the availability of the mouse genome sequence and advances in genetic manipulation has resulted in multiple initiatives , both in the public and private sector , to produce mouse mutants on a large scale .
coordinated efforts are on way to systematically knock out all mouse genes to provide researchers targeting constructs , vectors , live mice , and phenotypic data at a variety of level and detail .
the relationship between the effect of genetic manipulation and the resulting phenotype is a key component to establishing a fundamental understanding of molecular and cellular process .
parallel to the growth in mouse models to study disease patterns , rapid strides have been made in developing imaging instruments specifically targeted to the small animal .
instruments like mri , pet , spect , ct , in vivo fluorescence , in vivo bioluminescence , and intravital imaging allow one to visually , and sometimes quantitatively , evaluate biological processes at the cellular and subcellular level in a living subject .   however , the major challenge in successfully characterizing morphological features of transgenic mouse models has been the trade - off between resolution and field of view .
researchers particularly want the ability to detect gross structural change or , in the absence thereof , localize on subtle variations at microscopic level in 3d in tissue and cell structures .
thus the ideal system used in characterizing transgenics should offer multiscale 3d visualization at a very high native resolution . in this paper
we describe the application of cryo - imaging system and the multiscale approach to characterize a transgenic mouse .
our group has developed a cryo - imaging system , which provides contrast rich , bright field anatomical , and fluorescence cellular and molecular imaging of an entire mouse with micron - scale resolution .
the system acquires three - dimensional ( 3d ) , very high - resolution , large field of view ( fov ) , brightfield anatomy and fluorescence molecular image volumes from sequential images of the tissue block face by alternately sectioning and imaging . in an earlier report , we have demonstrated few applications of cryo - imaging ranging from high - resolution anatomical imaging of whole mouse , vascular imaging , and molecular fluorescence imaging of fluorescent mouse models and embryos .
the system has been used for validation of mr studies of human blood vessel lesions , ex vivo characterization of human atherosclerotic iliac plaque , and very recently for single cell detection of fluorescently labeled cancer and stem cells .
true color enhanced volume rendering techniques provide fast 3d visualizations of cryo - imaged samples .
cryo - imaging is unique among all 3d in vivo ( e.g. , micro - mri ) and microscopic techniques , because it allows micron resolution and information - rich color contrast mechanisms over very large 3d fields of view .
mri imaging of whole mouse can produce gray - scale images after administering contrast agents with significantly less resolution .
optical projection tomography is limited to small samples such as embryos and requires treatments to reduce scatter and increase transparency .
diffuse optical tomography while tackling the light scatter problem with advanced algorithms   can image a whole mouse albeit at limited resolution providing little anatomical detail .
johnson et al . used very high - resolution ct and osmium tetroxide staining to image tissues and embryos .
serial histology techniques to create a 3d volume involve serially cutting the sections , applying histological processing , mounting on glass slides , digitizing the slides , and then creating a 3d volume from the two - dimensional ( 2d ) images .
such systems have been used for a variety of biomedical applications [ 13 , 14 ] including characterizing phenotypical change in large histological mouse model datasets .
however , such processing suffers from tears , tissue shrinkage , errors in image alignment , and typically time - consuming manual editing of 3d reconstructions .
systems have been mostly applied for imaging excised organs such as brains [ 16 , 17 ] and embryos [ 18 , 19 ] with adult whole mouse block - face imaging being limited to low - resolution photographs of sections taken for autoradiography studies   and ct and block - face imaging for creating a mouse atlas .
we have developed the robotic 3d cryo - imaging system and first applied it to high - resolution tiled imaging of a wild - type mouse .
now we report , for the first time , morphological characterization of a transgenic mouse recently generated by researchers at case western reserve university .
this mouse , dubbed  supermouse  by popular press , contains a chimeric gene in which the cdna for pepck - c was linked to the -skeletal actin gene promoter , expressing pepck - c in skeletal muscle .
