The Digital Fish Library: Using MRI to Digitize, Database, and Document the Morphological Diversity of Fish Rachel M. Berquist1, Kristen M. Gledhill1, Matthew W. Peterson1, Allyson H. Doan1, Gregory T. Baxter1, Kara E. Yopak1, Ning Kang1, H. J. Walker2, Philip A. Hastings2, Lawrence R. Frank1,3* 1 Center for Scientific Computation in Imaging, University of California San Diego, La Jolla, California, United States of America, 2 Marine Vertebrate Collection and Marine Biology Research Division, Scripps Institution of Oceanography, University of California San Diego, La Jolla, California, United States of America, 3 Center for Functional Magnetic Resonance Imaging, University of California San Diego, La Jolla, California, United States of America Abstract Museum fish collections possess a wealth of anatomical and morphological data that are essential for documenting and understanding biodiversity. Obtaining access to specimens for research, however, is not always practical and frequently conflicts with the need to maintain the physical integrity of specimens and the collection as a whole. Non-invasive three- dimensional (3D) digital imaging therefore serves a critical role in facilitating the digitization of these specimens for anatomical and morphological analysis as well as facilitating an efficient method for online storage and sharing of this imaging data. Here we describe the development of the Digital Fish Library (DFL, http://www.digitalfishlibrary.org), an online digital archive of high-resolution, high-contrast, magnetic resonance imaging (MRI) scans of the soft tissue anatomy of an array of fishes preserved in the Marine Vertebrate Collection of Scripps Institution of Oceanography. We have imaged and uploaded MRI data for over 300 marine and freshwater species, developed a data archival and retrieval system with a web-based image analysis and visualization tool, and integrated these into the public DFL website to disseminate data and associated metadata freely over the web. We show that MRI is a rapid and powerful method for accurately depicting the in- situ soft-tissue anatomy of preserved fishes in sufficient detail for large-scale comparative digital morphology. However these 3D volumetric data require a sophisticated computational and archival infrastructure in order to be broadly accessible to researchers and educators. Citation: Berquist RM, Gledhill KM, Peterson MW, Doan AH, Baxter GT, et al. (2012) The Digital Fish Library: Using MRI to Digitize, Database, and Document the Morphological Diversity of Fish. PLoS ONE 7(4): e34499. doi:10.1371/journal.pone.0034499 Editor: Andrew Iwaniuk, University of Lethbridge, Canada Received August 30, 2011; Accepted March 2, 2012; Published April 6, 2012 Copyright: ß 2012 Berquist et al. This is an open-access article 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. Funding: The DFL received its primary funding from the Biological Infrastructure Program (Grant DBI-0446389) of the National Science Foundation (NSF: http:// www.nsf.gov/div/index.jsp?div = DBI) with additional funding provided by NSF award EF-0850369. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: [email protected] Introduction the significant recent advances in imaging, software, and computer hardware technologies to develop digital libraries [4–7]. In Natural history collections possess a wealth of anatomical, principal, a comprehensive digital library would provide a morphological, genetic and geographic data that are essential for complete set of tools and methods to collect, visualize, analyze, documenting and understanding biodiversity [1–3]. Although archive, and disseminate high resolution imaging data over the some of the materials housed in fish collections include dried hard web. In practice, developing a comprehensive program that tissue preparations, cleared-and-stained specimens, and tissues integrates all of these aspects is a significant technological and samples for molecular genetic analysis, the bulk of these collections organizational task. In this paper we describe our current progress typically contain intact ‘wet’ specimens that have been preserved on the Digital Fish Library project (DFL, http://www. in formalin and post-fixed in alcohol for long-term storage. digitalfishlibrary.org), which aims to develop such a program with However, broad access to this material is commonly impeded by a particular focus on magnetic resonance imaging (MRI) of fishes stringent storage requirements, which involve hazardous liquids, housed in the Marine Vertebrate Collection (MVC) of the Scripps fragile storage containers (glass), and cramped storage facilities. Institution of Oceanography (SIO). However, while we have Access is often further limited by the desire of curators to maintain focused on a particular imaging modality and a specific collection the long-term integrity of specimens, especially in the case of of specimens, the methods and integrated infrastructure, which we holotypes and species that are delicate, rare, or difficult to obtain. report here, are applicable to a wide range of applications. This is This can conflict with specimen usage, which often requires the demonstrated with examples of research projects that have dissection, and sometimes even the destruction, of specimens in developed from, and the use of the techniques developed for, order to reveal internal anatomical and morphological detail. the DFL [8–15]. Moreover, the DFL infrastructure serves as one As the cost of maintaining physical collections becomes successful model for digital library creation and maintenance. It increasingly expensive, the biology community is faced with the has great flexibility and can be used for data assimilation from a difficult problem of how to preserve these valuable research and variety of sources, including not only newly acquired specimens education resources. One approach to this problem is to leverage PLoS ONE | www.plosone.org 1 April 2012 | Volume 7 | Issue 4 | e34499 The Digital Fish Library from specific collections (e.g. the MVC), but incorporation of data strength of the magnetic field of the scanner and the ability of its acquired from other modalities (CT, x-ray, photography, genetics, hardware to detect the resulting MR signal in specimen tissues. etc), from other groups or projects, or from scanning specimens in Cloutier et al. [22] were the first to apply standard anatomical other taxa. This digital infrastructure also provides great flexibility MRI to postmortem fish, demonstrating that proton resonance in data exploration. T1, T2 and FLASH pulse sequences provided selective enhance- The primary goal of the DFL was to digitize a representative set ment of cranial tissues in a coelacanth, Latimeria chalumnae. These of fish species from the MVC because of the tenuous state of the methods distinguished cartilage, muscles, brain, and various future of these types of collections [1]. But beyond merely digitally connective tissues, confirming gross soft tissue anatomy previously preserving these specimens, digital imaging provides a means of described from dissection. Although other groups have since used visualizing and quantifying their anatomy, facilitating morpholog- similar methods to depict the internal soft tissue anatomy in a ical investigations without the need for invasive dissection. number of freshly collected and preserved species [23–32], Although there is a variety of modern high resolution scanning resulting image quality has been limited, with the application of methods of potential utility for imaging fish, the strength of MRI is high quality MRI for detailed anatomical studies of fishes for in its ability to image soft tissues, as it is sensitive to a broad range comparative morphology still not sufficiently demonstrated. This of physical parameters related to tissue physiology and architec- has been due in part to their use of MRI scanners (typically 1.5 ture. For anatomical imaging, MRI may be viewed as a Tesla (T) and 3T) and hardware designed for human clinical complimentary technique to computed tomography (CT), which studies, rather than those (typically 7T and 11.7T) developed for has poor specificity for contrasting soft tissues but is excellent for smaller animal research. Though much more widely available imaging hard tissues such as bone. Both CT and MRI are than high-field small animal systems, clinical scanners impose currently the most widely accessible technologies with the capacity limitations on the attainable MRI signal due to their larger bore to acquire high-resolution (i.e. sub-millimeter down to around sizes (and thus lower field strengths), and restricted gradient 15 mm) standardized 3D volumetric data from whole specimens settings necessary to ensure patient safety. Furthermore, MRI is encompassing the size range typical of most fishes (,1cmto extremely sensitive to the structural and chemical nature of tissue, .2 m), and have become familiar tools for imaging the in situ and therefore the physical condition of postmortem specimens and anatomy of animals [16]. However, the advantages of MRI come the environment to which they have been exposed have significant at a price - high resolution 3D MRI scanning often requires