In Vivo 3D Digital Atlas Database of the Adult C57BL/6J Mouse Brain by Magnetic Resonance Microscopy

In this study, a 3D digital atlas of the live mouse brain based on magnetic resonance microscopy (MRM) is presented. C57BL/6J adult mouse brains were imaged in vivo on a 9.4 Tesla MR instrument at an isotropic spatial resolution of 100 μm. With sufficient signal-to-noise (SNR) and contrast-to-noise ratio (CNR), 20 brain regions were identified. Several atlases were constructed including 12 individual brain atlases, an average atlas, a probabilistic atlas and average geometrical deformation maps. We also investigated the feasibility of using lower spatial resolution images to improve time efficiency for future morphological phenotyping. All of the new in vivo data were compared to previous published in vitro C57BL/6J mouse brain atlases and the morphological differences were characterized. Our analyses revealed significant volumetric as well as unexpected geometrical differences between the in vivo and in vitro brain groups which in some instances were predictable (e.g. collapsed and smaller ventricles in vitro) but not in other instances. Based on these findings we conclude that although in vitro datasets, compared to in vivo images, offer higher spatial resolutions, superior SNR and CNR, leading to improved image segmentation, in vivo atlases are likely to be an overall better geometric match for in vivo studies, which are necessary for longitudinal examinations of the same animals and for functional brain activation studies. Thus the new in vivo mouse brain atlas dataset presented here is a valuable complement to the current mouse brain atlas collection and will be accessible to the neuroscience community on our public domain mouse brain atlas website.


INTRODUCTION
Three-dimensional (3D) digital brain atlases have become indispensable tools for scientists in the neuroscience community. The 3D atlases are used to quantify accurately the volumes and shapes of brain structures, for statistical mapping of functional brain activation in a well-defi ned stereotaxic anatomical space, and for mapping of gene expression patterns (Brill, 2006;Ng et al., 2007;Schwaber et al., 1991;Zaborszky and Vadasz, 2001; http://www.brain-map.org/). The average human brain atlases that are based on hundreds of magnetic resonance imaging (MRI) scans of different normal individual brains and the probabilistic human brain atlases (Ahsan et al., 2007;Ashburner and Friston, 2005;Collins et al., 1995;Evans et al., 1993Hammers et al., 2003;Holmes et al., 1998;Mazziotta et al., 2001) have facilitated discoveries of new specifi c morphometric signatures in several diseases including Alzheimer's disease (Carmichael et al., 2005;Chetelat and Baron, 2003;Sowell et al., 2003;Thompson et al., 2001) as well as normal child brain development (Dager, 2007;Lee et al., 2007). These approaches may eventually become the gold standard for diagnosis as well as provide clues to alteration in neurocircuitry and gene expression patterns relevant for pathophysiology of a wide variety of diseases.
In parallel to human brain atlas neuroinformatics, morphometric studies of genetically engineered mouse models have also been undertaken to quantify and characterize brain anatomical 'signatures' under controlled genetic conditions. Until recently, morphometric data have most frequently been extracted by manually outlining regions of interest in the mouse brain MRI images and compared with corresponding data from wild-type controls. This approach has already lead to discoveries of gross abnormalities both in vivo and in vitro such as enlarged ventricles in vivo in a mouse model of transient cerebral ischemia (McDaniel et al., 2001), hydrocephalus in SPAG6-and SPAG16L-defi cient mice, a model of Knobloch syndrome (Zhang et al., 2007), and hippocampal atrophy in a PDAPP mouse model of Alzheimer's disease in vitro (Redwine et al., 2003) which was not confi rmed in vivo (Benveniste et al., 2007). Volume changes in vitro in amyotrophic lateral sclerosis (ALS) mouse models of genetic (Özarslan et al., 2006) or dietary origin (Petrik et al., 2007;Wilson et al., 2004) have also been demonstrated. Recently, Bock et al. (2006) reported reduced cerebellar volume and other changes in the inferior colliculus and the olfactory bulbs in cdf mutant mice using both in vivo MRI and atlas-based semi-automated image analysis, demonstrating the utility of atlas-based neuroinformatics for mouse brain phenotyping.
Several 3D mouse brain atlases derived from formalin-fi xed samples and in vitro magnetic resonance microscopy (MRM) technology with resolution below or near 100 µm in at least one image dimension (Benveniste and Blackband, 2002) have recently become available (Badea et al., 2007;Kovacevic et al., 2005;Ma et al., 2005;Mackenzie-Graham et al., 2004). These newly published atlases are now increasingly being used as templates for semi-or fully-automated brain structure segmentation (Ali et al., 2005;Badea et al., 2007;Chen et al., 2005) or for mapping of other rodent brain imaging modality data especially brain activation data acquired in vivo (Dorr et al., 2007;Mirrione et al., 2007;Zhang et al., 2007). However, the atlas templates derived from in vitro specimens are not ideal for the mapping of in vivo data due to postmortem and/or the formalin fi xation process which change brain shape and size. For example, the human average in vivo brain atlases (Montreal Neurological Institute brain or MNI brain) (Evans et al., 1993) were designed in part to replace the postmortem single brain template of the Talairach and Tournoux atlas (Talairach and Tournoux, 1988) for more accurate in vivo data interpretation. Thus, large discrepancies were found between the in vivo MNI average brain and the original Talairach coordinates (Brett et al., 2002;Chau and McIntosh, 2005;Lancaster et al., 2007;Toga, 2002;Uylings et al., 2005;Van Essen, 2002).
Similar to the human brain data it is reasonable to hypothesize that for analysis of in vivo mouse brain data, an in vivo atlas system would likely be more suitable. To our knowledge, there are no complete mouse brain atlas templates available that are derived from in vivo MRM images and there is also limited quantitative information in regards to morphological differences between in vivo and in vitro mouse brains. The general lack of in vivo mouse brain atlas templates is probably related to the challenges involved in acquiring in vivo MRM images with suffi ciently high SNR and CNR to identify subtle and small anatomical structures (Benveniste and Blackband, 2002). As a continuation of our previous mouse brain atlas work (Ma, et al., 2005), we have constructed an adult male C57BL/6J mouse brain atlas database derived directly from in vivo T2-weighted 3D MRM images. To optimize the quality of the MRM images we designed new MR hardware and determined the most favorable parameters for contrast and SNR trade-offs. The new in vivo data are quantifi ed and compared to our previous in vitro atlas data and demonstrate non-trivial and unexpected structural deformations caused by in vitro processing.

