REVIEW ARTICLE

Application of magnetic resonance imaging in zoology

Alexander Ziegler • Martin Kunth •

Susanne Mueller • Christian Bock • Rolf Pohmann •

Leif Schroder • Cornelius Faber • Gonzalo Giribet

Received: 26 July 2011 / Revised: 21 September 2011 / Accepted: 22 September 2011 / Published online: 13 October 2011! Springer-Verlag 2011

Abstract Magnetic resonance imaging (MRI) is a non-invasive imaging technique that today constitutes one of

the main pillars of preclinical and clinical imaging. MRI’s

capacity to depict soft tissue in whole specimens ex vivo aswell as in vivo, achievable voxel resolutions well below

(100 lm)3, and the absence of ionizing radiation have

resulted in the broad application of this technique both inhuman diagnostics and studies involving small animal

model organisms. Unfortunately, MRI systems are expen-

sive devices and have so far only sporadically been used toresolve questions in zoology and in particular in zoomor-

phology. However, the results from two recent studies

involving systematic scanning of representative speciesfrom a vertebrate group (fishes) as well as an invertebrate

taxon (sea urchins) suggest that MRI could in fact be used

more widely in zoology. Using novel image data derivedfrom representative species of numerous higher metazoan

clades in combination with a comprehensive literature

survey, we review and evaluate the potential of MRI forsystematic taxon scanning. According to our results,

numerous animal groups are suitable for systematic MRI

scanning, among them various cnidarian and arthropodtaxa, brachiopods, various molluscan taxa, echinoderms, as

well as all vertebrate clades. However, various phyla in

their entirety cannot be considered suitable for thisapproach mainly due to their small size (e.g., Kinorhyncha)

or their unfavorable shape (e.g., Nematomorpha), while

other taxa are prone to produce artifacts associated eitherwith their biology (e.g., Echiura) or their anatomy (e.g.,

Polyplacophora). In order to initiate further uses of MRI in

zoology, we outline the principles underlying variousapplications of this technique such as the use of contrast

agents, in vivo MRI, functional MRI, as well as magneticCommunicated by T. Bartolomaeus.

A. Ziegler (&) ! G. GiribetMuseum of Comparative Zoology, Department of Organismicand Evolutionary Biology, Harvard University,26 Oxford Street, Cambridge, MA 02138, USAe-mail: aziegler@fas.harvard.edu

G. Giribete-mail: ggiribet@oeb.harvard.edu

M. Kunth ! L. SchroderLeibniz-Institut fur Molekulare Pharmakologie,Robert-Rossle-Strasse 10, 13125 Berlin, Germanye-mail: kunth@fmp-berlin.de

L. Schrodere-mail: lschroeder@fmp-berlin.de

S. MuellerCentrum fur Schlaganfallforschung, Charite-UniversitatsmedizinBerlin, Chariteplatz 1, 10117 Berlin, Germanye-mail: susanne.mueller1@charite.de

C. BockAlfred-Wegener-Institut fur Polar- und Meeresforschung,Am Handelshafen 12, 27570 Bremerhaven, Germanye-mail: christian.bock@awi.de

R. PohmannMax-Planck-Institut fur Biologische Kybernetik,Spemannstr. 41, 72076 Tubingen, Germanye-mail: rolf.pohmann@tuebingen.mpg.de

C. FaberInstitut fur Klinische Radiologie, Universitatsklinikum Munster,Albert-Schweitzer-Campus 1, 48149 Munster, Germany

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Zoomorphology (2011) 130:227–254

DOI 10.1007/s00435-011-0138-8

resonance spectroscopy. Finally, we discuss how future

technical developments might shape the use of MRI for the

study of zoological specimens.

Keywords MRI ! High-throughput ! Noninvasive !Metazoa ! Three-dimensional ! NMR

Abbreviations2D Two-dimensional

3D Three-dimensionalBBB Blood–brain barrier

BOLD Blood oxygenation level-dependent

CA Contrast agentcLSM Confocal laser scanning microscopy

CSI Chemical shift imaging

CT Computed tomographyDTI Diffusion tensor imaging

DWI Diffusion-weighted imaging

FLASH Fast low-angle shotFMNH Field Museum of Natural History

fMRI Functional magnetic resonance imagingFOV Field of view

FR RARE factor

FSPGR Fast spoiled gradient echoMEMRI Manganese-enhanced magnetic resonance

imaging

MR Magnetic resonanceMRI Magnetic resonance imaging

MRS Magnetic resonance spectroscopy

NA Average numberNMR Nuclear magnetic resonance

OPT Optical projection tomography

PET Positron emission tomographyRARE Rapid acquisition with relaxation enhancement

SE Spin echo

SIO Scripps Institution of OceanographySNR Signal-to-noise ratio

TA Acquisition time

TE Echo timeTR Repetition time

TSE Turbo spin echo

lCT Micro-computed tomographyZMB Zoologisches Museum Berlin

ZMH Zoologisches Museum Hamburg

Introduction

Magnetic resonance imaging (MRI), a noninvasive imag-

ing technique based on the principle of nuclear magnetic

resonance (NMR), constitutes one of the main pillars ofpreclinical and clinical imaging (Baker 2010; Walter et al.

2010). The importance of MRI is based on its capacity to

depict hard and in particular soft structures at relatively

high resolutions (Tyszka et al. 2005), but without the useof ionizing radiation as would be the case in other

noninvasive imaging techniques such as computed

tomography (CT) or positron emission tomography (PET).Although various elements such as fluorine, carbon,

helium, or phosphorous can be detected using MRI,

hydrogen is by far the most frequent element to be targetedas it is abundantly present in all biological samples and

even in fossils (Mietchen et al. 2005, 2008a). Today’sbroad application of MRI in life sciences is reflected in the

recent publication of various books that summarize and

extend the current knowledge on specific MRI applicationssuch as, for example, magnetic resonance neuroimaging

(Modo and Bulte 2011), small animal imaging (Kiessling

et al. 2011), or in vivo NMR imaging (Schroder andFaber 2011).

Undoubtedly, MRI scanning of metazoans other than

humans focuses on model organisms. In the last two dec-ades, numerous animal species have become suitable

model organisms for MRI studies, both in vivo as well as

ex vivo. These organisms encompass almost exclusivelyvertebrate taxa, in particular the mouse (Mus musculusLinnaeus, 1758), rat (Rattus norvegicus (Berkenhout,

1769)), zebra finch (Taeniopygia guttata (Vieillot, 1817)),pig (Sus scrofa Linnaeus, 1758), zebra fish (Danio rerio(Hamilton, 1822)), as well as various smaller primate

species such as the rhesus macaque (Macaca mulatta(Zimmermann, 1780)), the capuchin monkey (Cebuscapucinus (Linnaeus, 1758)), and the marmoset monkey

(Callithrix jacchus (Linnaeus, 1758)). However, sometraditional invertebrate model organisms such as the fruit

fly (Drosophila melanogasterMeigen, 1830) and the honey

bee (Apis mellifera Linnaeus, 1758) have recently beenadded to this list (Haddad et al. 2004; Null et al. 2008).

Apart from its use in model organisms, the application

of MRI in studies with a primarily zoological focus datesback to the late 1980s when the first anatomical MRI

images of invertebrate specimens were published (Gassner

and Lohmann 1987; Lohmann and Gassner 1987; Conneret al. 1988). However, it took several years before

improved hardware and software permitted the successful

scanning of small animals at resolutions that would allow adifferentiation of anatomical structures in organisms of

only a few centimeters in size (Hart et al. 2003).

Due to the capacity of MRI to depict in particular softtissue with excellent contrast (Benveniste and Blackband

2006), MRI has seen application also in two other fields

involving the study of zoological specimens: veterinaryradiology and food science. For diagnostic purposes, MRI

is frequently used on animal patients such as dogs, cats,

and horses (Assheuer and Sager 1997; Gavin and Bagley2010; Elliott and Skerritt 2010; Murray 2011), but exotic

228 Zoomorphology (2011) 130:227–254

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pets are frequently scanned as well (Straub and Jurina

2001; Valente et al. 2006). In contrast to veterinary radi-ology, the use of magnetic resonance (MR) techniques in

food science is aimed at noninvasively assessing the

quality of preprocessed as well as processed food intendedfor human consumption. In the course of these studies,

primarily fish species such as the Atlantic herring (Clupeaharengus Linnaeus, 1758), Atlantic salmon (Salmo salarLinnaeus, 1758), Atlantic cod (Gadus morhua Linnaeus,

1758), and steelhead trout (Salmo gairdneri Richardson,1836) have been scanned (Webb et al. 2001; Belton et al.

2003, 2005; Farhat et al. 2007; Gujonsdottir et al. 2009;

Renou et al. 2011). As small animal scanners have becomemore widely distributed, smaller culinary resources such as

oysters (Crassostrea gigas (Thunberg, 1793) and Ostreaedulis Linnaeus, 1758) are being routinely investigatednow as well (Davenel et al. 2006, 2010; Pouvreau et al.

2006).

Despite a growing number of successful studies based onthe application of MRI in zoology, this technology cannot

yet be considered a mainstream tool in studies on non-

model animal organisms. The principal reason for this iscertainly the perceived limited access to scanning systems

in combination with often prohibitive costs of operation.

However, despite these obstacles, an increasing number ofMR studies with a strictly zoological background are being

undertaken. Even major logistic efforts such as the sys-

tematic scanning of representative species from two animalgroups, namely actinopterygiids and other fishes (The

Digital Fish Library, http://www.digitalfishlibrary.org) as

well as echinoids (Ziegler et al. 2008; Ziegler in press) havebeen performed using MRI, demonstrating that large num-

bers of specimens can be analyzed with this technique.

More importantly, these exemplary high-throughput pro-jects were performed by tapping into a vast, yet largely

under-utilized resource, the worldwide collections of alco-

hol-preserved museum specimens. MRI’s capacity to non-invasively provide spatial information of whole specimen

internal anatomy was key to the large-scale use of museum

material (Corfield et al. 2008; Chanet et al. 2009) andresulted in a broad taxon sampling, which would have been

impossible to achieve based on freshly collected material

alone.As the gathered MR image data are digital by nature,

advanced 3D visualization and modeling software can be

used to convey complex morphological structures in amore plastic manner (Walter et al. 2010, Boistel et al.

