although x - rays represent one of the most important diagnostic imaging methods for radiotherapy , they are associated with a number of limitations including insufficient spatial resolution , contrast , and quantitative scale . recently , computed tomography ( ct ) and magnetic resonance imaging ( mri ) have been found to produce higher quality results along with more information on internal structures , and can be used as non - invasive screening tools . however , these techniques are unable to detect micro - sized fine structures for the early diagnosis of tumors , vascular diseases , and other disorders . the advent of synchrotron radiation has added a new dimension to the use of x - rays for imaging and radiotherapy . synchrotron radiation has the potential to provide new solutions to the problems associated with other imaging modalities such as ct and mri . because of its high brilliance and laser beam - like directionality , synchrotron radiation can provide ultra - high resolution images using a small field of view . the use of synchrotron radiation sources may enable considerable improvements in some imaging techniques routinely used in medicine . synchrotron radiation has been used for several different techniques , including venous coronary angiography , digitized cerebral synchrotron radiation angiography , monochromatic x - ray computed tomography , monochromatic mammography , two wavelength digital subtraction angiography , phase - contrast / edge - enhancement imaging , diffraction - enhanced imaging , and microtomography , in several countries including the united states , germany , and japan [ 9,11,13 - 15,19,25 ] . in addition , synchrotron - based k - edge digital subtraction angiography is another imaging technique that is highly sensitive to low concentrations of contrast agents . it has been recently reported that k - edge digital subtraction angiography is particularly useful for studying neurovascular pathology in pigs . in teratologic studies , experiments investigating the effect of various teratogens ( including carbon monoxide , x - rays , or ethanol exposure ) on the developing embryo have found that these factors produce vertebral and other skeletal anomalies in the offspring of mice . these abnormalities of the fetus had been observed by fetal tissue double - staining using alizarin red and alcian blue , gross inspection , radiography , or mammography . however , the mouse fetus is only about 18~20 mm in size from crown to rump , making it extremely difficult to use these modalities for observing small lesions . in this study , we used synchrotron radiation to obtain a high definition x - ray image of the fetus in utero . the fetus removed from the icr mouse and the time at which the fetal skeleton could be observed was estimated . our results might be used to find new technologies that enable real - time observation of fine structures without staining . pregnant icr mice ( body weight 43.1~72.7 g ) were purchased from commercial breeders ( daehan biolink , korea ) and fed a standard chow ( labdiet , usa ) . the first day of gestation was designated as the day after observing the vaginal plug . the committee for the care and use of laboratory animals in yonsei university ( korea ) reviewed and approved the protocols for our study according to the guidelines for animal experiments edited by the korean academy of medical sciences . we examined the animals using a min - r screen - film system ( eastman kodak , usa ) and conventional mammography equipment ( performa ; ge healthcare , usa ) with 24 kvp , 60 ma and 0.5 sec of exposure time . the film was processed with and automatic processor ( model 2000 ; kodak , usa ) . the experiments were carried out on a 5c1 beamline at the pohang accelerator laboratory ( pohang university of science and technology , korea ) with a 2.5 gev , 150 ma storage ring current , and 1.32 t magnetic field . the electric field was used with s type of polarization in the plane of ring . since longitudinal coherence is not a stringent requirement for refractive index radiology , non - monochromatized ( " white " ) light was used with no optical elements except beryllium windows . after the beam went through the window into air , we introduced slit and attenuator systems to minimize damage associated with the scintillator and to prevent background image over - saturation . the detection system was based on a cdwo4 single crystal scintillator cleaved to a thickness of < 100 m of silica which was resistant to radiation damage and highly homogeneous . the high - resolution radiograph on the scintillator was magnified with an optical lens , captured by a commercial - grade ccd video camera ( ik-536 ; toshiba , japan ) , and recorded with a digital video recorder ( gv - d300 ; sony , japan ) . to prevent oversaturation , an nd4 filter and 52s polarizer ( kenko , japan ) 1 ) . the background image obtained just before fetus imaging was stored as a digital image and a temporal subtraction image using a computer . after synchrotron radiation imaging , one mouse was randomly selected and euthanized on gestation day 18 and its fetuses were removed for synchrotron radiation analysis . mice were anesthetized with intraperitonial injections of ketamine ( 22.5 mg / kg ; yuhan , korea ) and xylazine ( 1.75 mg / kg ; bayer , germany ) . two mice at embryonic day ( e)12 , four mice at e14 , two mice at e15 , and two mice at e18 were radiographed with conventional x - ray ( vpx-100a ; toshiba , japan ) and mammography equipment . to observe fetal anatomy , the pregnant mice were suspended around the rectangular positioner with surgical ties and placed in a vertical position ( fig . synchrotron radiation was derived from an accumulation ring of electrons with an accelerated energy of 2.5 gev . one mouse at e12 , e14 , e15 , and e18 was anesthetized after synchrotron radiation imaging and the fetuses were removed . each fetus was skinned and eviscerated for the bone and cartilage staining with alizarin red s and alcian blue . the staining solutions were composed of those two reagents and prepared as follows . the staining solution was mixed with 0.12% alizarin red s in 95% ethanol and 0.14% alcian blue in 75% ethanol . this solution was added into mixture of glacial acetic acid 10 ml and 70% ethanol 170 ml at room temperature for three days . specimens were macerated in 1% koh for two days at room temperature and then cleared through 1% koh in 20% glycerol . the specimens were then stored in 100% glycerol and photographed using a stereomicroscope ( stemi v6 ; zeiss , germany ) . pregnant icr mice ( body weight 43.1~72.7 g ) were purchased from commercial breeders ( daehan biolink , korea ) and fed a standard chow ( labdiet , usa ) . the first day of gestation was designated as the day after observing the vaginal plug . the committee for the care and use of laboratory animals in yonsei university ( korea ) reviewed and approved the protocols for our study according to the guidelines for animal experiments edited by the korean academy of medical sciences . we examined the animals using a min - r screen - film system ( eastman kodak , usa ) and conventional mammography equipment ( performa ; ge healthcare , usa ) with 24 kvp , 60 ma and 0.5 sec of exposure time . the film was processed with and automatic processor ( model 2000 ; kodak , usa ) . the experiments were carried out on a 5c1 beamline at the pohang accelerator laboratory ( pohang university of science and technology , korea ) with a 2.5 gev , 150 ma storage ring current , and 1.32 t magnetic field . the electric field was used with s type of polarization in the plane of ring . since longitudinal coherence is not a stringent requirement for refractive index radiology , non - monochromatized ( " white " ) light was used with no optical elements except beryllium windows . after the beam went through the window into air , we introduced slit and attenuator systems to minimize damage associated with the scintillator and to prevent background image over - saturation . the detection system was based on a cdwo4 single crystal scintillator cleaved to a thickness of < 100 m of silica which was resistant to radiation damage and highly homogeneous . the high - resolution radiograph on the scintillator was magnified with an optical lens , captured by a commercial - grade ccd video camera ( ik-536 ; toshiba , japan ) , and recorded with a digital video recorder ( gv - d300 ; sony , japan ) . to prevent oversaturation , an nd4 filter and 52s polarizer ( kenko , japan ) the background image obtained just before fetus imaging was stored as a digital image and a temporal subtraction image using a computer . after synchrotron radiation imaging , one mouse was randomly selected and euthanized on gestation day 18 and its fetuses were removed for synchrotron radiation analysis . mice were anesthetized with intraperitonial injections of ketamine ( 22.5 mg / kg ; yuhan , korea ) and xylazine ( 1.75 mg / kg ; bayer , germany ) . two mice at embryonic day ( e)12 , four mice at e14 , two mice at e15 , and two mice at e18 were radiographed with conventional x - ray ( vpx-100a ; toshiba , japan ) and mammography equipment . to observe fetal anatomy , the pregnant mice were suspended around the rectangular positioner with surgical ties and placed in a vertical position ( fig . synchrotron radiation was derived from an accumulation ring of electrons with an accelerated energy of 2.5 gev . one mouse at e12 , e14 , e15 , and e18 was anesthetized after synchrotron radiation imaging