Animals and preparation for imaging
Inbred C57BL/6J male mice, 12-14-week old, weighing 25-30 g were used (Jackson Laboratory, Bar Harbor, ME). All protocols for live animal experiments were approved by the Institutional Animal Care and Use Committee. For MR microscopy (MRM) the mice were initially anesthetized with an intraperitoneal injection of a mixture of Nembutal (50 mg/kg), glycopyrrolate (0.01-0.02 mg/kg) and 0.9% saline. A gas mixture of oxygen and isofl urane (1-2%) was used for anesthesia maintenance. The electrocardiogram (ECG), respiratory rate and body temperature were monitored and recorded constantly with an MR-compatible small animal monitoring system (PC_SAM, model #1025, SA Instruments). The body temperature of the mouse was kept at 36.5°C and the respiratory rate was maintained around 50-65 bpm by adjusting the concentration of the isofl urane gas mixture.

MR microscopy
All the in vivo mouse brain MRM images were acquired on a superconducting 9.4T/210 mm horizontal bore magnet (Magnex) controlled by an ADVANCE console (Bruker) and equipped with an actively shielded 11.6 cm gradient set capable of providing 20 G/cm (Bruker, Billerica, MA). A birdcage radio-frequency (RF) coil (inner diameter 72 mm) was used as the transmission RF coil and a 30-mm-diameter surface RF coil as the receiver coil. We designed a new animal positioning system which provided more robust support to the animal cradle as well as better support of the RF birdcage coil against acoustic vibrations generated by the gradients and other sources. The new design also allowed us to place consistently the region of interest (i.e. the brain of the mouse) within the homogeneous center of the magnetic fi eld (Figure 1). The system comprises a long bore tube, which is supported at both ends via brackets attached directly to the exterior of the magnet cryostat (Figure 1). Four foam 'donut' shaped gaskets within the bore help dampen acoustically induced vibrations of the bore tube ( Figure 1A). The second major part is the inner tube which holds the animal cradle and the surface coil mounted directly on fi xtures built into the animal cradle (shown assembled in Figures 1B,C). The mouse to be scanned is placed in the supine position with its head fi xed on top of the center of the RF surface coil by the head fi xture which also includes a mouth bar (for further details on the technical design see Smith et al., 2008).

MR image acquisition
T2-weighted MR data were generated with a 3D large fl ip angle spin echo sequence that shortens TR and the total scan time (Ali et al., 2005;Bogdan and Joseph, 1990;DiIorio et al., 1995;Elster and Provost, 1993;Ma et al., 1996). In pilot experiments, optimal SNR and tissue contrast was achieved at a fl ip angle of 145° (number of excitations = 1; TE = 7.5 ms; TR = 400 ms). The mouse brains were imaged under two different spatial resolution schemes: 100 µm isotropic resolution (scan time ∼2.8 hours) and 100 µm in-plane resolution with 200 µm slice thickness (scan time ∼1.5 hours). The latter was implemented to determine the spatial resolution effects on the image contrast-to-noise, segmentation conditions and the accuracy of the resulted quantitative morphometry with the overall goal of reducing scan time if at all possible for future studies. The longest dimension of the mouse brain was set as the frequency encoding direction, with the remaining orthogonal directions sampled by phase encoding techniques. Image reconstruction was performed using Bruker Paravision software (version 4.0). A 3D fast Fourier transform was applied without any apodization, fi ltering or zero-fi lling. In all cases, 3D datasets were reconstructed into 16-bit grayscale images.