the transgenic pepck - c mice were seven times more active than their controls in their cages , ran for up to 5  km at a speed of 20  m / min on a mouse treadmill ( control animals run for 0.2  km at this speed ) .
as reported earlier , the mice eat almost twice as much as controls but are half the body weight ; they also have an extended life span relative to the controls . in their seminal report
, the researchers have detailed the study of biochemical pathways to better understand the repatterning of energy metabolism .
the battery of tests involved assay of metabolites in blood and tissues , home cage activity testing , exercise capacity testing , respiratory quotient ( rq ) measurements during exercise , and histological and electron microscopy analysis of skeletal muscle .
characterization of the pepck - c mouse with a cryo - imaging , multiscale approach involves detailed high - resolution imaging of a whole mouse and a control mouse , processing and manipulations of enormous giga - byte sized data volumes , and enhanced 3d visualizations . in the following sections we describe the cryo - imaging setup , the imaging techniques , and enhanced visualizations in 3d at multiscale and qualitative and quantitative comparison of organs .
briefly , it consists of a cryo - microtome , a microscope imaging system , a robotic xyz positioner , and a computer control system .
the cryo - microtome is a motorized , large section , whole body cryo - microtome with section thickness adjustable between 240  m and a maximum specimen dimension of 250  mm  110  mm  50  mm .
an xyz robotic positioner carries the imaging system comprising of a stereo microscope , coaxial fluorescent attachment with multiple filter cubes , low light digital camera , and brightfield and fluorescent light sources .
the computer control system automatically pans the positioner over the specimen for a high - resolution tiled image acquisition and controls the sectioning and image acquisition sequence through the custom developed programmable sectioning and cryo - imaging ( prosci ) software .
the image processing and visualization system is a quad - core windows 64-bit pc with 32  gb of ram capable of handling large cryo - image volumes .
a suite of matlab custom programs and custom amira scripts are used for image preprocessing tasks and 3d visualization .
in order to acquire cryo - image data , mice covered under iacuc - approved projects , were euthanized in a method approved by case animal resource center which consists of either inhalation of carbon dioxide delivered from tank or anesthetization using an agent such as pentobarbital , at a dose prescribed by the case arc .
a cryo - embedding compound , oct ( optimal cutting temperature , tissue - tek , terrance , ca ) , was used to completely immerse the mouse in an aluminum foil mould .
the mould was then covered in more foil and placed inside a freezing chamber made of styrofoam and filled with liquid nitrogen .
after about 15 minutes , the mould assembly was removed from the liquid nitrogen bath and placed inside the cryo - microtome chamber to equalize the specimen temperature to the cryo - microtome temperature .
the mould was removed from the specimen after two to three hours and the frozen mouse block was mounted on the microtome stage using more oct . an initial  facing - off 
then the section thickness was set to 40  m , the imaging system was readied for a pixel size of 15.6  m , and the system was started to alternately slice and acquire tiled brightfield images of the block - face .
the system was run completely unattended with the user periodically alerted by the prosci program about imaging progress through email and cell phone text messages .
details of the transgenic mice are available elsewhere . the transgenic animal used in this study weighed 25.8  gm and was 421 days old when sacrificed .
once the mouse specimen was mounted on the stage , the operator selected the boundaries of the acquisition volume and the system calculated the number of tiles required per block - face taking into account the operator - entered minimum overlap zone between adjacent tiles , typically 15% .
the robotic system computed the coordinates of each tile based on the pixel size and automatically sectioned and imaged the entire volume .
brightfield images ( each 1360 by 1036 pixels ) were acquired in 6  4 tiling pattern to cover each block - face .
a total of 586 sections were needed for the transgenic mouse and 616 for the control . a metadata file stored the global coordinates , imaging parameters , and experiment parameters . following acquisition of 2d images on the imaging workstation ,
nonuniform illumination pattern was compensated using a reference image of a white card and compensated images for each tiled block - face were composited together to yield a typical 2d whole mouse section image of 6500 by 3600 pixels .