2011), for example by embedding fully interactive, labeled

3D models of zoological specimens into the publicationitself (Ziegler et al. 2010b, c, 2011). In addition, the

simultaneous display of several interactive datasets on a

computer screen has become possible, for example byusing the ImageJ (NIH, http://rsb.info.nih.gov/ij) Volume

Viewer plugin, thereby permitting real-time comparison of

structures between different species. This improved andaccelerated access to morphological data certainly influ-

ences the way in which anatomical information will be

processed in the future (Budd and Olsson 2007; Schmidt-Rhaesa 2009; Giribet 2010).

One of the aims of this review is to assess the general

suitability of higher metazoan clades for their systematicscanning by MRI. To this end, we provide—in many cases

for the first time—MRI data on numerous phyla and listthose species that have previously been shown to be suit-

able candidates. In order to maximize taxon coverage, this

study centers primarily on the use of freshly fixed andmuseum specimens. Furthermore, we outline the principles

of various MR applications and provide examples for their

use in zoology. These applications include the use ofcontrast agents, in vivo MRI, functional MRI, as well as

MR spectroscopy. In addition, we provide a section on

practical and logistic considerations as well as a section onartifacts that may occur during the study of zoological

specimens. We conclude by providing an outlook on how

technical improvements could shape MRI’s further use inthe study of zoological specimens, hoping that the reader

becomes inspired by the breadth of possible MR applica-

tions in zoology.

Some practical and logistic considerations

Clinical (i.e., human) MRI scanners can primarily be found

in hospitals and neurobiology research institutions, whilepreclinical (i.e., small animal) MRI systems are predomi-

nantly located in neurobiological, pharmacological, and

developmental research institutes. Figure 1 depicts a rangeof clinical and preclinical scanners with corresponding coils

(the coil surrounds the sample and receives the radio fre-

quency signal emitted after excitation of the targeted nucleiwithin the sample). Clinical (Fig. 1a) as well as preclinical

(Fig. 1c, e, i, j) scanners have been sold in the thousands

over the last decades. Some institutions have pooled theirMRI systems in a dedicated imaging facility on a fee-based

access scheme, whereas other facilities may provide access

free of charge, often after approval of a project proposal.Since MRI technology is complex, support from trained

MRI staff will be necessary in most instances, at least in the

starting phase of an imaging project. Depending on the typeof magnet used and the imaging equipment available (coils,

gradient systems, etc.), millimeter- to meter-sized speci-

mens can be imaged either in part or whole. Typically, thecoils used in preclinical scanners provide enough room to fit

entire mice or rats (Fig. 1f), whereas those found in clinical

scanners can accommodate human-sized samples or partsthereof (Fig. 1b). Some MRI facilities have the capacity to

Zoomorphology (2011) 130:227–254 229

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custom-build coils in order to provide the best possible

signal detection for a given sample (Fig. 1k). In case of

over-sized specimens, it might be valuable to look for early(and hence usually smaller) developmental stages.

While living specimens are usually scanned in their

natural medium (i.e., in air, fresh, or saltwater), ex vivostudies require specimen preparation prior to scanning,

mostly by placing the specimen in distilled water (Ziegler

and Mueller 2011) or in a hydrogen-free liquid such asFluorinert (van der Linden et al. 2009). Although the var-

ious fixation protocols used in zoology may influence

scanning results, MRI has been successfully performed onBouin-, formalin-, and ethanol-fixed material (Chanet et al.

2009). Depending on the sample and the scanning protocol,

specimens can be scanned in water, but also in formalin orsometimes even directly in 70% ethanol. Unfortunately, no

study has yet systematically addressed the effects of

varying fixation protocols on scanning results across taxa.In addition, as MRI has the capacity to modulate tissue

contrast depending on the chosen protocol, the choice of

scanning parameters is crucial for the success of the

experiment. However, only a handful of studies provide

visual examples of the effect of differing protocol usage onother than model organisms (Brouwer et al. 1992; Ziegler

et al. 2010a; Ziegler and Mueller 2011). This means that

each taxon will usually require protocol optimizationbefore systematic scanning is initiated.

The final voxel resolution achievable depends on a

number of factors, in particular the size of the specimen,the field of view (FOV), magnet strength, gradient strength,

and the type of protocol. On preclinical scanners, voxel

resolutions of (15 lm)3–(100 lm)3 are typically obtain-able, while clinical scanners provide about (90 lm)3–

(1 mm)3 voxel resolution. Higher in-plane (i.e., x- and

y-plane) resolutions may be achievable using 2D imagingprotocols, but this comes at the expense of resolution in the

z-plane, usually with a factor of 4–8. The selected resolu-

tion and protocol in turn define the duration of the scan. Forexample, 2D single-slice scans can be performed at con-

siderably high resolutions in less than 1 min, while 3D

a b c d

e f g h

k

i

j l m

Fig. 1 Overview of MRI hardware used in the course of this study.MRI scanners can be divided into clinical (i.e., human) andpreclinical (i.e., small animal) systems, with the latter being eitherbased on horizontal or vertical bore magnets. a Human 3 T MRIscanner (Philips Achieva) and b a corresponding head coil for adults.c Small animal 4.7 T MRI scanner (Bruker Biospec) including acustom-built swim tunnel for studies on marine organisms (d).e Small animal 7 T MRI scanner (Bruker Pharmascan) for studies onmodel organisms with (f) corresponding rat and mouse coils (3.8 cm

and 1.8 cm internal diameter, respectively). g Small animal 9.4 TNMR scanner (Bruker Avance) equipped for imaging and h acorresponding double-resonant 1H/129Xe birdcage coil (3 cm internaldiameter). i Small animal 9.4 T (Bruker Biospec) and j 16.4 T (BrukerBiospec) MRI scanners. k Custom-built surface coil with1.8 9 2.4 cm dimensions as used on the 16.4 T scanning systemshown in (j). l Small animal 17.6 T NMR scanner (Bruker Avance)equipped for imaging and m a corresponding coil with 5 mm internaldiameter

230 Zoomorphology (2011) 130:227–254

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isotropic scans of an entire sample are usually performed

overnight.Isotropic datasets in particular are suitable for 3D

reconstruction. However, in most cases, MRI image data

display too many varying gray-scale levels for simplethreshold-based rendering and will therefore require manual

segmentation efforts. Unfortunately, the fast, automated

modeling that can be performed on humans and selectedmodel organisms is based on machine-learning algorithms

that have been trained using thousands of exemplary data-sets—this amount of indispensable data is usually not

available in comparative zoological studies of non-model

organisms. But since future developments in computervisualization and pattern recognition might solve the cur-

rent problem of laborious manual 3D reconstruction, it is of

even greater importance to deposit the raw datasets andmake them accessible to the public. A number of web-based

voxel repositories are currently being developed, such as

the Digital Fish Library (http://www.digitalfishlibrary.org),the Digital Morphology Library (http://www.digimorph.org),

and the Morphological Database (http://www.morphdbase.de).

Most of the sea urchin MRI datasets, for example, areavailable for inspection and download at MorphDBase.

However, the zoological community as well as its major

funding sources has not yet agreed on a standardized pro-cess for voxel data deposition (see Ziegler et al. 2010b and

Rowe and Frank 2011 for discussion).

Suitability of higher metazoan taxa for systematic MRIscanning

Species from various metazoan clades have been scanned

in the past. Table 1 lists those species for which anatomicalMRI (as well as other NMR) data with varying degrees of

detail and quality are currently available. In addition to a

literature survey, we provide image data for numerousmetazoan taxa from Ctenophora to Vertebrata (Figs. 2, 3,

4, 5, 6, 7, 8, 9), some of which have never been analyzed

before using MRI. However, the images provided hererepresent only a single virtual 2D slice out of a multitude of

slices that were gathered for each specimen scanned in the

course of this study. These images therefore only convey arestricted impression of the structural complexity of the

specimens and more can be learned by applying the above-

mentioned interactive dataset manipulation software.Of the 65 higher metazoan clades listed here (Table 1),

fifteen were judged as unsuitable right away because of the

minute size (e.g., Kinorhyncha, Micrognathozoa, Tardi-grada) or the unfavorable shape (e.g., Nematomorpha) of

their species. Apart from size and shape considerations, the

problem of artifact-causing materials located inside speci-mens has to be taken into consideration as well. As outlined

in the following section, various artifacts can impede suc-

cessful imaging of otherwise suitable taxa (e.g., Echiura,Sipuncula, Polyplacophora). Here, we give an estimate of a

clade’s general suitability for systematic MRI scanning as

previously performed on actinopterygiids and other fishes(The Digital Fish Library) as well as echinoids (Ziegler

et al. 2008; Ziegler in press). However, based on a number

of unforeseeable factors, each individual species understudy may prove to be suitable or not. Our conclusions are

primarily based on the use of freshly fixed and museummaterial, but as outlined in one of the following sections,

further aspects have to be taken into consideration when

studying organisms in vivo using MRI.Unfortunately, MRI currently does not permit to gather

meaningful anatomical data from specimens smaller than

about 1 mm. This void in whole specimen imaging can befilled using other imaging techniques such as micro-com-

puted tomography (lCT), confocal laser scanning micros-

copy (cLSM), or optical projection tomography (OPT)(Walter et al. 2010; Boistel et al. 2011). However, in

contrast to MRI, soft tissue imaging using these techniques

may require considerable specimen preparation, forexample through staining (Metscher 2009). Nonetheless,

the zoologist nowadays has a continuum of imaging

modalities at hand that permits the imaging of wholezoological specimens of varying sizes. In this context, MRI

clearly serves the size spectrum of millimeter- to meter-

sized specimens.

Potential artifacts occurring during MRI of zoologicalspecimens

The techniques used to encode spatial information in MRIcan lead to undesirable effects that alter image information

or, even worse, may appear as faulty anatomical detail. If

possible, these artifacts should be avoided or at least becorrectly identified. Many of the artifacts occurring in MR

images are due to imperfections of the experimental pro-

tocol or the hardware employed. For instance, interferencewith radio frequency signals from other electronic devices

(e.g., when the door to the scanner room is left open) can

cause stripes in the image(s). Faulty positioning or sizing ofthe FOV can lead to aliasing, i.e., the appearance of signal

from outside of the targeted region within the image.