Creation of individualized atlases using a semi-automatic approach
In order to create individualized atlases, a fi rst step is to defi ne and segment the individual anatomical structures from the MRM images of the mouse brain. The segmentation procedure we used was essentially similar to that described previously (Ma et al., 2005) with the exception that the fi rst in vivo reference brain was segmented semi-automatically using our existing in vitro C57BL/6J mouse brain atlas (http://www.bnl. gov/ctn/mouse). The in vitro reference image was fi rst registered with the in vivo image to be segmented (referred to as 'target' image) using linear and non-linear transformations [using both RVIEW (Studholme et al., 1996) and AIR_5.2.5 software packages ]. The same transformation parameters were subsequently applied to bring the atlas of the in vitro brain into registration with the target in vivo brain hereby producing a pilot segmentation of the target image. Due to registration errors and especially due to inherent morphological differences between in vitro and in vivo mouse brains (Figure 6), the initial pilot segmentation did not match perfectly with the true structure boundaries in the in vivo image. Therefore the automatic segmentation result necessitated further smoothing and refi nement which was done semi-automatically using a commercial 3D visualization and modeling software package (Amira 3.1, TGS, San Diego, CA).
The fi rst segmented in vivo brain image was subsequently used as the new reference template to segment all other in vivo images since it registered better with the remaining in vivo group than the original in vitro reference brain. By repeating the above described processes a total of 12 in vivo mouse brain images were segmented. Each of the segmented in vivo data sets serves as an individualized atlas with 20 outlined brain structures which included: neocortex, hippocampus, amygdala, olfactory bulbs, basal forebrain and septum, caudate-putamen, globus pallidus, thalamus, hypothalamus, central gray, superior colliculi, inferior colliculi, the rest of the midbrain, cerebellum, the rest of the brainstem (i.e. pons and medulla), corpus callosum/external capsule, internal capsule, anterior commissure, fi mbria, and ventricles. Quantitative structural information such as the averaged volumes and surface areas of each of the 20 structures were also extracted.
Creating the average atlas, the probabilistic atlas and average voxel deformation maps A minimal deformation brain or an average brain is a simulated brain located in the geometric center of the population and represents the average shape and signal intensity of the group (Guimond et al., 2000;Ma et al., 2005). Therefore an average brain atlas is assumed to be a better representative of the whole group. The procedure of creating the average brain and atlas has been described in Ma et al. (2005). Initially, one of the individual brains and/or atlases was assumed to represent the group average. It was then iteratively updated based on the average transformation parameters of all the brains within the group and fi nally converged to the true geometric center of the group. As previously reported in Ma et al. (2005), we used the similarity index (SI) between two intermediate average atlases to monitor the convergence process. A similarity index close to 1 means little discrepancy between two intermediate average atlases and is an indication of convergence.
A probabilistic atlas contains structural spatial distribution information in the form of a probability within each voxel. The procedure for constructing the probabilistic atlas was the same as described in Ma et al. (2005). We used rigid-body transformation to align each individual in vivo brain image to the in vivo average brain template and then determined for each voxel the maximum probability of the structure spatial occupations. The probabilistic atlas is a powerful tool for phenotyping and provides a priori probabilistic information for Bayesian based automatic segmentation algorithms (Ali et al., 2005;Mazziotta et al., 1995Mazziotta et al., , 2001. The average voxel deformation/displacement map was constructed by an initial linear and subsequent non-linear registration of all the individual brain atlases to the average brain atlas during which the non-linear voxel deformation was measured and recorded. By initially normalizing all individual brains through linear transformations, the deformation map represents a higher order average local geometrical difference within the group and provides direct visualization of these variations. In displacement/deformation maps, structural geometries within or between groups are revealed at the voxel level because the color coding at each voxel represents the average distance that each homologous voxel in the individual brains travels in order to register with the template brain. For example, the brighter the color, the bigger the distance a given voxel traveled to register with the template brain. For further details of the procedure of creating these atlases see Ma et al. (2005).

Statistics
Statistical evaluations of volume and surface area measurements were conducted on the extracted data from the 3D images acquired from the 12 mice in vivo. The volume and surface data of each of the 20 structures measured in vivo were compared with previous acquired in vitro data (Ma et al., 2005) using an unpaired, two-tailed t-test. Intragroup data were analyzed using a paired t-test. A p-value < 0.05 was considered statistically signifi cant.