these stitched images were then automatically aligned to each other to correct for minor misalignments due to the repositioning error of the stage at the return point .
the total stitched 2d image stacks amounted to a data size of > 60  gb . since such a large dataset can not be loaded fully in the memory of the visualization workstation , we converted the stack of images into a large data access ( lda ) volume inside our customized amira visualization software .
lda allows us to subsample at various resolutions an entire mouse or organ , or to sample the data at full resolution within smaller cropped subvolumes . from a relatively low - resolution 3d mouse
the whole mice were segmented from the oct blocks through our fully automated volume visualization . since
cryo - image data is in color , unlike other gray - scale medical imaging data ( mri , ct , etc . )
we have designed color feature detectors which take into account the dominant color of a region to segment it .
examples of red , green , and blue ratio feature detectors ( fr , fg , fb ) are shown below , where r , g , and b refer to the 8-bit data for red , green , and blue channels , respectively , 
								 ( 1)fr = rr+g+b ,  fg = gr+g+b , fb = br+g+b . 
							
for instance , regions containing a high proportion of red such as liver , lungs , and vasculature can be extracted using the red feature detector fr . for detecting stomach and intestinal regions , which are predominantly brown in color , we exploited the fact that brown is composed of one part of   r , two parts of   g , and zero ( 0 ) parts of   b. a brown feature detector is therefore expressed as a weighted linear combination of red and green feature detectors : 
 ( 2)fbr=(0.33fr+0.67fg ) . 
							 for segmenting out the embedding compound oct , which has a uniform white color
a balanced color detector was used : 
								 ( 3)f=[abs(rg)+abs ( gb)+abs ( rb)]3imax  , where imax   = 255 . 
							
subsequent to feature detection , opacity transfer functions ( otfs ) were used to assign an appropriate  opacity value to each voxel .
below , we show an example of color - based step otf that we have employed in our visualizations :  
 ( 4)={255,f > t,0,ft , 
							 where t is an empirically determined threshold . in other algorithms
, we have optimized opacity by imposing several simultaneous constraints on an opacity function which maps each voxel to a custom opacity value .
enhanced volume rendering techniques for high - resolution color cryo - imaging data have been described elsewhere .        
individual organs such as the heart , brain , kidney , spleen , and glands were delineated using a combination of feature detection for initial automatic segmentation and then manually correcting the results . in organs such as the ovaries , where lack of uniform color and lack of high contrast between neighboring tissues prevented automatic approaches , manual segmentation was carried out in selected sections and 2d interpolation
volumes of organs and tissues were computed from pixel count of 2d segmentation , the 2d pixel dimension and section thickness .
the gross animal weights and brain volumes were used as normalizing factors for comparing organ volumes between the transgenic and the control .        
for comparing specific bone lengths such as the femur , digital sections through the axis of the bone were created through interactive multiplanar reformatting of the original 3d data volume .
measurement tools available in the software package were used to compute bone length and cross - sectional areas .
the length of the animal ( nose to base of tail ) was used as the normalizing factor for comparison across animals .
standard tools available in amira were used for volume presentation such as snapshot capture , and movie making .
the image acquisition and multiscale volume visualization is illustrated in figure 1 . from the stitched 2d image stack ( figure 1(a ) ) , a 60  gb 3d volume was created and visualized at multiple resolutions . in the down - sampled whole mouse rendering , major organs were clearly seen through transparent skin ( figure 1(b ) ) .
a bounding box was placed in the abdomen area to extract the region in highest resolution and organs such as the kidneys , adrenal glands , ovaries , and vasculature segmented and reconstructed in 3d ( figure 1(c ) ) .
for example , a section through the left ovary shows the inner medulla consisting of loose connective tissue and large blood vessels and a peripheral cortex region containing a number of ovarian follicles in different stages of development as small as 50  m in diameter .