However, some artifacts are caused by intrinsic propertiesof the hardware used or the sample itself.

A major source of artifacts is the movement of the

sample, whether in vivo or ex vivo. An MR image isgenerated by a large number of single scans that are

combined to form the final dataset. Any movement of the

sample during this series of scans causes blurring of theimage(s): it is important to note that both the sample and

Zoomorphology (2011) 130:227–254 231

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Table 1 Suitability of metazoan taxa for systematic scanning usingMRI. Taxon information includes the clade name, its vernacularname, the number of described or known species, and their size range(based primarily on Westheide and Rieger 2007, 2010). The species

analyzed so far using MRI (and other NMR techniques) are listed inalphabetical order within each taxon. Systematic arrangement ofclades was based on Dunn et al. (2008) and Edgecombe et al. (2011)

Taxon information Species studied so far using MRI (and otherNMR techniques)

Suitability for MRI

Ctenophora, comb jellies, *80 spp.,few mm to few m

Pleurobrachia pileus (this study): wholespecimen, ex vivo, Fig. 2a

Suitable, but many species toosmall

Placozoa, 1 sp. (many undescribed),few mm

– Unsuitable, too small

Porifera, sponges, *10.000 spp., few mmto few m

Axinella sp. (this study): whole specimen, exvivo, Fig. 2b; Lubomirskia baicalensis(Muller et al. 2006): whole specimen, exvivo; Suberites domuncula (Bringmannet al. 1999): whole specimen, in vivo;unknown species (Lee et al. 2010): wholespecimen, in vivo

Suitable, but many species toosmall

Cnidaria, anemones, jellyfishes, coralsand others (incl. Myxozoa), *10.550spp., several lm to few m

Actinia sp. (Ziegler and Mueller 2011, thisstudy): whole specimen, ex vivo, Fig. 2c;Aurelia aurita (this study): wholespecimen, ex vivo; Cyanea capillata(Blackband and Stoskopf 1990): wholespecimen, in vivo

Suitable, but some species toosmall or too large

‘‘Mesozoa’’, orthonectids andrhombozoans, *107 spp., few mm

– Unsuitable, too small

Xenoturbellida, 1 sp., few cm – Presumably unsuitable

Acoela, *380 spp., several lm to severalmm

– Unsuitable, too small

Nemertodermatida, 11 spp., several lm toseveral mm

– Unsuitable, too small

Chaetognatha, arrow worms, *150 spp.,several mm to several cm

– Some larger species likelysuitable, many too small

Nematoda, roundworms,[15.000 spp.,several lm to few m

Ascaris sp. (this study): wholespecimen, ex vivo, Fig. 2d

Some large forms suitable, butmost species too small

Nematomorpha, horsehair worms, *320spp., few cm to few m

– Unsuitable, too thin

Tardigrada, water bears, *1.030 spp.,several lm to few mm

– Unsuitable, too small

Onychophora, velvet worms, *200 spp.,few mm to several cm

Ooperipatellus sp. (this study): wholespecimen, ex vivo, Fig. 3a

Some larger forms suitable, butmany too small

Arthropoda (Chelicerata—Crustacea)

Chelicerata, arachnids and horseshoecrabs, *100.000 spp., several lm toseveral cm

Acanthoscurria geniculata (Lauridsen et al.2011): whole specimen, in vivo;Aphonopelma californica (Pohlmann et al.2007): opisthosoma, in vivo; Nephila sp.(this study): whole specimen, ex vivo,Fig. 3b

Many large forms suitable, butmany smaller species not

Pycnogonida, sea spiders, *1.000 spp.,few mm to several cm

Pycnogonum sp., (this study): wholespecimen, ex vivo, Fig. 3c

Only some large taxa suitable,body usually too small

Myriapoda, centipedes, millipedes,symphylans, and pauropods, *16.000spp., few mm to several cm

Scolopendra sp. (this study): proximal part,ex vivo, Fig. 3d

Many suitable, but many too small

232 Zoomorphology (2011) 130:227–254

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Table 1 continued

Taxon information Species studied so far using MRI (and otherNMR techniques)

Suitability for MRI

Hexapoda, springtails, proturans,diplurans, and insects, *1.000.000spp., several lm to several cm

Apis mellifera (Tomanek et al. 1996, Haddadet al. 2004): whole specimen and head, exvivo; Ascioplaga mimeta (Hornschemeyeret al. 2006): caput and thorax, ex vivo;Bombyx mori (Mapelli et al. 1997): larva, invivo; Canthon cyanellus cyanellus (Favilaet al. 2004): whole specimen, in vivo;Coccinella septempunctata (Chudek et al.1996, Geoghegan et al. 2000): wholespecimen, in vivo; Dinocampus coccinellae(Chudek et al. 1996, Geoghegan et al.2000): whole specimen, in vivo;Dinoponera quadriceps (Fresneau et al.1991, Hart et al. 2003): whole specimen, invivo; Drosophila melanogaster (Fresneauet al. 1991, Null et al. 2008, this study):whole specimen, in and ex vivo; Dytiscusmarginalis (Wecker et al. 2002): wholespecimen, ex vivo; Epiblema scudderiana(Mietchen et al. 2008b): whole specimen, invivo; Eurosta solidaginis (Mietchen et al.2008b): whole specimen, in vivo;Graphiphora augur (Goodman et al. 1995):whole specimen, in vivo; Manduca sexta(Conner et al. 1988, Michaelis et al. 2005,Watanabe et al. 2006, Hallock 2008): headand larva, in vivo; Periplaneta sp. (Zieglerand Mueller 2011, this study): proximal anddistal part, ex vivo, Fig. 3e; Periplanetafuliginosa (Takahashi et al. 1989): wholespecimen, in vivo; Pieris brassicae(Goodman et al. 1995): whole specimen, invivo; Plodia interpunctella (Chudek et al.1996): whole specimen, in vivo; Prionuscoriarius (this study): larva, ex vivo;Sarcophaga bullata (Jasanoff and Sun2002, Jasanoff 2005): head, in vivo;Sarcophaga peregrina (Price et al. 1999):whole specimen, in vivo; Schistocercagregaria (Gassner and Lohmann 1987):whole specimen, in vivo; Solenopsis invicta(Slowik et al. 1997): whole specimen, exvivo; Spodoptera litura (Skibbe et al.1995), thorax, ex vivo; Venturia cavescens(Chudek et al. 1996): whole specimen, invivo; Vespula vulgaris (Hart et al. 2003):whole specimen, in vivo

Many species suitable, but manymore too small

Crustacea, shrimps, crabs, lobsters, andbarnacles, *50.000 spp., several lm tofew m

Callinectes sapidus (Brouwer et al. 1992):whole specimen, in vivo; Cancer pagurus(Fernandez et al. 2000): whole specimen, invivo; Cherax destructor (Brinkley et al.2005): cephalothorax, in vivo; Majasquinado (Fernandez et al. 2000, Bock et al.2001a): cephalothorax, in vivo;Pachygrapsus marmoratus (Ziegler andMueller 2011, this study): whole specimen,ex vivo, Fig. 3f; Procambarus clarkia(Herberholz et al. 2004): whole specimen,in vivo

Many species are suitable, butmany will be too small

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Table 1 continued

Taxon information Species studied so far using MRI (and otherNMR techniques)

Suitability for MRI

Priapulida, penis worms, *20 spp.,several lm to several cm

Priapulopsis bicaudatus (this study): wholespecimen, ex vivo, Fig. 2e

Some large species suitable, butmany too small

Loricifera, *200 spp. (mostundescribed), several lm

– Unsuitable, too small

Kinorhyncha, mud dragons,[150 spp.,several lm to few mm

– Unsuitable, too small

Bryozoa, moss animals,[4.500 spp.,several lm to mm

Dendrobeania sp. (this study): colony, exvivo, Fig. 4a

Single organism too small, but thecolony can be suitable

Entoprocta,*150 spp., several lm to fewmm

– Unsuitable, too small

Cycliophora, cycliophorans, 2 spp.,several lm

– Unsuitable, too small

Annelida (Echiura—Clitellata)

Echiura, spoon worms, *150 spp., fewmm to few m

Thalassema thalassemum (this study): wholespecimen, ex vivo, Fig. 4f

Some species suitable, but mostare too small

Sipuncula, peanut worms, *150 spp.,few cm to several cm

Sipunculus sp. (this study): whole specimen,ex vivo, Fig. 4e

Some larger forms suitable, butmany species too small

Polychaeta, bristle worms, *9.000 spp.,several lm to few m

Nereis sp. (Ziegler and Mueller 2011, thisstudy): proximal part, ex vivo, Fig. 4g

Some taxa suitable, but manytoo small

Clitellata, earthworms and leeches,[4.000 spp., few mm to few m

Hirudo medicinalis (this study): proximalpart, ex vivo, Fig. 4h

Some taxa suitable, but manytoo small and too thin

Mollusca (Solenogastres—Cephalopoda)

Solenogastres, *250 spp., few mm toseveral cm

– Some species likely suitable, butmost are too small

Caudofoveata, *70 spp., few mm to fewcm

– Some species likely suitable, butmany are too small

Monoplacophora, 25 spp., several lm tofew cm

– Some taxa likely suitable, but mostare too small

Polyplacophora, chitons, *900 spp., fewmm to several cm

Acanthochitona sp. (this study): wholespecimen, ex vivo, Fig. 5a; 22 furtherspecies were scanned by us in 2009, alldisplaying strong susceptibility artifactssurrounding the ferromagnetic radula

Unsuitable because of magnetite-bearing radula

Gastropoda, snails and slugs, *100.000spp., several lm to several cm

Achatina sp. (Cooper 2011): whole specimen,in vivo; Aplysia californica (Hsu et al.1996, Grant et al. 2000, Neustadter et al.2002, Novakovic et al. 2006): buccal massand single neuron, in and ex vivo;Dendronotus sp. (Ziegler and Mueller 2011,this study): whole specimen, ex vivo,Fig. 5b