Anesthesia and animal stability during imaging
In spite of good fi xation of the mouse head in the cradle using the mouth bar, the respiratory pattern often induced severe motion artifacts and hence compromised MRM image quality especially for longer scans. Figure 2 displays the time course of the spontaneous respiratory rate of seven individual C57BL/6J mice anesthetized with Nembutal and supplementary isofl urane inhalant gas during a 4-hour MRM scanning procedure. The fi gure demonstrates that the respiratory rate of the mice was stable during the fi rst 2 hours (e.g. respiratory rate at 1 and 2 hours of scan time was 60.3 ± 9.6 bpm and 60.6 ± 9.6 bpm, respectively). However after 2 hours the respiratory rate trended towards being faster and more fl uctuating in the later scan period (e.g. at 3.3 hours the average respiratory rate had increased to 69.8 ± 18.8 bpm, p < 0.05). The relatively non-fl uctuating respiratory rate during the fi rst 2 hours could be due to more stable physiological conditions (e.g. open airways, better pulmonary function and less compromise of respiratory muscles) in this initial time period.

Effect of scan time and spatial resolution on segmentation
Considering the technical challenges and time demand involved in high resolution in vivo mouse brain imaging, we tested the effect of voxel size on MRM image segmentation. We acquired MRM images with a spatial resolution of 100 × 100 × 200 µm 3 requiring 1.5 hour scan time and 100 × 100 × 100 µm 3 requiring about 2.8 hours of scan time. Approximately 30% of the 3-hour higher resolution scans were discarded due to motion artifacts and/or animal death which we attribute largely to respiratory failure. In contrast, 95% of the lower resolution scans were successful. Figure 3 shows the clear difference in CNR between the low-and high-resolution scans. For example, the substructures in hippocampus such as the CA1, the hippocampal sulcus and the dentate gyrus were poorly defi ned on the low-resolution scans. Figure 4 shows the percentage difference between the 20 brain structure's volumes and surface areas derived from the lower resolution (n = 12) and the high resolution groups (n = 12). As can be observed, for smaller structures such as the fi mbria, the anterior commissure or the globus pallidus, the percentage difference in volume and surface area tended to be slightly larger (although not statistically signifi cantly). For example, the volume of fi mbria was 22.7% smaller measured from lower resolution scans than from higher resolution scans. In order to maximally reveal subtle structure details, we used the higher resolution scans to create the presented in vivo atlases. But for the majority of brain structures, the lower resolution scans are clearly adequate in volume and surface area measurements (Figure 4). This information is important when designing future in vivo phenotyping experiments where limiting scan time is desirable in physically fragile transgenic mouse models of human diseases.

The in vivo C57BL/6J atlas and database
In the following sections, for optimal data analysis and description, all visual and quantitative assessments of the new in vivo C57BL/6J mouse data were compared with previously published in vitro C57BL/6J mouse data (Ma et al., 2005). Figure 5A shows the representative axial, coronal and sagittal slices from an in vivo MRM C57BL/6J mouse brain dataset. Figures 5B-D display the corresponding reconstructed atlas surface in 3D. We were able to identify and segment 20 different anatomical regions from the in vivo images. However, certain parts including the boundaries of the posterior hypothalamic area, the globus pallidus, the caudate-putamen and the fi ne extensions of the white matter fi bre tracts were challenging to defi ne and we had to rely on parallel visual comparisons with existing 2D histologic atlases (Franklin and Paxinos, 1997;Hof et al., 2000) for complete segmentation of these regions. In Figure 6, we have compared representative sections from the new in vivo mouse brain data and the previous in vitro data (Ma et al., 2005) side by side. Apart from the obvious differences in overall CNR and SNR, the most signifi cant visual difference between the two types of images was the appearance and size of the ventricles. In vitro, the ventricles had shrunk to such a degree that most parts of the third and fourth ventricles were not identifi able (and consequently not included in Ma et al., 2005). Other structural differences between the in vivo and the in vitro brains were also indirectly revealed during the semiautomatic segmentation process. For example, a combination of an initial linear and a subsequent 169-parameter non-linear warping (in AIR_5.2.5) had to be applied to achieve an acceptable 'fi t' when  registering the in vitro templates with the individual in vitro brain images. In comparison, when registering two in vivo specimens, a rigid body registration based on mutual information (in RVIEW) alone would render an acceptable pilot segmentation.