a few fimbriae of the infundibulum of the uterine tube can also be seen . at this full resolution , these and other many small structures like 50  m villi of the small intestine , mesenteric blood vessels 20  m in diameter , thin walls of the vessels 30  m and skeletal muscle and nerve fibers were very clearly seen .
coronal section images composited from 24 (= 4  6 ) individual tiles are shown in figure 2 .
major organs like the heart , lungs , liver , salivary gland , stomach , spleen , pancreas , small intestine , colon and lymph node are visible .
the very visible peritoneum of the control mouse encases a large amount of visceral adipose tissue as compared to the pepck - c transgenic mouse .
this extraordinary difference in adipose tissue depots is a general finding for these animals . using semiautomatic segmentation techniques , we computed fractional vascularization in the visceral adipose tissue encased by the peritoneum in the lower abdomen area around the cervix .
the gross adipose tissue volume , estimated from voxels in 86 sections , was 38% more in the younger control mouse compared to the pepck - c transgenic mouse .
the percentage of tissue classified as vasculature within this limited adipose tissue mass was higher for the pepck mouse as compared to the control ( 2.5% vascularized in the transgenic animal ; 0.4% in control ) .
the highly vascularized pancreas extending from the spleen to the duodenum can be also seen in both the section images . in figure 3 ,
the dermal layers were distinct with an 83% thinner hypodermis for the transgenic as compared to the control ( 502  mm for control and 84  mm thick for the pepck mouse ) .
the epidermis of the control was 16% thinner than that of the transgenic ( 407  mm transgenic , 348  mm control ) .
the dermis was similar in thickness ( 578  mm transgenic , 621  mm control ) .
the parietal peritoneum , visible below the skin , was also measured and found to be same .  
cutaway through the 3d reconstructed hearts of the transgenic and control mice reveal the chambers of the heart , pulmonary trunk , and pulmonary veins .
manual segmentation of the heart from the 2d images also enabled computation of the heart volume .
the transgenic heart was 351.52  cubic  mm in volume while the control heart was 366.87  cubic  mm .
multiplanar reformatting allowed bone length comparison of femur and tibia of the pepck mouse and the control animal ( figure 5 ) .
the femur measured 11.46  mm ( transgenic ) and 12.41  mm ( control ) while the tibia measured 17.26  mm ( transgenic ) and 15.12  mm ( control ) . the body length from tip of nose to base of tail
was estimated to be 87.6  mm for pepck mouse and 83.8  mm for the control mouse .
when normalized to body length , the measures were similar for femur ( 13% for transgenic and 15% for control ) .
however the tibia was longer for transgenic ( 20% for transgenic and 18% for control ) .
the tibia to femur length ratio was calculated to be 24% higher for transgenic as compared to control .
cortical area was measured at right midtibial and midfemoral diaphysis by extracting digital planes transverse to the bone axes .
tibia cortical area was 0.74  mm for transgenic and 0.71  mm for control .
femora cortical area was 1.35  mm for transgenic and 1.38  mm for control .       
the pericardial fat around the heart was segmented from the 2d section images and volume visualized in 3d ( figure 6 ) .
the pericardial fat depots were more pronounced in the control mouse as compared to the pepck mouse which is visible readily in the 3d volume visualization . from the 2d segmentations ,
the major organs like brain , heart , kidney and spleen and glands such as pituitary , thymus and adrenal were segmented and volumes computed . the pericardial fat volume is also included . both absolute values as well normalized values by gross animal weights and brain volumes are presented .
the high - resolution color data volumes from the whole mouse that were obtained using the case cryo - imaging system and multiscale viewing provide researchers the ability to seamlessly bridge the requirements of resolution and fov .
most existing small animal imaging systems force the researchers either to scan the mouse at low - resolution to capture the entire animal in a single fov or to choose only a region of the whole mouse for investigation at higher - resolution .
as we have shown in this application , with cryo - imaging , image data is acquired at a very high - resolution , and using multiscale viewing , it is possible to view in 3d or 2d at the whole body level , zoom to a region at higher - resolution , zoom to an organ , and finally zoom to examine tissues at the highest resolution .