Many species likely suitable, butmany are too small

Scaphopoda, tusk shells, *600 spp., fewmm to few cm

– Some taxa suitable, but many aretoo small

234 Zoomorphology (2011) 130:227–254

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Table 1 continued

Taxon information Species studied so far using MRI (and otherNMR techniques)

Suitability for MRI

Bivalvia, bivalves, *15.000 spp., severallm to few m

Anodonta sp. (Synthesys NA F Pilot Study,http://www.synthesys.info/na_f.htm):whole specimen, in and ex vivo; Arcopsisadamsi FMNH 3 (this study): whole spec-imen, ex vivo; Argopecten purpuratus (vonBrand et al. 2009): whole specimen, invivo; Barbatia cancelaria FMNH 348 (thisstudy): whole specimen, ex vivo; Brachi-dontes exustus FMNH 243 (this study):whole specimen, ex vivo; Cerastodermaedule (this study): whole specimen, ex vivo,Fig. 5c; Corbicula fluminea FMNH 242(this study): whole specimen, ex vivo;Crassostrea gigas (Davenel et al. 2006,Pouvreau et al. 2006): whole specimen, invivo; Crassostrea virginica (Lannig et al.2008, Lee et al. 2010): whole specimen, invivo; Cyrenoida floridana FMNH 6 (thisstudy): whole specimen, ex vivo; Elliptiocomplanata (Holliman et al. 2008): wholespecimen, in vivo; Gemma gemma FMNH357 (this study): whole specimen, ex vivo;Histella arctica FMNH 264 (this study):whole specimen, ex vivo; Margaritiferaauricularia (Synthesys NA F Pilot Study,http://www.synthesys.info/na_f.htm):whole specimen, in and ex vivo; Mytilusedulis (this study): whole specimen, exvivo; Ostrea edulis (Davenel et al. 2010):whole specimen, in vivo; Solemya velumFMNH 358 (this study): whole specimen,ex vivo; Teredo clappi FMNH 2 (thisstudy): whole specimen, ex vivo; Yoldialimatula FMNH 359 (this study): wholespecimen, ex vivo

Many species are suitable, butsome will be too small

Cephalopoda, cephalopods, *1.000 spp.,few cm to several m

Loligo pealeii (Mooney et al. 2010): wholespecimen, in vivo; Lolliguncula brevis(Gozansky et al. 2003): receptor organ, exvivo; Sepia sp. (this study): wholespecimen, ex vivo, Fig. 5d; Sepia officinalis(Quast et al. 2001): brain, ex vivo

Mostly suitable, but a few taxa willbe too small

Nemertea, ribbon worms, *1.100 spp.,few mm to several m

Lineus sp. (this study): whole specimen, exvivo, Fig. 4c; Dinonemertes cf.investigatoris (this study): median part, exvivo

Some large forms suitable, butmany species too small

Brachiopoda, lamp shells, *380 spp.,few mm to few cm

Terebratalia transversa (this study): wholespecimen, ex vivo, Fig. 4d

Suitable, some species will be toosmall

Phoronida, horseshoe worms, *14 spp.,few mm to several cm

– Some taxa likely suitable, butsome species too small

Gastrotricha, hairy backs,[500 spp.,several lm to few mm

– Unsuitable, too small

Platyhelminthes, flatworms, cestodes,trematodes, and others,[22.120 spp.,few mm to several m

Fasciola hepatica (this study): wholespecimen, ex vivo, Fig. 4b

Some species suitable, but manytoo small

Gnathostomulida, jaw worms,\100 spp.,several lm to few mm

– Unsuitable, too small

Micrognathozoa, 1 sp., several lm – Unsuitable, too small

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Table 1 continued

Taxon information Species studied so far using MRI (and otherNMR techniques)

Suitability for MRI

Rotifera (incl. Acanthocephala), wheelanimals and thorny-headed worms,*3.100 spp., several lm to few cm

– Typical rotifers are too small, butsome acanthocephalans may besuitable

Myzostomida, myzostomids, *140 spp.,few mm

– Unsuitable, too small

Echinodermata (Crinoidea—Echinoidea)

Crinoidea, feather stars and sea lilies,*620 spp., few cm to few m

Antedon mediterranea ZMH E6859 (Zieglerin press, this study): whole specimen, exvivo, Fig. 6a

Partly suitable, but body will betoo small in many species

Asteroidea, sea stars, *1.600 spp., fewcm to few m

Acanthaster planci (this study): wholespecimen, in and ex vivo, Fig. 6b; Asteriasrubens (this study): whole specimen, exvivo; Asterina gibbosa ZMH E1195(Ziegler in press, this study): wholespecimen, ex vivo

Suitable, but some species will betoo small

Ophiuroidea, brittle stars, *2.000 spp.,few mm to several cm

Ophiocoma nigra ZMH E2025 (Ziegler inpress, this study): whole specimen, ex vivo,Fig. 6c

Suitable, but some taxa will be toosmall

Holothuroidea, sea cucumbers, *1.200spp., few mm to few m

Aslia lefevrei (Ziegler in press, this study):whole specimen, ex vivo, Fig. 6d;Echinopsolus acanthocola (this study):whole specimen, ex vivo

Suitable, but some will be toosmall

Echinoidea, sea urchins, sand dollars,and heart urchins, *950 spp., few mmto several cm

94 Species: Ziegler (in press) provides a fulllist; Clypeaster rosaceus ZMB Ech 2520(this study): whole specimen, ex vivo,Fig. 6e

Suitable, but some taxa showstrong artifacts (sediment)

Hemichordata, acorn worms andpterobranchs, *101 spp., few mm tofew m

Harrimania kupferi ZMB Ent 1579 (thisstudy): whole specimen, ex vivo, Fig. 6f

Many suitable, but some will betoo small

Urochordata, tunicates, salps, and others,*2.120 spp., few mm to several m

Ciona intestinalis (Ziegler and Mueller 2011,this study): whole specimen, ex vivo,Fig. 6g

Many suitable, but some will betoo small or too large

Cephalochordata, lancelets, 29 spp., fewcm

Branchiostoma lanceolatum (this study):whole specimen, ex vivo, Fig. 7a

Suitable, some are too small

Vertebrata (Myxini—Mammalia)

Myxini, hagfish, *60 spp., several cm tofew m

Eptatretus stoutii (The Digital Fish Library):whole specimen, ex vivo, Fig. 7b

Suitable

Petromyzontida, lampreys, 42 spp.,several cm to few m

Petromyzon marinus (The Digital FishLibrary): whole specimen, ex vivo, Fig. 7c

Suitable

Chondrichthyes, cartilaginous fishes,[1.100 spp., several cm to several m

46 species: The Digital Fish Library;Chiloscyllium arabicum (Blackband andStoskopf 1990): torso, in vivo; Isurusoxyrinchus (Perry et al. 2007): torso, exvivo; Lamna ditropis (Perry et al. 2007):torso, ex vivo; Squalus acanthias (thisstudy): head, ex vivo; Triakis semifasciata(this study): whole specimen, ex vivo,Fig. 7d

Suitable

Sarcopterygii, lobe-finned fishes, 8 spp.,few m

4 species: The Digital Fish Library;Latimeria chalumnae (The Digital FishLibrary): head and torso, ex vivo, Fig. 7e

Suitable

236 Zoomorphology (2011) 130:227–254

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Table 1 continued

Taxon information Species studied so far using MRI (and otherNMR techniques)

Suitability for MRI

Actinopterygii, ray-finned fishes,*30.000 spp., several mm to few m

222 species: The Digital Fish Library;Acanthostracion quadricornis (Boistel et al.2011): whole specimen, ex vivo; Agonuscataphractus (this study): head, ex vivo,Fig. 7f; Alloclinus holderi (Rowe and Frank2011): whole, ex vivo; Clupea harengus(Veliyulin et al. 2007): thorax, ex vivo;Crassius sp. (Blackband and Stoskopf1990): torso, in vivo; Cyprinus carpio (Vander Linden et al. 2004, Van den Burg et al.2006, Chanet et al. 2009): whole specimenand head, in and ex vivo; Danio rerio(Kabli et al. 2006): whole specimen, in andex vivo; Gadus morhua (Bock et al. 2002,Lannig et al. 2004): whole specimen, invivo; Hyporhamphus australis (Butcheret al. 2009): whole specimen, in and exvivo; Lampris guttatus (Runcie et al. 2009):brain, ex vivo; Monopterus albus(Rasmussen et al. 2010): proximal part, exvivo; Pachycara brachycephalum (Market al. 2002, Van der Linden et al. 2004):whole specimen, in vivo; Porichthysnotatus (Forbes et al. 2006): wholespecimen, in vivo; Salmo gairdneri (Nottet al. 1999): head, ex vivo; Salmo salar(Veliyulin et al. 2006): thorax, ex vivo;Sebaster sp. (Rogers et al. 2008): wholespecimen, ex vivo; Toxotes jaculatrix(Chanet et al. 2009): whole specimen, exvivo; Zoarces viviparus (Bock et al. 2001b,2002): whole specimen, in vivo

Suitable, but some species will betoo small

Amphibia, amphibians, *6.300 spp., fewmm to few m

Bombina orientalis (this study): wholespecimen, ex vivo, Fig. 8a; Rana sp. (thisstudy): whole specimen, ex vivo; Ranaesculenta (Sbarbati et al. 1992): head, invivo; Rana sylvatica (Rubinsky et al.1994a): whole specimen, in vivo; Xenopuslaevis (Lee et al. 2007): embryo, in vivo

Suitable, but some species will betoo small

Testudines, turtles, *280 spp., few cm tofew m

Caretta caretta (Valente et al. 2006): wholespecimen, in and ex vivo; Chrysemys pictamarginata (Rubinsky et al. 1994b): wholespecimen, in vivo; Geochelone pardalispardalis (Raiti and Haramati 1997): wholespecimen, in vivo; Kinixys sp. (Straub andJurina 2001): whole specimen, in vivo;Testudo graeca, T. hermanni, T. horsfieldii(Straub and Jurina 2001): whole specimen,in vivo; Trachemys scripta (Blackband andStoskopf 1990, Straub and Jurina 2001,Stecyk et al. 2009, Rasmussen et al. 2010,Lauridsen et al. 2011): whole specimen andheart, in and ex vivo; Trachemys scriptaelegans (this study): head, ex vivo, Fig. 8b;unknown species (Kuoni et al. 1993): wholespecimen, in and ex vivo

Suitable

Sphenodontida, tuataras, 2 spp., severalcm

– Presumably suitable

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the coil surrounding the sample are immobile for the

duration of the scan. Movement artifacts are especiallyproblematic for in vivo MRI, where body motion has to be

suppressed by tight fixation and/or the use of anesthesia. It

is, however, not possible to completely avoid motion arti-facts in body regions that display movement due to, for

example, heart beat or breathing. In these cases, special

measures like synchronizing the image acquisition withmovement or the use of additional navigator scans to detect

motion are necessary. Even when scanning samples postmortem, care has to be taken to keep the sample from

moving, for example caused by its flotation in the sur-

rounding fluid or by scanner vibration caused by the gra-dient system. Low-melting agarose can be used to embed

tiny or fragile specimens for scanning in order to avoid this

type of artifacts. In addition, glass or plastic rods can beemployed to immobilize fixed specimens within the sample

container (Ziegler and Mueller 2011).