The individual atlases.
The average mouse brain/atlas and derived voxel deformation maps. Figure 7 shows an axial slice from the constructed average in vivo MRM brain with its corresponding atlas superimposed. As expected the average brain has better SNR than any of the individual in vivo brain    The signal-to-noise ratio (SNR) is clearly superior to the single brain atlases (compare with Figures 5 and 6).
MRM images (compare with Figures 3, 5 and 6). During the iterative construction process of the average in vivo atlas, the convergence index reached 0.99 to 1 for all structures at the third iteration whereas for the corresponding in vitro average brain atlas (Ma et al., 2005), an overall convergence index of 0.98 to 1.0 required fi ve iterations. The fact that less iteration were needed for convergence among the in vivo brains is suggestive of less general geometric variability of the in vivo group compared to the in vitro group.
To further quantify and visualize the geometrical differences between the in vivo and in vitro brains at the voxel level, we created a series of voxel displacement/deformation maps within and between the two groups. Figure 8 shows the resulted average voxel deformation maps from (1) mapping each of the ten in vitro brains to their average in vitro atlas template (Figures 8A,D); (2) mapping the 12 in vivo mouse brains to their average in vivo brain atlas (Figures 8B,E,G) and (3) mapping each of the 10 in vitro brain to the in vivo average brain (Figures 8C,F). We wish to point out that the deformation/displacement values were only measured during the 169 parameter non-linear warping in AIR_5.2.5 (i.e. the voxel displacements occurred during rigid-body or affi ne transformations in the initial registration process were not included) in order to exclude the linear variations such as scale and orientations of individual brain images. Also, the distance maps bear unavoidable registration errors especially for the smaller structures which are harder to be correctly registered, such as external/internal capsules, ventricles and fi mbria. Therefore care has to be taken when interpreting the deformation maps at these locations.
When visually comparing the geometrical deformation map of the in vitro group (Figures 8A,D) with that of the in vivo group (Figures 8B,E), it is clear that for both conditions (formalin-perfusion/in vitro versus in vivo), the cerebellum, neocortex, olfactory bulbs and the brainstem contained surfaces with the largest displacements whereas structures located more centrally displayed the least. Furthermore, the in vitro group displayed more areas with quantitatively higher displacement values than the in vivo group, especially at the level of the cerebellum and olfactory bulbs. For both conditions, displacements greater than 0.5 mm mostly occurred at the level of the brain stem (Figure 8). In vitro, the brain stems were curved much more ventrally (Figures 8A,D); in vivo, the curvature of the brain stems also varied greatly probably due to different positioning of the animal's cervical spine which would affect the shape of brain stem. Interestingly, the in vivo displacement map revealed a slight left-wards neocortical asymmetry ( Figure 8B). Figures 8C,F show (on a voxel by voxel level) the local deformations the in vitro brains had to undergo when 'conforming' themselves to the average in vivo brain and thus directly display the geometric alterations between the two groups in 3D. Figure 8B can therefore be directly compared to 8C because both groups were mapped to the same in vivo average brain template. It is evident from the color coding that the areas in vitro associated with largest deformations when matching to the in vivo average brain were located frontally and caudally. We measured the statistical signifi cance of the different local deformation characteristics of both groups (Figures 8B,C) by using a voxel-wise unpaired student t-test. Figure 9 shows the result of the t-test and confi rms that the brain stem, olfactory bulb, and cerebellum display signifi cantly larger deformations in vitro compared to in vivo. Furthermore, structures located ventrally show larger in vitro ∼ in vivo differences than structures located dorsally. Table 1 lists the average volume and surface areas of each of the 20 segmented regions of the new in vivo brain data as well as quantitative comparisons with the previous collected in vitro data (Ma et al., 2005). Please note that in Table 1, the structure names marked with a '*' signifi es that the percentage changes were measured from the in vitro average atlas instead of from the individual in vitro brains in order to reduce the effects of the brain stem (which is often damaged in vitro) and account for the slightly different olfactory bulb partitioning during the construction of the in vivo atlases in which the piriform cortex was included as part of the olfactory bulb (the in vitro average atlas has been updated to be consistent with the partitioning of the in vivo atlases). We also computed the total brain volume and surface area with or without including the brain stem and both measurements show signifi cant total brain volume reduction in vitro (6.5% reduction measured without brain stem or 10.1% reduction with brain stem, p < 0.01).