skeletal muscle fibers in the mouse , typically 45  m in diameter , were seen very clearly in cryo - images . in earlier work involving fluorescently labeled stem and cancer cells , we showed that one can zoom down to single cell level .
the multiscale approach provides an efficient way of handling extremely large whole mouse data sets . a tiled color bright field acquisition with an adult whole mouse using a 6  4 tiling configuration and 40  m section thickness generates > 60  gb of color image data   which is a prohibitively large size for volume rendering on a workstation with a conventional , single graphics processor .
if fluorescence acquisition is added , it imposes an even greater burden due to increased data size . as a remedy to the extreme data problem , we designed the multiscale volume rendering approach which greatly improves the visualization experience .
. some of the important criteria to consider during multiresolution volume rendering are data access time and graphics hardware limitations on the target machine .
data access time can be greatly reduced by employing modern solid state disk ( ssd ) hard drives , which generally have higher data read and write speeds as compared to conventional hard drives . as for graphics hardware , the larger the amount of graphics ram , the smaller the decimation that needs to be applied to produce high - resolution renderings of subregions within the large data sets .
first , it offers a convenient method of scanning an entire mouse since no special preparation involving stains are required .
second , a low - resolution view of the entire mouse with volume visualization of internal organs is available through automatic segmentation of oct and automatic 3d rendering .
third , the organs and tissues are displayed in true color providing a virtual necropsy experience .
fourth , data can be seamlessly explored at every level through user - friendly select - and - view bounding boxes .
semi - automatic segmentation through algorithmic extraction of features followed by interactive manual editing provides visualization and quantification options .
while this application has only utilized brightfield imaging , the system is capable of obtaining molecular fluorescence image data as well , thereby rendering unique versatility to the system .
the featuresets of the system make it useful for applications such as anatomical phenotyping . a particularly interesting approach for identifying mutant outliers was described by kovacevic et al .
where an annotated 3d atlas of the average mouse brain with estimates of average and variability was created from mri images , against which mutants could be compared .
given the ability to segment organs in color , cryo - imaging will be well suited for such analyses for transgenic mice .
the published work on pepck - c transgenic mouse contained histology of excised skeletal muscle tissue and low - resolution whole body mr imaging .
cryo - imaging of this transgenic strain and its control provides the first glimpse at characterizing some of the morphological difference between the pair .
the high amount of total body fat in the control , clearly visible in the 2d sections , is consistent with the published mr results .
however , unlike mri , the high - resolution of cryo - imaging allows distinction between various fat depots and color differences . as an example , we computed and compared the pericardial fat volumes and fractional vascularization in a region of the visceral fat depot in the abdomen .
we were also able to detect various organs and separate the dermal layers similar to histology analysis .
it has been observed that these transgenic mice live longer and are more energetic at an older age .
we chose regions and organs of the two mice to compare few standard ageing biomarkers .
we note that the older pepck mouse has less pericardial fat , less visceral and subcutaneous fat , thinner hypodermis and thicker epidermis as compared to the control animal .
skin thickness measurements are widely accepted as a biomarker of ageing while visceral and pericardial fat deposits are biomarkers of potential metabolic disorders .
the thymus and spleen were chosen as ageing biomarkers since the thymus is a primary lymphoid organ considered to be consistently sensitive to morphological effects and the spleen is a secondary lymphoid organ .
characterization of thymic and splenic weights in age - related studies in nonhuman primates has shown that the thymus becomes smaller with age while the splenic changes were not statistically significant . in this study
, we found the older pepck - c mouse has a larger thymus while the spleen is smaller than noted with the control animal .
we also noted a difference in overall tissue coloration between the pepck mouse and control . in our experience from cryo - imaging wild type mice ,
younger mice have an overall brighter tissue color . in this regard , the pepck - c mouse looks 
adipose tissue is pinker , consistent with our observation of increased vascularity . through observations such as these , cryo - imaging can point researchers to examine certain tissues in more detail .