Another major source of artifacts are local variations of

magnetic susceptibility within the sample, which maycause distortions of the highly homogeneous magnetic field

required for MRI. These distortions may lead to image

deformations or even to dropouts that may extend muchfurther than the actual size of the artifact-causing sub-

stance. The most common reason for this type of artifact is

air within or surrounding the sample, but even strongersusceptibility artifacts are caused by ferro- or paramagnetic

materials. In zoological specimens, these materials may bepresent in the form of buckshot, complex mineralized

structures, or ingested magnetic sediments (Elliott and

Skerritt 2010; Gavin and Bagley 2010). For ex vivo spec-imen scanning, immersion of the entire sample in distilled

water, agarose, or any fluid with susceptibility close to that

of the tissue can help to avoid the susceptibility variationsat the tissue/air interfaces. Air bubbles inside the sample

should, if possible, be removed by degassing (Ziegler and

Table 1 continued

Taxon information Species studied so far using MRI (and otherNMR techniques)

Suitability for MRI

Squamata, scaled reptiles,[6.000 spp.,few cm to several m

Euleptes sp. (this study): frontal part, ex vivo,Fig. 8c

Suitable, some species may be toolong

Crocodilia, crocodilians, 24 spp., few mto several m

Alligator mississippiensis (this study): wholespecimen, ex vivo, Fig. 8d

Suitable, but some species may betoo large

Aves, birds,[10.000 spp., few cm tofew m

Apteryx australis, A. haastii, A. mantelli, A.rowi (Corfield et al. 2008): brain, ex vivo;Ciconia ciconia (Berthold et al. 2001):thorax, in vivo; Serinus canaria(Tindemans et al. 2003): brain, in vivo;Sylvia borin (Czisch et al. 2001):reproductive organs, in vivo; Taeniopygiaguttata (Poirier et al. 2008, this study):head, ex vivo, Fig. 8e

Suitable, but some species may betoo large

Mammalia, mammals, 5.416 spp., few cmto several m

Numerous mammalian model organisms aswell as pets have been scanned using MRI;Daubentonia madagascariensis (Kaufmanet al. 2005): brain, ex vivo; Delphinusdelphis (Marino et al. 2002): brain, ex vivo;Homo sapiens (Ziegler et al. 2011): head, invivo, Fig. 9c; Kogia simus (Marino et al.2003b): brain, ex vivo; Macaca mulatta(Saleem et al. 2002): brain, in vivo; Musmusculus (this study): whole specimen, exvivo, Fig. 9a; Orcinus orca (Marino et al.2004): brain, ex vivo; Oryctolaguscuniculus (Wang et al. 2005): brain, exvivo; Papio papio (Liu et al. 2008): womb,in vivo; Phocoena phocoena (Marino et al.2003a): brain, ex vivo; Platanista gangetica(Endo et al. 1999): torso, ex vivo; Susscrofa (Rasmussen et al. 2010, this study):heart and head, ex and in vivo, Fig. 9b;Tursiops truncatus (Marino et al. 2001,Ridgway et al. 2006): head and brain, inand ex vivo; various primate species(Hopkins et al. 2000): brain, in vivo

Suitable, but some species may betoo large

238 Zoomorphology (2011) 130:227–254

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Mueller 2011). During in vivo measurements, the presence

of air-filled structures is usually unavoidable and can onlybe treated by adapting the sequence so as to minimize their

negative effects, although some image distortions or

dropouts may remain. Unfortunately, susceptibility arti-facts cannot always be avoided, and several examples are

presented in some of the figures in this article. These

include artifacts caused by air bubbles within (Figs. 2b, 4d,8a) or surrounding the sample (Figs. 2b, 3c, 6g) as well as

ferromagnetic inclusions (Figs. 2e, 4e, f, 5a, c, 6f). Theseinclusions are potentially present in all sediment-ingesting

animals (e.g., Echiura, Sipuncula, some Echinoidea, some

Holothuroidea, some Bivalvia, and some Gastropoda), butalso in taxa that feed on sediment-eaters (e.g., Crustacea,

Vertebrata). Ways to tackle this problem may be to starve

living specimens, or, in the case of museum material, to

systematically look for specimens that display the least orno artifacts. In some instances, a surgical removal of the

artifact-causing structure, for example in the case of the

strongly ferromagnetic radula found in Polyplacophora(Fig. 5a), might be an option.

Darker regions within the image dataset may also be

caused by variations of the fields of the radio frequencycoils. In particular, the use of small surface coils for signal

reception can lead to inhomogeneous image intensity(Fig. 8b, e). In addition, examining relatively large samples

at higher field strengths (i.e., 7 T and above) can lead to

strong inhomogeneities of the transmit field. These inho-mogeneities appear when the wavelength of the MR signal

is smaller than the object size, leading to strong variations

a b c

d e

1 cm

1 cm 1 cm

1 cm

5 mm

cr

ph

me te

bd

mf

bmph

ep

ut

incu

prin

tr

ca

ac

ba

lm

ep

cu

rm

ep

Fig. 2 Results from MRI scans of various metazoan taxa fromCtenophora to Priapulida. a Horizontal section through an entirecomb jelly (Pleurobrachia pileus (O.F. Muller, 1776), Ctenophora).Note the massive shrinkage artifact caused by too rapid rehydration.b Vertical section through an entire sponge (Axinella sp., Porifera).The black spots within and surrounding the specimen are presumablysusceptibility artifacts caused by tiny air bubbles. c Vertical sectionthrough an entire sea anemone (Actinia sp., Cnidaria). Note themovement artifacts (caused by scanner vibration) indicated by theslight blur occurring around the tentacles. d Horizontal sectionthrough the frontal and caudal areas of a large roundworm (Ascaris

sp., Nematoda). e Horizontal section through an entire penis worm(Priapulopsis bicaudatus (Danielssen, 1868), Priapulida). The darkareas are susceptibility artifacts caused by ferromagnetic sedimentingested by the animal. Please refer to Table 2 for an overview ofscanning parameters, while Table 1 provides assessments of thesuitability of metazoan taxa for systematic MRI. ac aboral canal, babase, bd basal disc, bm basilar muscle, ca caudal appendage, cr combrow, cu cuticle, ep epidermis, in intestine, lm longitudinal retractormuscle, me mesohyl, mf mesenterial filament, ph pharynx, prproboscis, rm retractor muscle, te tentacle, tr trunk ring, ut uterus

Zoomorphology (2011) 130:227–254 239

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in image intensity and contrast (van de Moortele et al.

2005). Specialized hardware and transmit technologies are

necessary to compensate for these effects.Further relevant artifacts are caused by chemical shift

differences, especially between water and fat. Here, fatty

regions are shifted in one dimension relative to the watersignal. Often, this type of artifact can be avoided by

experimentally suppressing the fat signal, usually resulting

in longer scan times. Finally, so-called Gibbs ringingconsists of shifted repetitions of sharp edges in the image.

This is an inherent effect of the spatial encoding technique

and can best be removed by retrospective multiplication ofthe raw data with a filter function. However, while this

effectively removes the artifact, it also somewhat degrades

the spatial resolution of the dataset.

Applying contrast agents in MRI of zoologicalspecimens

For MRI of humans and other animals, a variety of contrastagents (CA) that modulate tissue contrast are available.

Most of these substances comprise molecules with para-

magnetic properties that either increase (T1 agents) ordecrease (T2 agents) the local MRI signal in their vicinity.

T1 agents often contain paramagnetic Gd3? (gadolinium) or

Mn2? (manganese) ions, while T2 agents are usuallycomposed of iron oxide nanoparticles (Shapiro et al. 2004).

Common to all CAs is their property of having a specific

influence on a given tissue, which originates from proper-ties of those molecules that bind the surroundings of

paramagnetic cores. These molecular properties change the

a

5 mm

db

e f1 cm

1 cm

1 cm

1 cm

c

1 cm

lm

op

bl

ss

lm

tr

pr

in

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lm

fb

in

hclm

cs

he gi

le

hd

ps

pe mu

an

le

an

os

Fig. 3 Results from MRI scans of various panarthropod taxa fromOnychophora to Crustacea. a Vertical section through an entire velvetworm (Ooperipatellus sp., Onychophora). This specimen showsstrong susceptibility artifacts presumably related to an unsuitablefixation protocol. b Horizontal section through an entire spider(Nephila sp., Chelicerata). Note the strong signal originating from thepaired book lungs. c Virtual horizontal section through body and legsof a sea spider (Pycnogonum sp., Pycnogonida). This specimen showsstrong artifacts presumably related to air bubbles attached to thecuticula. d Horizontal section through the anterior segments of a

centipede (Scolopendra sp., Myriapoda). e Horizontal section throughthe anterior half of a cockroach (Periplaneta sp., Hexapoda).f Horizontal section through the carapace of a crab (Pachygrapsusmarmoratus (Fabricius, 1787), Crustacea). Please refer to Table 2 foran overview of scanning parameters, while Table 1 provides assess-ments of the suitability of metazoan taxa for large-scale MRI. anantenna, bl book lung, cs cardiac stomach, fb fat body, gi gills, hchindgut cecum, hd head, he heart, in intestine, le leg, lm leg muscle,mu musculature, op opisthosoma, os ostium, pe pericardium, phpharynx, pr proboscis, ps prosoma, ss sucking stomach, tr trunk

240 Zoomorphology (2011) 130:227–254

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relative contrast of tissues and make organs distinguishable

in MR images (Weinmann et al. 2003).There are CAs that do not penetrate the blood–brain

barrier (BBB) and may therefore be specifically used to

detect blood leakage in patients with a brain tumor or otherpathologies. Some CAs may remain intravascular and can

thus provide contrast to perform angiography (Fig. 8d),

perfusion measurements, or estimations of vessel densityand size, while other CAs specifically enter neurons via

calcium channels and allow visualizing neuronal tracts.CAs used in molecular MRI either target specific mole-

cules or can be activated by enzymes, a property that can

be exploited to image gene expression (Louie et al. 2000).However, the most common use of CAs is currently their

systemic application in order to achieve visualization of

pathological conditions or a better delineation of morpho-

logical detail.Such systemic CAs may also be applied ex vivo when

studying fixed specimens. Staining protocols have been

published for a number of species and organs such as therat fetus (Petiet et al. 2007), the mouse brain (Kim et al.