Figure 8. The average local deformation maps of: (A) 10 in vitro individual mouse brains mapped to the average in vitro brain. (B) 12 in vivo individual mouse brains mapped to the average in vivo brain. (C) 10 in vitro individual mouse brains mapped to the average in vivo brain. (D), (E) and (F): The ventral views of (A), (B) and (C), respectively. (G) A cross sectional view of (B) and (E).
Note: the images are not strictly proportional. The color represents the average distance a given voxel underwent following registration of each of the individual brains to the average brain template. Table 1 shows that the standard deviations and thus intragroup variability of the in vivo brains are very minor with individual regional volume standard deviation ranging from 0.2 mm 3 (internal capsule and anterior commissure) to 7.5 mm 3 (neocortex). Importantly Table 1 also demonstrates that among all of the in vitro structures, the ventricles exhibited the most signifi cant shrinkage (78.6% decrease in volume and 68% decrease in surface area with p < 0.01) compared to the in vivo data, which is consistent with previous visual observation. Additionally, the following structures showed signifi cant volume and/or surface area reduction in vitro versus in vivo: the brain stem, the cerebellum, the olfactory bulbs, the hypothalamus, the superior colliculi, the inferior colliculi, the hippocampus, the basal forebrain and the septum. Interestingly, as further noted in Table 1, the following structures show expansion in vitro including the caudate putamen, the amygdala, the central gray, the globus pallidus, the internal capsule and the anterior commissure. It is at present unknown why some structures are more affected and shrink while others are unaffected or expand. For smaller structures such as the internal capsule, the anterior commissure, the fi mbria and the external capsule, the results may be unreliable due to segmentation errors secondary to inferior SNR, spatial resolution and volume averaging in vivo compared to in vitro. Alternatively, the various brain regions may be more or less affected by the ventricular space collapse and undergo different shear stresses in vitro, resulting in either expansion or shrinkage. This might for example explain why the caudate putamen expands because it is located adjacent to the ventricles. However we have not attempted to model or test this theory. The regional differences in myelin content might also play a role but we have no systematic measure for how this relate to the volumetric changes at this time. Figure 10 shows the in vivo probabilistic atlas in 3D and in cross-section. The bright red color corresponds to the probability of 1 indicating that all the 12 brains have the same structure and/or shape   423.9 ± 9.9 −6.5 0.00 1191.8 ± 30.3 1.1 0.47

* indicates that the values were measured from the in vitro average atlas instead of the individual brains to reduce the effects of tissue damage in brain stem and the slightly different olfactory bulb partitioning of the in vitro atlases (the in vitro average atlas has been updated to be consistent with the partitioning of the in vitro atlases which includes piriform cortex as part of the olfactory bulb). The total brain volume and surface areas were measured
with and without inclusion of the brain stem. at this location. The lower probabilities are represented by green and blue colors indicating higher group variability. As can be observed, the variability was generally very small inside each structure. Higher variability typically occurred at structure boundaries such as towards the cortical surface, the surface of the cerebellum, brain stem and smaller structure boundaries such as the external capsule, the internal capsule and the anterior commissure. In addition, hard-to-defi ne boundaries such as those between the hypothalamus and the thalamus or between the superior and inferior colliculi were also prone to low probability values which could indicate errors caused by possible segmentation inconsistencies at these locations. Furthermore, it is well-known that registration errors affect smaller structures more than larger structures which could also contribute to the lower probabilities observed at the boundaries of small and narrow structures such as white matter tracts. The probability atlas also shows that the brain stem displays a lower interior probability than any other structure primarily due to the inconsistent positioning of the animal's cervical spine during MRM scans.

DISCUSSION
The major results of this study are (1) the presentation of an in vivo C57BL/6J brain atlas derived from MRM images with 20 different anatomical structures identifi ed; (2) quantitative meta data (e.g. volumes and surface areas of the 20 structures) demonstrating minimal intragroup variability among the 12 in vivo genetically identical C57BL/6J mouse brains; (3) an average in vivo MRM atlas template of the C57BL/6J mouse brain (n = 12); (4) a corresponding in vivo C57BL/6J probabilistic atlas; (5) a local group deformation map based on the 12 in vivo C57BL/6J mice brains demonstrating the intragroup voxel-wise geometrical variations and fi nally (6) quantitative data defi ning differences between in vitro and in vivo C57BL/6J mouse brain morphometry.

