To understand the biological foundations of skin and hair pigmentation and associated diseases such as albinism or melanoma, scientists and physicians need quantitative and three-dimensional information on architecture, content and location of pigment cells. Researchers at Penn State College of Medicine have developed a new technique that allows scientists to visualize every cell containing melanin pigment in 3D, in an entire zebrafish.
The study of melanin is difficult because it blocks the light used in traditional microscopy. The researchers therefore turned to X-ray imaging, which can pass through an optically opaque material like melanin.
In 2019, a team led by Dr Keith Cheng, distinguished professor of pathology, pharmacology, biochemistry and molecular biology, developed “X-ray histotomography” – a cellular form of CT imaging, as a method of study of the 3D architecture of cells and tissues in biological samples with unprecedented resolution and clarity. Spencer Katz, a student in Cheng’s team’s MD / PhD Medical Scientist Training Program, modified this micro-CT technique to specifically study melanin, a pigment that scientists are studying in body color research. human skin and melanoma, in whole zebrafish.
Melanin is a brown to black pigment that gives zebrafish its characteristic stripes and the dark skin, hair and eyes of humans. Over 15 years ago, Cheng and his lab discovered a key gene in the evolution of fair skin color in humans by studying a particular line of mutant, golden zebrafish that has lighter stripes. . This discovery demonstrated the relevance of zebrafish models for studying critical questions about human biology and diseases, including albinism and melanoma.
Micro-CT, like human CT, uses a series of x-rays taken at slightly different angles to calculate or reconstruct 3D representations of the original object. For micro-CT, the samples are smaller and the resolution developed by Cheng’s team is 2000 times higher. Katz used silver to stain melanin, which allowed researchers to determine the 3D location and density of melanin from scans of whole zebrafish.
To perform the imaging, the Cheng Lab partnered with Dilworth Parkinson at the Lawrence Berkeley National Laboratories Advanced Light Source in Berkeley, Calif., Which is home to one of America’s most powerful synchrotron X-ray sources, where he runs a micro-CT resource. suitable for Cheng X-ray histotomography. The lab’s new X-ray detection system was designed to achieve unprecedented resolutions for whole zebrafish-sized samples or human biopsies. The team scanned the zebrafish with normal and altered pigmentation, including golden.
The researchers were able to visualize each cell containing melanin, called melanocytes, in fish, and map each of their positions in 3D. In addition, they could obtain quantitative measurements of their melanin content, allowing for the first time a direct comparison of the melanin content between normal and mutant fish. They published the results of their study in eLife.
This work laid the groundwork for further research into melanin-containing cancers, or melanomas, which are generally categorized by the depth of tumor cell invasion. According to Cheng, a researcher at the Penn State Cancer Institute, a number of models of melanoma in zebrafish exist and can be studied using the new technique. Katz and Cheng said human melanomas can be silver stained and imaged in the same way to more fully characterize tumor cells and their arrangements in tumors. They predict that scientists will be able, for example, to count the number of tumor cells of different characteristics and more definitively study invasion, a central feature of cancer, helping doctors make prognostic and treatment decisions.
In the future, the Cheng Lab will continue to develop new staining and optical methods to expand the applications of histotomography. This study represents proof of principle on how full-body 3D computer analysis of organisms and tissues can be performed using micro-CT which can provide a much more complete understanding of gene function.
Maksim Yakovlev, Daniel Vanselow, Yifu Ding, Alex Lin, Victor Canfield, and Khai Chung Ang of Penn State College of Medicine also contributed to this research. The new lens system used in the study was designed and built by Yuxin Wang of Mobile Imaging Innovations Inc. The authors declared no competing interests.
This research was supported by the National Institutes of Health, the Pennsylvania Department of Health Tobacco CURE Funds, the Jake Gittlen Laboratories for Cancer Research at Penn State College of Medicine, the Penn State Huck Institutes of the Life Sciences, and the Penn State Institute for Computational and Data. Sciences.
This study used the College of Medicine’s zebrafish functional genomics core and represents the activities of the Penn State Center for Artificial Intelligence Foundations and Scientific Applications and computational phenomena initiatives.