2009), or whole zebra fish specimens (Ullmann et al.

2010). However, the specific protocols in general dependon both the CAs used and the sample under investigation

and must therefore be optimized individually. When usingGd-based T1 agents, CA concentrations of 2–10 mM are a

good starting point. Specimens should be incubated in CA-

containing buffer for at least 2 h per millimeter samplethickness (personal observation), and imaging should be

performed in the same solution to avoid dilution and

1 cm

a b c d

e f g h1 cm 1 cm 1 cm

1 cm 1 cm 1 cm 5 mm

os

vsut

vgin

in

epde

loes

go bi

pe

ne

cu

in

vm

in

epin

pr as

ep

lm

cr

ph

pa

bv

brma

sm

gu

tr

ca

Fig. 4 Results from MRI scans of various metazoan taxa fromBryozoa to Clitellata. a Vertical section through a colony of mossanimals (Dendrobeania sp., Bryozoa). The three-dimensional orien-tation of the sample did not permit to obtain a well-aligned virtualsection. b Horizontal section through the proximal part of a flatworm(Fasciola hepatica Linnaeus, 1758, Platyhelminthes). c Sectionthrough an entire ribbon worm (Lineus sp., Nemertea). The knot-like habitus of this specimen did not permit to obtain a symmetricalvirtual section. d Horizontal section through an entire lamp shell(Terebratalia transversa (Sowerby, 1846), Brachiopoda). The twoblack spots inside the animal are susceptibility artifacts originatingfrom trapped air bubbles. e Vertical section through an entire peanutworm (Sipunculus sp., Sipuncula). Particularly, the proximal areas aredisfigured by susceptibility artifacts originating from ingested sedi-ment. f Horizontal section through an entire spoon worm (Thalassema

thalassemum (Pallas, 1774), Echiura). The dark areas within thespecimen are susceptibility artifacts derived from ingested sediment.g Horizontal section through the anterior part of a bristle worm(Nereis sp., Polychaeta). h Horizontal section through the proximalpart of a leech (Hirudo medicinalis Linnaeus, 1758, Clitellata). Pleaserefer to Table 2 for an overview of scanning parameters, whileTable 1 provides assessments of the suitability of metazoan taxa forlarge-scale MRI. as anterior sucker, bi blind intestine, br singlebryozoan, bv blood vessel, ca cecum, cr crop, cu cuticle, de dermis, epepidermis, es esophagus, go gonad, gu gutter, in intestine, lmlongitudinal muscle, lo lophophore, ma mantle, ne nephridium, osoral sucker, pa parapodium, pe pedicle, ph pharynx, pr proboscis, smspindle muscle, tr trunk, ut uterus, vg vitelline gland, vm ventralretractor muscle, vs ventral sucker

Zoomorphology (2011) 130:227–254 241

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therefore loss of contrast over acquisition time. Table 2

lists those figures that present specimens scanned after

application of a systemic CA.It is important to note that in certain specimens, contrast

between soft tissues is not always increased when CAs are

applied (Ziegler and Mueller 2011). However, contrast willalways increase significantly between soft tissue and cal-

cified or mineralized structures, such as components of an

endo- or exoskeleton. The effect of CAs in soft tissue isthat the relevant time constant T1 may be reduced to about

1% of its original value (Petiet et al. 2007), potentially

resulting in a signal increase of two orders of magnitude.To take full advantage of this gain, appropriate imaging

sequences have to be used since losses from the concom-

itant reduction in the time constant T2 have to be mini-mized. In particular, gradient echo sequences with very

short echo and repetition times (e.g., FLASH) allow full

exploitation of this gain in contrast, while coincidentally

reducing imaging times up to two orders of magnitude.

Shortened scan times can then either be used to increase

throughput in systematic morphological studies (Zhanget al. 2010), or, alternatively, to increase spatial resolution

for individual specimen imaging. The gain in signal-to-

noise ratio (SNR) may consequently allow improvement inisotropic resolution by a factor of two, resulting in a

reduction in voxel size by a factor of eight. Thus, MRI of

fixed specimens can always benefit from the use of T1 CAs.

In vivo MRI of zoological specimens

Apart from its usage on fixed specimens, MRI can also be

used to study living animals. Besides taking live images offunctioning organs, this approach minimizes misinterpre-

tations due to structural changes following fixation and

preservation (Natt and Frahm 2005). A number of

1 cm

a

1 cm 1 cm

1 cm

b

dc

angi

fo

ma

bpst

bm

go

fo

fo

pm amve ey te

cu

ma

dgol

pg

vm

vmpf

ma

in

lp sh

go

Fig. 5 Results from MRI scans of various molluscan taxa fromPolyplacophora to Cephalopoda. a Horizontal section through anentire chiton (Acanthochitona sp., Polyplacophora). The strongsusceptibility artifact in the proximal part is caused by the magnetite-and cobalt-bearing radula. b Vertical section through an entirenudibranch (Dendronotus sp., Gastropoda). c Vertical section throughan entire bivalve (Cerastoderma edule (Linnaeus, 1758), Bivalvia).The slight susceptibility artifacts are caused by ingested para- orferromagnetic particles within the digestive tract. d Horizontal section

through an entire squid (Sepia sp., Cephalopoda). Please refer toTable 2 for an overview of scanning parameters, while Table 1provides assessments of the suitability of metazoan taxa for large-scale MRI. am anterior adductor muscle, an anus, bm buccal mass, bpbranchial plume, cu cuttlebone, dg digestive cecum, ey eye, fo foot, gigills, go gonad, in intestine, lp labial palp, ma mantle, ol optic lobe, pfposterior foot retractor muscle, pg pallial groove, pm posterioradductor muscle, sh systemic heart, st stomach, te tentacle, veventricle, vm visceral mass

242 Zoomorphology (2011) 130:227–254

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prerequisites are necessary in order to obtain in vivo MRI

data with a similar resolution as would be the case withMRI of preserved samples (Benveniste and Blackband

2002).

In general, a satisfactory resolution for morphologicalMRI can only be achieved by using comparatively long

scanning times. Unfortunately, body movement and natural

organ function in living animals will almost certainly leadto imaging artifacts. Therefore, such samples need to be

mechanically fixed, chemically relaxed, or even fullyanesthetized. In the case of crustaceans or bivalves, for

example, mechanical fixation can easily be achieved by

attaching the carapace or the shell to the experimentalchamber. Such a setup was successfully applied under

defined physiological conditions for several days inside a

seawater flow-through chamber (Fig. 1d) located withinthe MRI scanner (Bock et al. 2001a; Lannig et al. 2008). In

these studies, an in-plane resolution of (200 lm)2–

(500 lm)2 was typically achieved, permitting the differ-entiation of various internal organs such as leg muscles,

hepatopancreas, gonads, gills, and heart in the Europeanspider crab Maja squinado (Herbst, 1788) as well as fast

d gfe

b ca

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in

lm

pb

es

go

tr

copr

in

asma

tu

bp

ra

go

dm

os

Fig. 6 Results from MRI scans of various basal deuterostome taxafrom Crinoidea to Tunicata. a Vertical section through the central partof a feather star (Antedon mediterranea (Lamarck, 1816), ZMHE6859, Crinoidea). In order to achieve a higher local resolution, thearms of the specimen were not scanned in their entirety. b Horizontalsection through an entire starfish (Acanthaster planci (Linnaeus,1758), Asteroidea). c Horizontal section through the central part of abrittle star (Ophiocoma nigra O.F. Muller, 1789, ZMH E2025,Ophiuroidea). This specimen had been slightly damaged duringpreparation. d Vertical section through an entire sea cucumber (Aslialefevrei (Barrois, 1882), Holothuroidea). e Horizontal section throughan entire sand dollar (Clypeaster rosaceus (Linnaeus, 1758), ZMBEch 2520, Echinoidea). The sediment inside the gut of this specimenis only slightly ferromagnetic and therefore causes almost no artifacts.f Vertical section through an entire acorn worm (Harrimania kupferi(von Willemoes-Suhm, 1871), ZMB Ent 1579, Enteropneusta). The

internals of this animal are filled with artifact-causing sediment.g Vertical section through an entire tunicate (Ciona intestinalis(Linnaeus, 1767), Tunicata). The tunica of the animal traps tinyartifact-inducing air bubbles. Accession numbers refer to theZoologisches Museum Berlin (ZMB) and the Zoologisches MuseumHamburg (ZMH). Please refer to Table 2 for an overview of scanningparameters, while Table 1 provides assessments of the suitability ofmetazoan taxa for large-scale MRI. Image (b) courtesy of Robert Sigland Hannes Imhof (Munchen, Germany). am ampulla, as atrialsiphon, bp branchial pharynx, bt branchial tentacle, bu bursa, cocollar, cs cardiac stomach, dm dilator muscle, es esophagus, go gonad,in intestine, lm lantern muscle, ma mantle, mo mouth, os oral siphon,pb pillar bridge, pc pyloric cecum, pr proboscis, ra radial canal, riring canal, rm retractor muscle, st stomach, tr trunk, tu tunica, vevertebra

Zoomorphology (2011) 130:227–254 243

123

and catch muscle, pericardium, gills, and heart in the

Eastern oyster Crassostrea virginica (Gmelin, 1791).As an alternative to permanent mechanical fixation,

cold- or freeze-tolerant ectotherms like insects, amphibi-

ans, or reptiles can be immobilized and thus more easilystudied by exposing them to temperatures near or even

below their freezing point, likely in combination with non-

permanent mechanical fixation. Such approaches have beensuccessfully applied to the study of anuran (Rubinsky et al.