a careful 3d volume measurement was carried out to compare the heart volume of this mouse with the control . while the absolute cubic volume was nearly the same , the transgenic heart volume was 40% greater when normalized by body weight .
when normalized by brain volume this difference reduced . since cardiac volume estimates derived from the cryo - images
are not correlated with the cardiac phase at the time of freezing , we estimated another measure of comparison .
voxels belonging to the blood pool in the cardiac chambers and vessels in the hearts were semiautomatically identified through the color detector scheme and were excluded from cardiac volume measurement .
the resulting cardiac tissue mass , when normalized by body weight , was found to be 22% more for pepck - c mouse than control .
comparison of other organ volumes such as brain and kidney and glands such as pituitary and adrenal showed a general trend of larger organs for the pepck - c mouse . in order to reduce the variability due to animal size and weight ,
organ volume - to - animal weights were computed . to reduce the variability due to lean body mass , organ - to - brain volume normalization was used . in the case of bone lengths ,
digital resectioning along arbitrary planes allowed bone morphometric data to be computed from the cryo - image volume .
measurements such as length and cortical area for tibia and femur were made for two comparisons .
it has been reported that short - term induced exercise in mice leads to differences in bone properties such as length and cortical area for the femur and tibia [ 2628 ] .
further , such changes have also been recorded in ageing studies [ 29 , 30 ] .  
the pepck - c mice are highly active and can endure sustained activity at maximal speed like running on mouse treadmills .
the particular mouse we cryo - imaged had participated in numerous unrelated high - activity studies and was chronologically older than the control mouse . although no longitudinal study was performed on either specimen , nonetheless , we were interested in comparing the normalized values .
the pepck - c mouse had a slightly higher value for tibia and lower for femora leading to a significantly higher tibia to femur ratio of 1.5 .
the ratio of 1.2 for the control mouse is similar to ratios derived from published reports .
the ratio of cortical area to bone length is considered to be indicative of bone growth .  
this ratio was similar for both the femora and tibia for both animals perhaps suggesting that ageing does not significantly deteriorate bone biomechanics for the transgenic .           in this study
, we have characterized the pepck - c  supermouse  and demonstrated that the case 3d cryo - imaging system can be adopted as a morphological phenotypic screen for transgenic animals .
the multiscale approach offers researchers tissue information at the most appropriate resolution traversing down the scale from mouse to organ to tissue to single cells .
the ability of being able to visualize structures in 3d and extract 2d sections along any arbitrary plane makes comparisons easy and focused .
further , visualizations do not involve laborious manual segmentation and in most cases are achieved semiautomatically through the novel color feature detectors .
the results of such phenotyping effort will provide a roadmap for adopting cryo - imaging for cataloging the plethora of transgenic mouse models .
 we have developed , for the case 3d cryo - imaging system , a specialized , multiscale visualization scheme which provides color - rich volume rendering and multiplanar reformatting enabling one to visualize an entire mouse and zoom in to organ , tissue , and microscopic scales . with this system , we have anatomically characterized , in 3d , from whole animal to tissue level , a transgenic mouse and compared it with its control . the transgenic mouse overexpresses the cytosolic form of phosphoenolpyruvate carboxykinase ( pepck - c ) in its skeletal muscle and is capable of greatly enhanced physical endurance and has a longer life - span and reproductive life as compared to control animals . 
 we semiautomatically analyzed selected organs such as kidney , heart , adrenal gland , spleen , and ovaries and found comparatively enlarged heart , much less visceral , subcutaneous , and pericardial adipose tissue , and higher tibia - to - femur ratio in the transgenic animal . 
 microscopically , individual skeletal muscle fibers , fine mesenteric blood vessels , and intestinal villi , among others , were clearly seen . 
1. Introduction
2. Cryo-Imaging System
3. Methods
4. Results
5. Discussion and Conclusion