ColorBones: Full step-by-step protocol for the visualization and identification of bone discolorations using the ImageJ© plugin DStretch® v1
Authors: Göldner Dominik 
Published: November 2023

Fresh, undecayed, and healthy bone in animals and humans typically exhibits a greasy, yellowish-white to yellowish-brown color (Dupras and Schultz, 2013). However, their color can undergo alterations, especially in archaeological or forensic contexts, when exposed to environmental factors. In such cases, bones may display various types of discoloration. These color changes can be attributed to different taphonomic agents during the postmortem deposition of the deceased body. Still, they can also occur during the organism's lifetime and under specific conditions, such as medical treatment or nutrition. Depending on the cause, bone staining can be superficial, but it can also penetrate deeper into the bone structure. Discoloration may be confined to a single bone, parts of it, or it can extend over multiple bones (e.g., in joint areas) or even the entire skeleton. Additionally, discolorations can be (more or less) monochromatic or polychromatic, depending on the number of taphonomic events causing staining, their sequence, and the physicochemical agents involved. Hence, diverse coloration processes can either conceal previously present colors or collaboratively create new color combinations. Furthermore, different processes may result in similar discolorations, complicating the making of a differential diagnosis regarding their origin. Conversely, colors can fade and lose vibrancy over time, posing challenges to their recognition. Examining color patterns in human skeletal remains as part of taphonomic processes is crucial for collecting and reconstructing information about their depositional history. Ultimately, this information can assist in identifying unknown individuals in medicolegal cases or contribute to the study of human history. A notable hue in bone staining is the appearance of green colors. This can be induced by the growth of plants, algae, and moss on bones when they are positioned on or near the terrestrial surface (Dupras and Schultz, 2013). Additionally, distinct green discolorations on bones can result from corroding copper-containing artifacts, such as grave goods (e.g., earrings, fingerings, jewelry, weapons, dishes, etc.) or clothing items (e.g., modern zippers and buttons) (Dupras and Schultz, 2013). In rare instances, green bone staining can occur during the lifetime of patients due to prolonged intake of tetra- and minocycline antibiotics (Judge et al., 2018). Typically, green discolorations tend to be more vivid and distinguishable from the surrounding bone colors. However, if only very small areas are affected, their identification can become challenging for the human eye, depending on factors such as intensity or the contrast with neighboring or underlying colors. This difficulty persists even when using conventional imaging techniques like photographic documentation in the visible light spectrum. While chemical analyses can unveil the composition and cause of such discolorations, the sampling and analytical process are invasive and expensive, often rendering them impractical. Alternatively, more advanced imaging techniques, such as multispectral cameras, could be considered. Nevertheless, this option relies on costly equipment and skilled operators and may not be widely available. Nonetheless, there are cost-effective and user-friendly methodological alternatives available for enhancing and identifying (green) bone discolorations through standard digital photography. One such option relies on a digital image enhancement technique called decorrelation stretching. Originally developed and utilized in remote sensing by NASA (Gillespie et al., 1986), the decorrelation stretch algorithm was later implemented by Jon Harman (2005) as a plugin called DStretch® for the open-source image processing tool ImageJ© (Schneider et al., 2012). Initially designed to aid the visualization of faded cave and rock art, DStretch® has become widely established in cave and wall art archaeology (e.g., Evans and Mourad, 2018). In recent years, researchers have further explored its alternative applications, e.g., by successfully applying DStretch® to painted pottery (Gonzáles et al., 2019), metal armor (Emmit et al., 2021), or tattooed skin of mummified human remains (e.g., Austin, 2022; Göldner and Deter-Wolf, 2023). The minimum requirements for utilizing DStretch® are a good camera and a desktop computer or laptop. It's worth noting that theoretically, a modern mobile phone or tablet with an integrated camera could also suffice, as there is a DStretch® app available. However, this may not meet the officially required documentation standards. Overall, DStretch® can be considered a noninvasive, relatively inexpensive, easy, and fast alternative for enhancing and recognizing (green) bone discolorations. To be able to reproduce the DStretch® procedure on discolored bones, I will present here a full step-by-step protocol based on digital photographs of an archaeological example of a female skull with copper-induced discolorations originating most likely from copper earrings. Note that the application of DStretch® only aims at the visualization, identification, and documentation of bone discolorations. It cannot reveal the exact cause or taphonomic agents involved in the development of these color changes. Also note that while this protocol is exemplified on green bone discolorations only, it can also be applied to other hues as well. However, if applied to colors other than green, defined parameters within the DStretch® application must be adjusted to produce desirable results. To facilitate the reproduction of the DStretch® procedure on discolored bones, I will present a comprehensive step-by-step protocol using digital photographs of an archaeological example: a female skull with copper-induced discolorations, likely stemming from copper earrings. It's crucial to understand that the application of DStretch® is focused solely on visualizing, identifying, and documenting bone discolorations. It does not provide insight into the exact cause or taphonomic agents involved in the development of these color changes. Additionally, it's important to note that while this protocol is demonstrated using green bone discolorations, it is equally applicable to other hues. However, when applied to colors other than green, certain parameters within the DStretch® application must be adjusted to achieve optimal results. Refer to the content below for additional background information on this protocol. Also, be aware that I have recently co-published a DStretch® protocol on protocols.io specifically for identifying hard-to-see tattoos on mummified human skin (Göldner and Deter-Wolf, 2023). This initial DStretch® protocol includes valuable information that may be of interest to you, encompassing the mathematical background of the algorithm and more general tips.