Rachel M. Berquist; Kristen M. Gledhill; Matthew W. Peterson; Allyson H. Doan; Gregory T. Baxter; Kara E. Yopak; Ning Kang; H. J. Walker; Philip A. Hastings; Lawrence R. Frank

“ Sophisticated data analysis tools provide methods for computing a wide range of metrics with which to assess morphology and which can provide significant insight even in the presence of a certain amount of tissue shrinkage or distortion. ”

“ Both CT and MRI produce 3D volumetric image data that are composed of individual volume elements (voxels) over which the local signal is averaged, an important distinction from imaging methods (e.g., X-ray and light photography) that simply produce 2D picture elements (pixels) . While voxel data are frequently viewed as 2D slices of pixels, it is important to understand that the information in a voxel is an averaged physical quantity that depends upon the particular acquisition method and the subsequent influence of the tissue on the signal. ”

“ However, we recognize that using museum specimens for quantitative analyses of soft tissues raises some concerns, since their tissues typically exist in a range of qualitative states, as discussed above. Inconsistencies in preservation histories and exposure of specimens to alcohol often result in undesired changes to soft tissue morphology, primarily from tissue shrinkage and hardening, which may compromise their suitability for comparative morphology and quantitative analyses derived from segmentation. ”

“ 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. ”

“ 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. ”

“ But these benefits come at the cost of increasingly sophisticated (and expensive) imaging devices that often require specialized facilities and staff, meticulous specimen preparation methods designed to enhance tissue contrast while mitigating imaging artefacts, exceedingly complex volumetric data that require advanced computational methods for analysis and visualization, and sophisticated data archival schemes that facilitate efficient data storage, search, and retrieval. ”

“ However, image quality can be compromised when scanning specimens that have not been optimally preserved for MRI, as is the case for virtually all fish collection specimens. Since the magnitude of the MR signal depends on the local state of tissue water and its surroundings, the fixation and preservation histories of individual specimens impacts the MRI properties of their tissues, and thus the outcome of MRI, as we observed with DFL fishes. ”

“ However, for individual studies requiring high quality quantitative imaging of specific anatomy in certain species, it may be preferable to scan specimens whose soft tissues have been specifically preserved and prepared for MRI prior to being catalogued in a collection. Our digital library infrastructure incorporates any of these possibilities and thus offers users flexibility in addressing basic research questions and addressing issues relating to the creation, continuation, and dissemination of digital library materials. ”

“ In principal, a comprehensive digital library would provide a complete set of tools and methods to collect, visualize, analyze, archive, and disseminate high resolution imaging data over the web. In practice, developing a comprehensive program that integrates all of these aspects is a significant technological and organizational task. ”

“ However, the most obvious advantage that volumetric imaging (i.e. MRI) offers over dissection, is that it keeps the relative morphological architecture of the specimen intact, thereby eliminating problems of geometric distortion caused by dissection itself. ”

“ This can conflict with specimen usage, which often requires the dissection, and sometimes even the destruction, of specimens in order to reveal internal anatomical and morphological detail.
As the cost of maintaining physical collections becomes increasingly expensive, the biology community is faced with the difficult problem of how to preserve these valuable research and education resources. One approach to this problem is to leverage the significant recent advances in imaging, software, and computer hardware technologies to develop digital libraries [3] – [4] . ”

“ In this paper we describe our current progress on the Digital Fish Library project (DFL, http://www.digitalfishlibrary.org) , which aims to develop such a program with a particular focus on magnetic resonance imaging (MRI) of fishes housed in the Marine Vertebrate Collection (MVC) of the Scripps Institution of Oceanography (SIO) . However, while we have focused on a particular imaging modality and a specific collection of specimens, the methods and integrated infrastructure, which we report here, are applicable to a wide range of applications. ”

“ Alcohol can also promote soft tissue shrinkage resulting in physical damage to specimens. For example, we frequently observed specimens where tissue shrinkage had caused muscles to pull away and detach from bones. As was demonstrated in Figure 2C, the presence of alcohol in specimen tissues also produces a significant residual MR signal intensity artefact which degrades T1-weighted images at 7T. Although not shown here, we experienced similar results at 3T when imaging fishes directly from alcohol. ”

“ Enormous potential will then emerge to rapidly, and objectively, digitize, quantify, and analyze vast arrays of anatomical and morphological features, in a vast and extremely diverse range of species. The goal of the DFL is therefore to develop the critical technological methods for scanning, visualizing, analyzing, archiving and disseminating high quality zoological digital imaging data, primarily focused on specimens housed in museum collections. ”

“ T1-weighted imaging also lends itself well to 3D isotropic imaging, which facilitates re-slicing of volume data sets along any orientation perpendicular to the acquisition direction without changing the in-plane resolution. The ability to re-slice data also minimizes complications when performing sophisticated 3D volumetric image analysis, such as image segmentation, where non-cubic voxels can result in directionally varying image distortions. ”

“ Ideally, a standardized protocol for preserving fishes for MRI could be adopted by collection managers, with selected specimens made available for imaging prior to exposure to alcohol. For instance, immediately fixing freshly collected fishes in generous quantities of 10% neutral-buffered formalin, including the injection of liberal amounts of fixative into body cavities, would greatly improve specimen fixation and stabilize tissue pH and tonicity. ”

“ For a growing number of specimens, annotated images of select MRI cross-sections and segmented renderings showing the identity of particular soft tissue structures have also been uploaded, providing the first instance of anatomical data available in many of our imaged species. ”