and the fetuses were removed . each fetus was skinned and eviscerated for the bone and cartilage staining with alizarin red s and alcian blue . the staining solution was mixed with 0.12% alizarin red s in 95% ethanol and 0.14% alcian blue in 75% ethanol . this solution was added into mixture of glacial acetic acid 10 ml and 70% ethanol 170 ml at room temperature for three days . specimens were macerated in 1% koh for two days at room temperature and then cleared through 1% koh in 20% glycerol . the specimens were then stored in 100% glycerol and photographed using a stereomicroscope ( stemi v6 ; zeiss , germany ) . ten fetuses were visualized at e12 , e14 , e15 , and e18 after gestation using a synchrotron radiation system . 3a shows representative fetal ribs at e14 ; some portions of the ribs could not be viewed because they overlapped the mother 's femur . fetal bones were visualized in three out of four mice examined at e14 using the synchrotron radiation system . as shown in fig . however , other bony structures were not visualized at this period by the synchrotron radiation system . 3c and d show that fetal ribs were more apparent and adjacent thoracic vertebrae were identified at e18 . at e15 and e18 , data use to identify fetal bony structures using synchrotron radiation , conventional radiography , and mammography equipment are shown in table 2 . in conventional radiography , fetal bony structures were first visualized in one mouse at e18 . this method enabled visualization of the uterus containing several fetuses and radiodense fetal bony structures ; however , it was difficult to specifically identify fetal anatomic structures because of low resolution ( fig . fetal bony structures were first visualized with mammography equipment in one mouse at e15 and two mice at e18 . as shown in fig . 5c , fetal bony structures were better defined at e18 and the skull , ribs , vertebrae , and extremities were identified . however , images of fetal bony structures from the mammography equipment were less resolved compared to those produced by synchrotron radiation ( figs . 4b and 5b ) . double - staining the fetal mice with alcian blue and alizarin red s showed that rib were not identifiable at e14 although a lack of staining does not necessarily indicate a lack of rib formation ( fig . 4a ) . however , calcified ribs and thoracic vertebrae were stained red at e18 ( fig . ossified metatarsus , tibia , fibula , pelvis , scapula , humerus , ulna , radius , and skull were also observed at this stage in the double - stained fetal mice ( fig . synchrotron radiation imaging of the fetus removed from the uterus at e18 showed the ossified bony structures similar to the ones observed by double - staining an age - matched mouse fetus ( fig . in this study , the mouse fetal skeleton was first visualized in a pregnant mouse by synchrotron radiation at e14 . this was a day earlier than first visualization by mammography equipment ( e15 ) and conventional radiography ( e18 ) . fetal ribs appeared faintly on images of three out of the four mice examined with synchrotron radiation at e14 . this was probably due to minimal calcification of the skeleton at this time point as observed in double - stained mouse fetuses . although images of fetal skeletons visualized by the mammography equipment demonstrated that this modality is sufficient for examining the whole mouse skeleton , mammography might not be suitable for visualizing small bony structures due to the small size of fetus . at e18 , fetal skeletons were more apparent and various bony structures such as ribs and vertebrae were visualized by synchrotron radiation with high magnification and resolution . however , since the field of view for synchrotron radiation was small ( 2  3 mm ) , it was difficult to define the anatomy of overlapping fetuses located in the uterus . for the data acquisition , we can use a highly collimated and bright x - ray beam associated with refractive edge enhanced contrast and requiring a short exposure time . this enables us to obtain images of excellent quality with high resolution and video - rate acquisition . when transiting the sample , the beam obtains information on sample image contrast through two mechanisms : the attenuation coefficient and/or the path difference by refraction and/or diffraction . however , the density of a biological sample is too low to produce sufficient contrast . therefore , the refractive edge enhanced contrast mechanism is dominant . image information is obtained by the edge of an object having a different refractive index , which x - ray beam transmits to the scintillator , is partially converted into visible light , magnified by optical lens , and transferred to a computer by the ccd video camera . in our experiment , we set a