lighting on land (described in Hackerott et al, 2025). Use with other species or under other conditions may require additional validation.
Materials
- Digital camera
- White background (e.g., dive slate)
- Red, green, and blue color standards (e.g., electrical tape)
- Computer with ImageJ software
- Analysis pipeline datafiles and scripts
Troubleshooting
Safety warnings
This specific method was validated using Acropora cervicornis, staghorn corals, under artificial lighting on land (described in Hackerott et al, 2025). Use with other species or under other conditions may require additional validation.
Before start
To maximize comparability, use the same color standards (e.g., same rolls of red, green, and blue electrical tape) across measurement sessions (e.g., sites, timepoints, species, etc.).
Include comparison groups (e.g., control vs treatment, pre- vs post-heating) to evaluate color changes as a metric of bleaching severity.
While color scores are quantitative, they are also relative. Therefore, it is important to conduct the PCA-LDA approach on the entire dataset in which comparisons will be made (e.g., comparing bleaching severity across species or sites).
Photo Capture
Place the coral fragment on the white background with the RGB color standards visible. Ideally, position the coral to maximize the fragment surface area that is visible.
Avoid shadows or glares on the coral or the color standards. It is critical that the coral fragment is under the same light conditions as the color standards.
Limit water droplets on the color standards.
Capture the photo from in front of the coral fragment. If bleaching is occurring in patches, it may be appropriate to capture photos from additional angles or faces for an averaged color score across the full fragment.
Image Processing
Open the ImageJ software. Select File > Open and select the first image to be processed.
Select Analyze > Histogram and a third window with the intensity histogram will appear. Select the Live button to activate.
Use the cursor to select a square that encompasses most of the red color standard tape. Click the RGB button until the intensity spectrum being shown is Red only. Record the mean value as the Red Standard value for that photo.
Repeat Step 7 for the green and blue color standard tapes, making sure to click the RGB button to record the corresponding color intensity (i.e., green intensity for the green standard, blue for blue).
To measure the coral, select the Freehand Selections button. Trace the entire coral fragment. Avoid including borders, shadows, and non-coral sections (e.g., crimps, monofilament, plugs, etc.). It is better to be slightly inside the border of the fragment rather than including outside border colors (such as the white background) or shadowed edges in the measurement area.
After the entire fragment is traced, click through the RGB options and record the mean red, green, and blue intensities as the Red, Green, and Blue Coral values.
Data Analysis
Calculate the standardized colors for each coral by dividing the Coral value by the corresponding Standard value (e.g., Red Coral / Red Standard).
Obtain the color score using the PCA-LDA approach detailed in this GitHub repository. Briefly, perform a PCA on the standardized colors, then use PC 1, 2, and 3 as predictors of the treatment group (e.g., control vs heated, baseline vs bleaching, etc.). Save the LD1 scores as the quantitative color score.
Color scores may need to be inverted in their signs (* -1) for intuitive interpretation (i.e., control > heated) and an adjuster constant may need to be added to make all scores positive (e.g., add 20 to all scores).
Color scores can then be visualized and analyzed using similar methods as those used for other bleaching metrics (e.g., cell density, chlorophyll concentration, etc.) to compare levels of bleaching severity between treatment groups.
Protocol references
Hackerott, S., Gregory, L.E., Howard, J.M. et al. Picture of health: evaluating an accessible method for quantifying coral thermal tolerance using photographic color analysis. Coral Reefs 44, 1327–1340 (2025). https://doi.org/10.1007/s00338-025-02686-x
Edmunds, P.J., Gates, R.D. & Gleason, D.F. The tissue composition of Montastraea franksi during a natural bleaching event in the Florida Keys. Coral Reefs 22, 54–62 (2003). https://doi.org/10.1007/s00338-003-0278-5
Emma L. Strand, Kevin H. Wong, Alexa Farraj, Sierra Gray, Ana McMenamin, Hollie M. Putnam; Coral species-specific loss and physiological legacy effects are elicited by an extended marine heatwave. J Exp Biol 1 June 2024; 227 (11): jeb246812. doi: https://doi.org/10.1242/jeb.246812
Acknowledgements
This material is based upon work supported by the National Science Foundation under Grant No. HRD-1547798 and Grant No. HRD-2111661. These NSF Grants were awarded to Florida International University as part of the Centers of Research Excellence in Science and Technology (CREST) Program. Additional support was also provided by NSF grant 1921402 (JEL). This work was also funded by Iberostar Wave of Change Rebuilding Coral Reefs Scholarship, American Museum of Natural History Richard Gilder Graduate School Lerner Gray Memorial Fund for Marine Research, Friends of Gumbo Limbo Graduate Research Grant, Judith Evans Parker Travel Scholarship, Florida International University Women Explorers Award, and support by Florida International University’s Institute of Environment, CREST Center for Aquatic Chemistry and the Environment, and University Graduate School awarded to SH. We would like to thank Francesca Virdis of Reef Renewal Foundation Bonaire (RRFB) along with RRFB and Buddy Dive Resort staff for valuable logistical support.