“ Although other groups have since used similar methods to depict the internal soft tissue anatomy in a number of freshly collected and preserved species [14] – [15] , resulting image quality has been limited, with the application of high quality MRI for detailed anatomical studies of fishes for comparative morphology still not sufficiently demonstrated. This has been due in part to their use of MRI scanners (typically 1.5 Tesla (T) and 3T) and hardware designed for human clinical studies, rather than those (typically 7T and 11.7T) developed for smaller animal research. ”

“ However, implementing these for efficient on-line usage is significantly more complicated than their implementation on the users' host computer. Improvements on porting these tools to web-based tools will be an ongoing endeavor. We emphasize, however, that we have developed a computational infrastructure in which visualization and analysis are directly and automatically linked to the database and accessible via the web. This infrastructure will facilitate future improvements to the web based tools. ”

“ Although these powerful 3D MR imaging technologies may never truly replace the resolution of traditional histology, or EM microscopy, improvements in temporal sampling and spatial resolution with each advancement in technology and image enhancement methods, means resolutions of less than 100 μm are becoming routine, with those less than 10 microns potentially viable [8] . ”

“ Our T1-weighted imaging protocol thus offered the best results for depicting the gross anatomy of major internal organs such as those of the alimentary tract, reproductive and excretory systems, etc, and provided sufficient image intensity and contrast for visualizing gross soft tissue anatomy in a diverse array of preserved fishes. ”

“ MRI Data Processing and Archiving
Raw data from MRI scanners is digital, and is thus easily stored in both its original (original 32-bit integer) format as well as in any number of commonly accepted standardized medical imaging formats. DFL data are stored both in raw and standard medical imaging DICOM (http://medical.nema.org/) and NIfTI (http://nifti.nimh.nih.gov/) formats which are commonly accepted formats for numerous image registration, segmentation, visualization and image analysis programs. ”

“ Recent developments in software methods have enabled the creation of a range of single-species digital atlases and databases based on 3D volumetric image data [43] , [40] , [70] – [71] and large-scale voxel-based imaging libraries, such as The Digital Morphology Library [72] and the DFL presented here [73] . However, with the enormous volumes of quantitative data that 3D imaging technologies inevitably generate, there is a critical need for computational tools that provide users with the ability for efficient data mining and analysis of these very large image datasets [74] – [75] . ”

“ As a result, anatomical details of the brain, sense organs, musculature, alimentary tract, etc, were more apparent, with cartilage particularly well contrasted, thereby aiding anatomical identification, segmentation, and morphological analysis. A T1-weighted pulse sequence was therefore used as the primary pulse sequence for acquiring general anatomical data for the DFL on both 3T and 7T scanners. Figures 2C and 2D illustrate the increase in image quality achieved by clearing post-fixative alcohol from specimens prior to scanning. ”

“ Both MRI and CT data were acquired for this island kelpfish, Alloclinus holderi (SIO 67-272, SL 94 mm) . A selection of soft tissue structures were segmented from MRI data and are color-coded as follows: blue = eyes; green = brain; red = alimentary tract; light brown = ovaries; purple = liver. Segmented soft tissues were co-registered with a volumetric rendering of CT imaged hard tissue structures acquired by The University of Texas High-Resolution X-ray Computed Tomography Facility (UTCT) (slice thickness 42 μm) . ”

“ This has resulted in obvious damage to some anatomical structures and the inevitable loss of MR signal and contrast apparent in this image. Figure 1C presents MRI data illustrating the adverse affects that freezing and thawing, and in addition, long-term storage in alcohol, can have on specimens. In this image of the coelacanth (Latimeria chalumnae) , the brain has visibly degraded (asterisk) and the muscles of the trunk have a ‘flaky’ appearance (arrow) indicating extensive tissue shrinkage and degradation over the 40 years or more that it has been preserved in the collection. ”

“ Scanning preserved fishes, rather than live or fresh post-mortem specimens, significantly enhances attainable image quality, as it stabilizes tissues and produces datasets devoid of motion, facilitating much longer scan times with higher achievable SNR, and thus considerably higher image resolutions. ”

“ Although many of the DFL fishes were well matched for our circular or tubular-shaped 3T and 7T RF coils, e.g. bulky, cylindrical shaped fishes (such as sharks and tunas) , and ball-shaped fishes (such as puffers and anglerfishes) , most species had more compressed bodies (such as most perciforms and flatfishes) or thin, elongated bodies (such as eels) . ”

“ Also, since the T1 of muscle in preserved fishes is approximately double that of fat [22] , muscle characteristically appears darker in contrast to more fatty tissues such as those of the brain and liver.
Common problems encountered when scanning museum fishes.Specimens were prepared for imaging and scanned as described in the Methods. Specific details for each species can be found in the DFL. (A) 3T horizontal image slice (937 μm3 resolution) of an oarfish, Regalecus glesne (SIO 96-82; SL 730 cm) demonstrating the effects of delaying soft tissue preservation. ”

“ SL 68 mm) exhibiting a prominent magnetic susceptibility artefact caused by gases trapped in its gut. (G) 7T sagittal and horizontal slices (60 μm3 resolution) of a benttooth bristlemouth, Cyclothone acclinidens (SIO 07-166; SL 53 mm) exhibiting very poor image contrast caused by its extreme low body mass leading to an insufficient signal loading of the RF coil. (H) 7T sagittal and horizontal slices (100 μm3 resolution) of a glass catfish, Kryptopterus bicirrhis (SIO Uncat; SL 45 mm) showing problems with slice plane misalignment associated with scanning very flat or thin-bodied fishes. ”