mirror between the scintillator and optic lens and therefore used a reflective mode rather than a transmission mode . thus , we intended to minimize the damage of the optic lens by receiving only visible light by selectively excluding x - rays [ 5 - 7 ] . x - rays and double - staining techniques have been used as important tools for laboratory animal research . these imaging systems permit researchers to screen animal models for mutations or pathologies , and to monitor disease progression and response to therapy . for example , carbon monoxide has been shown to be teratogenic in mice using mammography , and an animal model of lumbosacral agenesis was non - invasively studied with radiography . double - stained mouse fetus was also used for studying the influence of the p53 tumor suppressor gene on mouse skeletal development . however therefore , it is impossible to observe sequential development ( or ontogeny within a single organism ) of the fetus with this technique . additionally , the size of a typical mouse fetus is about 18~20 mm from crown to rump . this small size makes it impossible to observe small lesions in these structures using traditional x - ray - based techniques such as mammography . also , micro - ct is a non - invasive method and useful tool for assessing skeletal microstructures . the advantages of micro - ct include the ability to quantify soft tissues , generate 3d images , decrease the time for cesarean section or any staining , reduce waste production associated with staining , and potentially evaluate multiple stages of skeletal changes . however , the micro - ct system has a number of limitations associated with spatial resolution required for examining an individual fetus and grayscale resolution for detecting adjacent objects with similar densities . this system can be exploited to generate micron - level properties of blood vessel in real - time without the use of contrast agents . because the projection of synchrotron radiation should be vertical to the object , the mouse being examined had to be positioned in an upright posture . therefore , one mouse was sacrificed on e18 and the litter was examined with synchrotron radiation to observed mouse fetal anatomy initially depicted by synchrotron radiation . synchrotron radiation imaging of the fetus that was removed from the uterus at e18 showed ossified bony structures . although synchrotron radiation enabled visualization of the mouse fetal skeleton at e18 with high resolution , a greater beam size is desirable for imaging integrated bony structures of the mouse fetus such as the vertebral column , skull , and extremities . the main purpose of this study was to evaluate the use of synchrotron radiation as a means of detecting fine bony structures . the usefulness of this technique was determined by visualizing fetal mice skeleton and estimating the time when the fetal skeleton could be observed . results from the present study show that synchrotron radiation enables the visualization of mouse fetal skeleton with sufficient image definition at e18 . in conclusion , synchrotron radiation systems are expected to be an effective tool for real - time visualization of micro - sized fine bony structures with high magnification . this technique could be used to make these observations at earlier gestation stages compared to other conventional methods . synchrotron radiation may be a powerful tool for identifying for mutations or pathologies in mouse fetal skeletons as well as monitoring disease progression and response to therapy .
the x - ray synchrotron is quite different from conventional radiation sources . 
 this technique may expand the capabilities of conventional radiology and be applied in novel manners for special cases . to evaluate the usefulness of x - ray synchrotron radiation systems for real time observations , 
 mouse fetal skeleton development was monitored with a high resolution x - ray synchrotron . 
 a non - monochromatized x - ray synchrotron ( white beam , 5c1 beamline ) was employed to observe the skeleton of mice under anesthesia at embryonic day ( e)12 , e14 , e15 , and e18 . at the same time 
 , conventional radiography and mammography were used to compare with x - ray synchrotron . 
 after synchrotron radiation , each mouse was sacrificed and stained with alizarin red s and alcian blue to observe bony structures . 
 synchrotron radiation enabled us to view the mouse fetal skeleton beginning at gestation . 
 synchrotron radiation systems facilitate real time observations of the fetal skeleton with greater accuracy and magnification compared to mammography and conventional radiography . 
 our results show that x - ray synchrotron systems can be used to observe the fine structures of internal organs at high magnification .