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Seeing Through the Skin: New Imaging Technique to Study Melanin
Researchers have developed a new way to image whole organisms in 3D, bringing key skin colour pigments into focus at cellular resolution.

In humans, melanin is produced in skin melanocytes and transferred to keratinocytes. Although melanin is best known as the main determining factor of skin and hair colour, melanin also plays a major role in human health and disease as it helps to prevent UV-based DNA damage. Dysfunction of human pigmentation can result in diseases such as albinism and vitiligo, and the overgrowth of melanocytes can lead to melanocytic nevi or malignant melanoma with darkly pigmented lesions. In addition, neuromelanin, which is produced as a by-product of dopamine synthesis, is also believed to be related to Parkinson’s disease as brain cells containing this pigment undergo selective degeneration.

To better understand the biological underpinnings of skin and hair pigmentation, scientists require quantitative, three-dimensional information about the architecture, content, and location of pigment cells. However, most developmental and functional studies done to assess the roles of pigmentation patterns and cell morphology have been largely descriptive due to the challenges of melanin quantification at cellular resolution, particularly in whole animals.

In a recent study by researchers from Penn State College of Medicine, they have developed a novel technique that allows for 3D visualisation of melanin-containing cells in whole zebrafish, an important vertebrate model organism for developmental biology. Because melanin blocks the light used in traditional microscopy, the researchers turned to micro-CT, an X-ray imaging technique, which can pass through optically opaque matter like melanin instead. Micro-CT, like human CT, uses a series of X-rays taken at slightly different angles to compute or reconstruct 3D representations of the original object.

To develop this novel technique, Spencer Katz, the first author of the study, drew inspiration from “X-ray histotomography”— a cellular form of CT imaging used to investigate the 3D architecture of cells and tissues in biological samples at high resolution and clarity—which was developed in 2019 by scientists led by Dr. Keith Cheng, a distinguished professor of pathology, pharmacology and biochemistry and molecular biology. Katz then modified the technique to specifically investigate melanin in whole zebrafish.

More than 15 years ago, Cheng and his lab discovered a key gene in the evolution of light skin colour in humans by studying a particular mutant zebrafish line, referred to as golden, that has lighter stripes. The discovery demonstrated the relevance of zebrafish models to study human skin colour, human biology, and diseases like melanoma and albinism.

In the current study, the Cheng Lab partnered with Dilworth Parkinson at the Advanced Light Source at the Lawrence Berkeley National Labs in Berkeley, California, where the team was able to access a micro-CT resource suitable for Cheng’s X-ray histotomography. To study melanin in the zebrafish, Katz used silver to stain the melanin. The team then scanned the zebrafish with normal and altered pigmentation, including zebrafish with the “golden” mutation.

The resulting images showed every cell containing melanin, called melanocytes, in the whole-body fish. They were also able to map each of their positions in 3D and obtained quantitative measurements of the melanin content, allowing direct comparison of melanin content across normal and mutant fish for the first time. These findings are expected to lay the groundwork for further research on melanomas, which are typically graded by the depth of tumour cell invasion. In fact, according to Cheng, a researcher at Penn State Cancer Institute, a number of zebrafish models of melanoma can be studied using the new technique.

Besides zebrafish, Katz and Cheng believe that human melanomas can also be stained with silver and imaged in the same way. If successful, they may be able to characterise tumour cells and their arrangements more completely. Furthermore, it is predicted that the technique may allow scientists to count the number of tumour cells of different characteristics and more definitively study invasion, thereby assisting doctors with prognostic and treatment decisions.

The silver staining of melanin for micro-CT provides proof-of-principle for whole-body, 3D computational analysis of organisms and tissues. In future, the Cheng Lab plans to continue the development of new staining and optical methods to expand the applications of histotomography, and advance studies in whole-organism, high-resolution phenotyping to a paint clearer picture of the phenotypic effects of environmental, disease, and genetic variables.


Source: Katz et al. (2021). Whole-organism 3D quantitative characterization of zebrafish melanin by silver deposition micro-CT. ELife, 10.

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