1994a), chelonian (Rubinsky et al. 1994b; Stecyk et al.2009), hexapod (Hart et al. 2003), and aranean (Pohlmann

et al. 2007; Lauridsen et al. 2011) species. For example, the

following morphological features could be depicted in thecase of chelonians: carapace, gall bladder, gut, lungs, liver,

plastron, and ventricle (Stecyk et al. 2009).

For in vivo MRI of organisms that possess delicate skinsand where mechanical fixation of the body or any other form

of restraining is not desirable, as for example in the case of

cephalopods, amphibians, and mammals, adequate anes-thetics need to be applied instead. Typical anesthetics for

water breathers are ethanol and MS 222 (tricaine methane-sulfonate). For many invertebrates, MgCl2 (magnesium

1 cm

b

10 cm

1 cm

1 cm

d

c

a phno

mu

gs ey

brto

mu

br

mu

no

li

ey

mu

gi

he

hd

oo

10 cm 1 cm

fe

ey

mo

no

eygi

brst

sb

Fig. 7 Results from MRI scans of deuterostome taxa from Cepha-lochordata to Actinopterygii. a Vertical section through the proximalpart of a lancelet (Branchiostoma lanceolatum (Pallas, 1774),Cephalochordata). b Vertical section through an entire hagfish(Eptatretus stoutii (Lockington, 1878), SIO 87-125, Myxini). c Ver-tical section through the frontal part of a lamprey (Petromyzonmarinus Linnaeus, 1758, SIO 74-134, Petromyzontida). d Horizontalsection through an entire leopard shark (Triakis semifasciata Giard,1855, Chondrichthyes). e Vertical section through the head of acoelacanth (Latimeria chalumnae Smith, 1939, SIO 75-347,

Sarcopterygii). f Horizontal section through the head of a scorpaeniidfish (Agonus cataphractus Linnaeus, 1758, Actinopterygii). Acces-sion numbers refer to the Scripps Institution of Oceanography (SIO),La Jolla. Please refer to Table 2 for an overview of scanningparameters, while Table 1 provides estimates of the suitability ofmetazoan taxa for large-scale MRI. Images (b, c, and e) courtesy ofLawrence R. Frank (San Diego, CA, USA). br brain, ey eye, hd head,he heart, gi gill, gs gill slit, li liver, mo mouth, mu musculature, nonotochord, oo olfactory organ, ph pharynx, sb swim bladder, ststomach, to tooth

244 Zoomorphology (2011) 130:227–254

123

chloride) is an easy-to-handle, reversible muscle relaxant

(Cooper 2011)—concentrations of 2% in salt or freshwaterhave led to good initial results (personal observation). Vol-

atile narcotics like isoflurane and its derivatives are com-

monly applied for anesthesia in small mammals. However,the effects of all these anesthetics are time-dependent and

require accurate dosage as well as extensive physiological

monitoring of the animal under study. Furthermore, in par-ticular for smaller organisms, anesthesia might influence

routine metabolism and therefore may result in potentialmisinterpretations of in vivo conditions and regulatory

processes (Iwama et al. 1989).

In addition to anatomical information, in vivo MRI alsohas great potential to display dynamic information through

the use of angiography and other flow-sensitive MR tech-

niques that can be used to study the function of the heart orthe cardiovascular system both in vertebrates and inverte-

brates. For example, apart from morphological information

obtained on vessel architecture in M. squinado, MR angi-

ography was used to measure the ventilatory performancein this water breather (Bock et al. 2001a).

Functional MRI of zoological specimens

Functional MRI (fMRI) describes a set of techniques usedto visualize neuronal as well as muscular activity in vivo

(Ulmer and Jansen 2010). It is now a common tool in theimaging of human and small animal brain activity (van der

Linden et al. 2007, 2009). fMRI studies in animals may

provide insight into the principles of brain activity, thepathophysiology of brain functions, as well as brain func-

tional reorganization and plasticity. Currently, the most

commonly used methods in fMRI are the blood oxygena-tion level-dependent (BOLD) contrast imaging and the

manganese-enhanced MRI (MEMRI) techniques.

rca

10 cm

1 cm

1 cm

1 cm1 cm

b c

ed

ey

bcli

lu

fb

in

eymu

br

ey

he

br

lu

mu

heao

mu

to

ce

fe

beco

po

mu

Fig. 8 Results from MRI scans of vertebrate taxa from Amphibia toAves. a Vertical section through the frontal part of a toad (Bombinaorientalis (Boulenger, 1890), Amphibia). Slight susceptibility arti-facts are presumably caused by trapped air. b Horizontal sectionthrough the head of a turtle (Trachemys scripta elegans (Wied-Neuwied, 1839), Testudines). The reduced signal intensity toward theedges of the image is caused by the use of a surface coil. c Horizontalsection through the frontal part of a gecko (Euleptes sp., Squamata).Slight susceptibility artifacts occurring on the scaled skin are causedby tiny air bubbles. d Horizontal section through an entire alligator(Alligator mississippiensis (Daudin, 1801), Crocodilia). e Vertical

section through the head of a bird (Taeniopygia guttata (Vieillot,1817), Aves). Reduced signal intensity at the lower right edge of theimage is caused by the use of a surface coil. Please refer to Table 2for an overview of scanning parameters, while Table 1 providesassessments of the suitability of metazoan taxa for large-scale MRI.Image (d) courtesy of Kasper Hansen, Henrik Lauridsen, MichaelPedersen, and Tobias Wang (Aarhus, Denmark). ao aorta, bc buccalcavity, be beak, br brain, ce cerebellum, co cortex, ey eye, fb fat body,fe feather, he heart, in intestine, li liver, lu lung, mu musculature, popons, to tongue

Zoomorphology (2011) 130:227–254 245

123

BOLD contrast imaging was first described by Ogawaet al. (1990) and reflects hemodynamic changes in response

to neuronal activity. Here, hemoglobin is used as an

endogenous contrast agent by relying on the difference inthe magnetic properties of oxyhemoglobin (diamagnetic)

and deoxyhemoglobin (paramagnetic). BOLD contrast

imaging takes advantage of the fact that paramagneticsubstances act as MR-detectable CAs, which induce a

signal loss in magnetic-susceptibility-sensitive MRI. Dur-

ing neuronal activity, both the metabolic rate as well as thedemand for oxygen rises, resulting in an increase in the

regional blood flow. This leads to the presence of more

oxygen than neurons can dissipate, in turn decreasing therate of deoxyhemoglobin in the activated area, and thereby

causing an increase in the MRI signal intensity. Unfortu-

nately, fMRI using BOLD cannot be applied to metazoansthat use hemocyanin instead of hemoglobin for oxygen

transport (Bock et al. 2007).

For event-related BOLD fMRI, an appropriate stimulus(whether somatosensory, visual, auditory, or olfactory) has

to be provided. In order to avoid motion artifacts as well as

stress for the animal under study, fMRI investigations onzoological specimens have to be performed under anes-

thesia. Here, it is important to choose fMRI-compatible

anesthetics like a-chloralose or medetomidine (at least invertebrates), which do not reduce the coupling between

functional activation and hemodynamic response (van der

Linden et al. 2007). As the changes in the MRI signalinduced by fluctuations in the blood oxygenation level

are relatively small, the stimulus detection phases and

subsequent recoveries have to be measured in an alternateand repetitive manner using ultra-fast T2

*-weighted

sequences. Due to the complexity of the procedure, fMRI

datasets have to be analyzed with dedicated statistical tools(Lazar 2008).

In contrast to the indirect approach using BOLD contrast

imaging, Lin and Koretsky (1997) and Silva et al. (2004)demonstrated that MEMRI enables direct imaging of neu-

ronal activity using T1-weighted MRI. Mn2? ions possess

an ionic radius similar to that of Ca2? (calcium) ions,which act as mediators for the release of neurotransmitters

and neuronal signal transduction in metazoans. Mn2? ions

can therefore enter these excitable cells through voltage-gated calcium channels as well. As Mn2? ions have para-

magnetic properties and shorten the T1 relaxation time of

hydrogen protons, their presence leads to increased signalintensity in T1-weighted MRI. In addition to activation-

induced imaging, MEMRI can also be used to trace specific

neuronal connections such as olfactory, visual, andsomatosensory pathways (Watanabe et al. 2006). Further-

more, manganese can act as a whole-brain CA after sys-

temic administration (Silva et al. 2004; van der Lindenet al. 2007).

One of the advantages of imaging with Mn2? ions is that,

once they accumulate within a cell, their excretion rate isrelatively low, in the order of weeks. This allows admin-

istration of the CA outside the scanner, while the animal is

being stimulated and still awake. Mn2? ions are delivered asa solution of MnCl2 (manganese chloride). Here, the cel-

lular toxicity of Mn2? ions and their compatible dose have

a b c

1 cm 5 cm 10 cm

ey

ve

br

lu

liin

ey

mu

co

lv

nonoey

ea

ea

coce

di

Fig. 9 Results fromMRI scans of various mammalian taxa. aVerticalsection through an entire juvenile mouse (Mus musculus Linnaeus,1758, Rodentia). b Horizontal section through the head of a mini pig(Sus scrofa Linnaeus, 1758, Artiodactyla). c Horizontal sectionthrough the head of a human (Homo sapiens Linnaeus, 1758,

Primates). Please refer to Table 2 for an overview of scanningparameters, while Table 1 provides assessments of the suitability ofmetazoan taxa for large-scale MRI. br brain, ce cerebellum, co cortex,di diaphragm, ea ear, ey eye, in intestine, li liver, lu lung, lv lateralventricle, mu musculature, no nose, ve vertebra

246 Zoomorphology (2011) 130:227–254

123

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Zoomorphology (2011) 130:227–254 247

123

to be considered (Silva et al. 2004). For activity-induced

MEMRI in vertebrates, in some cases, an opening of theBBB is required, which can be achieved using a 25%

hypertonic solution of D-mannitol (Silva et al. 2004). If

manganese is administered in small doses, opening the BBBmay not be necessary. However, for neuronal tract tracing,

MnCl2 has to be introduced directly into the nervous tissue

using the olfactory, intravitreal, or retinal pathways (Linand Koretsky 1997), while systemic administration of

MnCl2 can be performed intraperitoneally, subcutaneously,or intramuscularly (Silva et al. 2004). Although the main

application of fMRI is in studies on vertebrate model

organisms and humans, fMRI has previously been per-formed on flies (Jasanoff and Sun 2002).