In vivo MRM mouse brain images
Choice of T2-weighted pulse sequence. In this study our goal was to create an in vivo C57BL/6J mouse brain atlas using MRM technology. We therefore strived to create the best possible MRM images from the point of view of SNR and brain anatomical CNR, while at the same time considering the necessary tradeoffs to be taken to reduce the total scan time. We chose to use a T2-weighted 3D MRM sequence with a large fl ip angle which would reduce TR and therefore scan time while at the same time preserving its T2-weighting (Ali et al., 2005). T2-weighted MR contrast is advantageous for imaging rodent brain anatomy in comparison with T1-weighted sequences (Benveniste et al., 2000). The shortened TR allowed us to achieve a spatial resolution of 100 µm in less than 3 hours. Several other time-effi cient T2-weighed sequences [e.g. T2-weighted rapid acquisition with relaxation enhancement (RARE) pulse sequence] are available which also provide high tissue CNR, but our initial pilot tests showed the images with RARE factors greater than 8 were more subject to motion artifacts causing lower success rates, i.e. inferior SNR and CNR (data not shown). Reducing the RARE factor will typically increase scan time (from increased signal averaging) in order to achieve similar image quality. For example using a 3D RARE sequence with the following parameters: TR of 1.2 s, RARE factor of 4, isotropic resolution of 100 µm and two signal averages would require about 4.2 hours of scan time, assuming that no anti aliasing factors would be required in the two phase encoding directions.
Physiological and non-physiological motion. MRM images acquired in vivo are affected by the physiological motion from the beating heart, the diaphragm and involuntary refl exes from the airways. The image artifacts from physiological motion can be minimized to some extent by immobilizing the head of the animal and/or using pulse sequences that are acquiring data synchronously with the ventilation and cardiac activity. We did immobilize the head of the animal using our custom build animal cradle but did not implement scan-synchronous ventilation as the total scan time would have increased dramatically. For example, with a spontaneous respiratory frequency of 60 bpm the effective TR would have increased from 0.4 s to 1 s and consequently increased scan time from 2.8 hours to 7 hours). The animals had to be anesthetized, immobile and physiologically stable for the scan duration to minimize motion artifacts. Our anesthetic regimen included the hypnotic pentobarbital (Nembutal) and supplemental isofl urane inhalant gas administered to the spontaneous breathing mouse as needed to maintain the depth of anesthetic state. The time course of the breathing pattern over the 3-4 hours experimental period revealed a stable breathing pattern during the fi rst 2 hours and a more fl uctuating one in the later period of the scanning (Figure 2). The more erratic breathing pattern after 2 hours could be contributed to by different anesthesia effects and/or general compromise of the respiratory function. In rodents the anesthesia duration with 50 mg/kg Nembutal administered i.p. is about 30-60 minutes (Laber-Laird et al., 1996). Therefore, in order to maintain the respiratory frequency around 60 bpm, isofl urane was typically added on in escalating concentrations after 30-60 minutes when Nembutal was wearing off. However, both Nembutal and isofl urane interfere with the animal's respiratory effort by decreasing the tidal volume as well as the respiratory frequency. Therefore the more irregular breathing pattern was more likely due to general respiratory compromise secondary to prolonged spontaneous breathing under anesthesia in the supine position. For example, during normal spontaneous ventilation, the rib cage and the diaphragm contribute more to the thoracic expansion. With induction of anesthesia, the functional residual capacity decreases as the diaphragm fl attens which contributes to the formation of atelectasis (Miller, 2005). Furthermore, most anesthetics also affect pulmonary and laryngeal stretch receptors leading to tachypnea and low tidal volumes, which can inevitably lead to an unstable respiratory pattern (Miller, 2005). We are currently working on refi ning our anesthesia technique to further decrease this problem for future studies.
Non-physiological motion originating from the MR instrument itself in the form of vibrations (acoustical) from the activity of strong gradients can also interfere with image quality. We designed and implemented a new animal cradle/holder and positioning system to circumvent this problem. The new positioning system stabilized and maintained the position of the anesthetized animal and at the same time also secured the two RF coils' positions in relation to the area of interest (e.g. the mouse brain). In contrast to most commercial MR instruments where the animal positioning devices are designed as cantilevers attached to a table in front of the magnet, our system (i.e. the outer tube of the positioning system) is supported at both ends and fi xed to the outside of magnet. It is thus 'free-fl oating' inside the magnet bore and isolated from the gradient inserts which helps dampen the vibration noise generated by the gradients during imaging (Smith et al., 2008).

Mouse brain anatomy revealed on the T2-weighted MRM images.
The 3D T2-weighted MRM in vivo images which we acquired were of suffi cient quality to allow the identifi cation of 20 different mouse brain structures, but there were several structures and anatomical borders which were hard to defi ne on the in vivo images. In our previous in vitro mouse brain atlas MRM study, we were also able to identify 20 brain regions on the T2*-weighted high resolution images (47 µm isotropic) which were acquired on a 17.6T MR instrument and these images clearly had superior CNR compared to the in vivo images acquired here (cf. Figure 6). Previously, the anatomical segmentation of the in vitro images was also associated with diffi culties especially when defi ning certain areas such as the boundaries of the posterior hypothalamic area, the boundary of the thalamus and pretectum as well as the fi ne extension of white matter tracts (Ma et al., 2005). Some of the same areas which could not be accurately defi ned on the in vitro images were also diffi cult to defi ne in vivo, for example the borders of the posterior hypothalamic area and white matter tracts. On the other hand, additional areas were prone to segmentation inaccuracies in vivo including the globus pallidus and the caudate-putamen which were more clearly visualized in vitro (Ma et al., 2005), demonstrating the different CNR characteristics of in vitro and in vivo studies.