Magnetic resonance spectroscopy as well as non-protonand diffusion MRI of zoological specimens

In addition to MRI based on hydrogen nuclei, NMR tech-

niques can also be used for magnetic resonance spectros-

copy (MRS) as well as non-proton and diffusion MRI. Inprinciple, these applications allow for the study of physi-

ological function both ex vivo and in vivo.

MRS is used to retrieve information about the molecularenvironment of the element under investigation: due to the

influence of neighboring atoms, hydrogen nuclei in dif-

ferent molecules are subject to a slightly different magneticfield, which can lead to a change in their Larmor frequency,

a phenomenon known as chemical shift. This can be

employed to noninvasively analyze a broad range ofcomplex samples. As MRS in its most simple form

acquires data from within the entire sample, no spatial

information is given. To overcome this deficiency, local-ized spectroscopy methods have been developed and they

can be divided into single- and multi-voxel techniques. In

single-voxel localization, the main benefit is that themagnetic field can be optimized better than compared to

bigger volumes, which in turn further improves spectral

resolution. Various techniques are based on leaving thesignal unperturbed either inside or outside the voxel of

interest (Granot et al. 1986, Ordidge et al. 1986; Bottomley

1987; Frahm et al. 1989; Garwood and De la Barre 2001).However, one disadvantage of single-voxel localized

spectroscopy is its potential to miss important areas of the

sample. In addition, in order to fill a 2D matrix of spectraand to overlay these data with an anatomical image, a long

acquisition time is inevitable. Despite such drawbacks,

MRS techniques have been successfully applied to thestudy of numerous vertebrate and invertebrate species

(Lohmann and Ratcliffe 1988; Hart et al. 2003).

As an extension to MRS, chemical shift imaging (CSI)combines spectroscopic with spatial information, thereby

offering to obtain metabolic profiles from multiple spatial

positions within a sample. In the classical CSI method, onespectrum is acquired at each phase-encoding step

(Maudsley et al. 1983). However, classical CSI is still

relatively time-consuming, as the acquisition time increa-ses with the number of points in the spectral dimension, but

more importantly in the three spatial dimensions. Hence,

there always remains a trade-off between sufficient sensi-tivity due to averaging and short acquisition times (Poh-

mann et al. 1997). Still, versatile CSI applications arepossible ex vivo as well as in vivo, for example pH-metry

(Gallagher et al. 2008), biopsies of cerebral lesions while

avoiding healthy tissue (Hermann et al. 2008), or measur-ing the oxidative capacity among leg muscles in humans

(Forbes et al. 2009). Apart from applications in humans,

CSI has been successfully performed with marine species(Lee et al. 2010). While metabolites of interest may be

abundantly present in living specimens, their concentration

in fixed tissue may differ drastically. Many metabolites willlikely have been replaced by the fixative, whereas, for

example, a comparison between fat and water will still be

feasible.In addition to hydrogen MRS or MRI, other nuclei such

as 3He (helium), 13C (carbon), 17O (oxygen), 19F (fluorine),31P (phosphorus), and 129Xe (xenon), as long as they arepresent in sufficient concentration, can be used to probe the

sample. Such methods are referred to as non-proton or

x-nuclei MRI. Since hydrogen protons have a high naturalabundance in organic substances, there is also a strong

hydrogen background signal from all areas within biolog-

ical samples. This can make it beneficial to scan differentnuclei, as they are usually less abundant than hydrogen and

in turn also have a weaker background signal. In addition,

the polarization of some of these nuclei, in particular 3He,13C, and 129Xe can be drastically increased by hyperpo-

larization (Walker and Happer 1997; Golman et al. 2001;

Ardenkjar-Larsen et al. 2003; Schroder et al. 2006). Thiseffect may lead to an improvement in signal strength of

four orders of magnitude, which can be used to detect very

low concentrations. For instance, hyperpolarized 3He hasbeen successfully used to image void spaces inside verte-

brate lung tissue, something not feasible with hydrogen

MRI.In many biological objects, the movement of molecules

occurs along fibrous structures such as blood capillaries

and neural or muscular tissue. These structures can bemade visible by MR techniques such as diffusion-weighted

imaging (DWI) (Le Bihan et al. 1986) or diffusion tensor

imaging (DTI) (Basser et al. 1994). Diffusion measure-ments by NMR are based on signal loss, as random motion

of particles causes the MR signal to attenuate during the

scan. In particular, DTI techniques have found applicationon typical MRI model organisms such as monkeys

248 Zoomorphology (2011) 130:227–254

123

(Pierpaoli and Basser 1996), zebra finches (De Groof et al.

2006), and mice (Aggarwal et al. 2009), but also on humantissue (Jellison et al. 2004). Interestingly, DTI can in

principle be applied both in vivo as well as ex vivo.

Potential artifacts that may occur during diffusion MRI arediscussed by Le Bihan et al. (2006).

The future of MRI

MRI and NMR research have been a huge success story

over the last 60 years, with fundamental breakthroughs in

the field continuously increasing the range of applicationsin the life sciences. With such strong momentum, it is

expected that future discoveries will bring further

improvements. The major driving force in MRI is certainlythe domain of clinical imaging, with most hardware solu-

tions adapted to the size and specific morphology of

humans. Some promising recent innovations in humanMRI may also be relevant for the study of smaller animals,

whereas others mainly address problems that are not

encountered when studying objects significantly smallerthan humans.

In general, the physical limitations—in particular with

regard to resolution—of MRI strongly depend on the typeof application, as this determines the hardware used and the

protocol chosen. For example, dynamic studies in living

animals require adequate temporal resolution and thus limitthe time to acquire detailed spatial information. More

importantly, the smaller the specimen, the smaller the

signal-generating volume elements as higher resolutionswill be necessary. This potential conflict has recently been

termed the ‘‘Bermuda triangle of small animal MRI’’ (Ja-

kob 2011), and the understandable desire of zoomorphol-ogists for maximum achievable resolution is not easy to

meet. Glover and Mansfield (2002) quote the resolution

limit of MRI due to molecular diffusion in the case ofhydrogen protons to be approximately 8 lm. Movement or

flow artifacts in living organisms might cause even more

severe restrictions on resolution. Some of these problemscan be tackled by proper preparation and fixation of the

sample. However, manipulating the molecular dynamics of

a sample may also influence relaxation behavior of thetissue under study. Hence, physical limitations on the

microscopic level like, for example, molecular diffusion

and T2 relaxation can usually not be corrected, in particularwhen studying biological samples that cannot be manipu-

lated internally.

On the other hand, approaches involving hardwareimprovements, for example the use of phased array coils

(Blamire 2008) or cryo probes (Baltes et al. 2009), can

increase the available SNR by a factor of 2–3. Furtherprogress can be seen in the development of sequences with

ultra-short echo times. Such improved acquisition schemes

increase the available signal or even make tissues that wereinvisible with conventional techniques now detectable, for

example tendons and cortical bone (Blamire 2008).

Although these approaches do not bypass the fundamentallimit in resolution, they at least allow approaching it and

may enable a considerable reduction in image acquisition

time.In summary, the micrometer scale represents the current

physical limit for conventional MRI applications. Near-future developments will predominantly reduce acquisition

times rather than yield major benefits in spatial resolution.

The more distant future will show how much of the newideas from other specialized fields can be imported into life

sciences to further improve MRI and its multiple applica-

tions in zoology.

Conclusions

We are convinced that the morphological study of zoo-

logical specimens can considerably profit from an extendeduse of MRI. The potential of this technique for zoology lies

particularly in the capacity of MRI to reveal gross mor-

phology in situ, both in vivo and ex vivo. Furthermore,systematic scanning efforts will provide a boost to zoology

by triggering new research, especially so when MRI is used

in combination with advanced 3D modeling and visuali-zation techniques as well as dataset deposition in voxel

repositories (Giribet 2010; Ziegler et al. 2010b; Rowe and

Frank 2011). The density of adequate MRI scanners iscurrently high enough to provide the logistic framework for

such studies, although basic zoological research using MRI

systems can be impeded by the sometimes prohibitive costof scanner access. Nevertheless, previous comprehensive

studies on vertebrates as well as invertebrates have dem-

onstrated that there may be ways to tackle this problem.Numerous metazoan clades have been shown to be suitable

for systematic MRI analysis and we hope that the multitude

of further NMR applications will inspire the members ofthe zoological community to increase the use of these

exciting technologies in their field.

Acknowledgments We would like to thank Thomas Bartolomaeus(Bonn, Germany), Sven Gemballa (Tubingen, Germany), MatthiasGlaubrecht (Berlin, Germany), Alexander Gruhl (London, UnitedKingdom), Alexander Haas (Hamburg, Germany), Markus Koch(Bonn, Germany), Janina Lehrke (Bonn, Germany), Carsten Luter(Berlin, Germany), Christian Muller (Frankfurt, Germany), ThomasStach (Berlin, Germany), and Esther Ullrich-Luter (Berlin, Germany)for specimen supply. We also gratefully acknowledge editorial sup-port by Christopher Witte (Berlin, Germany). Bivalve research wassupported by AToL grants from the National Science Foundation,USA, to Rudiger Bieler (#0732854), Paula M. Mikkelsen (#0732860),and Gonzalo Giribet (#0732903). We are grateful to Thomas

Zoomorphology (2011) 130:227–254 249

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Bartolomaeus, Doug Eernisse and one anonymous reviewer for theirsupportive comments on this manuscript.

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