In vivo compared to in vitro
Our idea to create an in vivo C57BL/6J mouse brain atlas database including an average brain atlas originated from the human brain neuroinfomatics literature demonstrating that an average in vivo human brain atlas (e.g. the MNI brain atlas) derived from hundreds of different MRI brain scans is a better anatomical representation of the general population and from a statistical point of view therefore is more accurate for interpreting in vivo data (Chau and McIntosh, 2005;Lancaster et al., 2007). In contrast to the MNI brain atlas derived from MRIs of many individual brains with different genotypes and environmental exposures, the 12 C57BL/6J mice used in this study were genetically identical and had been exposed to the same environment during their lifespan. It is therefore not surprising that our in vivo data demonstrated minimal group variability. Similar to the human literature we documented morphometric differences between the in vivo brains and the formalin-fi xed in vitro brains. First, the statistical test on the voxel-wise displacement maps derived from in vitro and in vivo datasets demonstrated signifi cantly larger deformations in vitro in the areas of the brain stem, the olfactory bulbs, cerebellum and frontal cortex (Figure 9). Second, our volumetric data analysis demonstrated that many gray matter structures shrink, while other structures expand in vitro in comparison to in vivo (Table 1). Although we speculated that different shear stresses of each structure secondary to the collapse of the ventricles in vitro might be partly explaining the volumetric changes, myelin water fraction differences and segmentation errors are other possible answers. Further studies will be required to better understand and explain these variations.
Further, from direct visual observation of the MRI images (Figure 6) and the inter-group voxel-wise deformation maps (Figures 8 and 9), it can be seen that non-linear deformations occurred between in vitro and corresponding in vivo structures. It was also noted that in the pilot segmentation process, a 169-parameter non-linear warping (AIR_5.2.5) was usually required to achieve a good pilot segmentation for the in vitro brains whereas for an in vivo pilot segmentation only a rigid-body alignment was needed. This implied indirectly that the in vitro brains inherently had larger higher order non-linear intragroup variations. In other words, from a mathematical point of view the morphological differences between the in vitro target brains and the in vitro reference brain were best described by higher order non-linear deformations whereas variations amongst in vivo brains were best described by simple linear transformations. Third, in the process of constructing the average in vivo atlas, the similarity index between successive intermediate average brains reached 0.99 to 1 for all the 20 structures after only three iterations while fi ve to six iterations were needed for the in vitro group, which also indicated the diffi culty to register individual in vitro brains due to higher order morphological variations. All these factors will clearly affect correct registration between in vivo and in vitro data. This might be especially true for mapping data from other modalities such as PET or fMRI, for which only linear or rigid body transformations are often used during image registration (Vaquero et al., 2001). In these cases, in vivo atlases or templates certainly would be preferable. However, in vitro atlases (47 µm isotropic) have better spatial resolution than the in vivo atlases presented here (100 µm isotropic) and consequently reduced the partial volume effects especially when segmenting smaller brain regions. Further systematic studies are needed to investigate whether the in vitro atlases are advantageous to serve as the templates for other in vitro data such as histology slices since they may share similar deformations caused by the extra tissue processing and handling. For this reason, the in vitro and in vivo atlases are both valuable and necessary tools to the neuroscience community.
All our quantitative analysis presented in this paper was based on comparisons made between our new in vivo atlas data and the previous in vitro data in Ma et al. (2005). It is possible however that the results and conclusions we reached here would change if we compared with other available in vitro mouse brain atlas data that was processed differently. For example, the in vitro MRM C57BL/6J mouse brains atlas recently developed by Badea et al. (2007) was based on six different mouse brains imaged in situ (in the cranium) in order to reduce deformation seen with exercised brains. Interestingly, for most internal brain structures (e.g. hippocampus, thalamus), the volumes measured from Badea's atlas are in good agreement with those measured from the in vitro atlas data of Ma et al. (2005). Therefore for most structures, the above comparison between the in vivo and in vitro volumes also holds for Badea's data (Badea et al., 2007).

Feasibility of using lower spatial resolution images for general in vivo volumetric analysis
We also investigated the accuracy of neuroanatomical segmentation in MRM images acquired at a lower spatial resolution which would be useful to improve the overall imaging throughput for future in vivo phenotyping studies requiring high resolution 3D imaging. Our results showed that even with unavoidable partial volume effects in the lower resolution images, it was still feasible to defi ne the 20 brain regions. The lower resolution scans only required 1.5 hour scan time and resulted in overall fewer animal fatalities, an increased number of successful scans and accurate volume/surface area measurements. Thus for future mouse brain morphometric studies using MRM in vivo we recommend using the lower resolution sequence which is advantageous especially for mouse models of diseases and aging, as the mice are often physiologically weakened from alterations related to the genetic manipulation.

CONCLUSION
In summary, the in vivo 3D MRM based digital atlases of the C57BL6/J mouse brain constructed here provides a new computational framework for future mouse brain morphology and functional neuroimaging studies. The in vivo atlases not only provide the users with a comprehensive platform for analyzing in vivo neurological data, they also provide the necessary framework to compare in vitro and in vivo studies. The in vivo mouse brain MRI template is inherently the natural template for longitudinal MRM studies, which are usually carried out at a similar or a lower spatial resolution level. Therefore the in vivo atlases will have wide usage in computational morphometry and quantitative phenotyping of mice. Using our templates for pilot segmentation, users can also easily modify and create their own mouse brain atlases to meet their own special needs. Our online database (http://www.bnl.gov/ctn/mouse) can be accessed for downloading and visualizing our new in vivo atlases.