In vivo PET/MRI Imaging of the Chorioallantoic Membrane

The Hen's Egg Test Chorioallantoic Membrane (HET-CAM) of fertilized chick eggs represents a unique model for biomedical research. With its steadily increasing use, non-invasive in ovo imaging for longitudinal direct quantification of the biodistribution of compounds or monitoring of surrogate markers has been introduced. The full range of imaging methods has been applied to the HET-CAM model. From the current perspective, Magnetic Resonance Imaging (MRI) and Positron Emission Tomography (PET) appear promising techniques, providing detailed anatomical and functional information (MRI) and excellent sensitivity (PET). Especially by combining both techniques, the required sensitivity and anatomical localization of the signal source renders feasible. In the following, a review of recent applications of MRI and PET for in ovo imaging with a special focus on techniques for imaging xenotransplanted tumors on the CAM will be provided.


INTRODUCTION
The Hen's Egg Test Chorioallantoic-Membrane (HET-CAM) of fertilized chicken eggs represents a unique model for biomedical research. During the development, the mesodermal layers of the allantois and chorion form the chorioallantoic membrane (CAM). This structure forms a rich vascular network enabling to study tissue grafts, tumor growth, metastasis formation, wound healing, drug delivery, toxicologic analysis, angiogenic and anti-angiogenic molecules [1].
The HET-CAM represents a relatively simple, quick, and low-cost model that allows screening of a large number of pharmacological samples in a short time. It has been successfully used to study cancer progression and its pharmacological treatment [2][3][4][5][6][7][8], angiogenesis [9], pharmacokinetics [10], properties of novel nanomaterials [9,11], or as a model system to study microsurgical instruments and techniques [12]. Especially for xenotransplantation tumor models, the HET-CAM offers various advantages in comparison to the murine models. Since the development of the lymphoid system starts in the late stage of incubation, the HET-CAM model represents a naturally immunodeficient host, enabling xenotransplantation of many kinds of tumors without species-specific limitations [11]. The blood vessel network of the CAM thereby provides an excellent environment for primary tumor formation and a basis for angiogenic blood vessel formation [12]. Human cell line derived [13] xenografts are considered an increasingly valuable tool in oncology potentially providing biologically models of many different cancer types. Where immunodeficient rodent models pose barriers to widespread application due to cost and efficiency constraints, the HET-CAM model renders as efficient model especially for initial testing of tumor progression [10,[14][15][16], in many countries not requiring any approval for animal experiments, if sacrificed before hatching.
Considering the capability of the HET-CAM model for monitoring the growth and progression of xenotransplanted tumors rises a huge potential for its use in the evaluation of the biodistribution of new compounds, especially in combination with specific targeting strategies. In this context the combination of the multi-contrast capabilities of MRI with the outstanding sensitivity of positron emission tomography (PET) appears promising. In the following a review of recent applications of MRI and PET for in ovo imaging with a special focus on techniques for imaging xenotransplanted tumors on the CAM will be provided.

THE HET-CAM MODEL
In preclinical research and drug development can-cer cell lines (CCL) are frequently used. However, in culture, CCLs often fail to retain morphology, cellular heterogeneity, and molecular profiles of the donor tissue [72,73], and drug performance in xenografts may not perfectly reflect clinical efficacy [74]. The success of new drugs in oncology requires preclinical models that render the full heterogeneity and pathophysiology of patient tumors, and CCL or patient-derived xenografts (PDX) may mimic physiological drug effects [75,76].
A broad range of applications for CCL and PDX have been reported using rodent models as host and their efficient use in prediction of response, development of biomarkers, and monitoring and identification of efficient treatment regimens has been proven [77]. Despite their frequent use, rodent models have practical and scientific limitations. In many applications, rodent models have shown a very limited success rate in engraftment. Successful engraftment often takes several months. Further high, often prohibitive costs and resources are required for keeping rodents in an appropriate facility under proper hygienic conditions. They are labor-intensive, time-consuming, and require ethical approval by the regulatory authorities.
The HET-CAM model represents a well-established alternative in vivo assay. It presents a highly vascularized extra-embryonic membrane, which is connected to the embryo through a continuous circulatory system. Even though T and B cells can be detected in the chick embryo immune system by embryo development day (EDD) 11 and 12, full immune competence is not developed until EDD 18 [16]. The HET-CAM is a low-cost model with the limita-tion of developing a nonspecific inflammatory response after EDD 15 [1]. Xenotransplantation and growth of cancer cell lines on the CAM is well-established (Figure 1) and has amongst others been applied for initial assessment of the efficacy of anticancer drugs [78]. Compared to the rodent models, tumor formation on the CAM is fast, with graft vascularization and thus interface to the chick embryo vascular system normally established already after 2-5 days [79].
For xenotransplantation, fertilized chicken eggs e.g., White Leghorn (Gallus domesticus) are purchased from a hatchery and maintained at 37.8 • C and a 60% relative humidity atmosphere for the whole incubation period. Upon arrival the eggs are carefully cleaned (e.g., by 70% ethanol solution) and incubation is started (EDD 0). After 4 days (EDD 4) of incubation the eggs are fenestrated and analyzed for fertilization by visual assessment of the CAM vascularization and heartbeat of the embryo. The shell access window is sealed to prevent contamination and the egg placed back into the incubator. Cancer cells are seeded on the CAM and a solid tumor, well interfaced to the extra-embryonic vascular system, forms within few days [1,16]. The viability of the embryos needs to be monitored daily by checking the CAM vasculature for blood flow, physiological embryo movement, and the growth of the chick embryo according to Hamburger and Hamilton [80].

MAGNETIC RESONANCE IMAGING (MRI)
With its versatile image contrast, MRI raised interest in imaging of chick embryos already in the 80s. In 1986, Bone et al. [81,82] reported first three-dimensional MR microscopy on the live chick embryo. At a 1.5T prototype system with dedicated gradient system and receive coil, they achieved a spatial resolution of 200 x 200 x 1200 µm 3 with T1 and T2 weighting, applying partial saturation (PS) and spin echo (SE) 3D-Fourier imaging techniques (3D-FT). To minimize motioninduced image artifacts, the chick embryo was immobilized by placing the egg in ice chips between 20 (EDD 11) and 90 (EDD 15) minutes. Imaging was performed at room temperature. Even though the spatial resolution and hence the fidelity of the anatomical details was still limited, Bone et al. clearly showed the potential combination of in ovo imaging and MRI as basis for further MRI studies. Improvements in spatial resolution were reported by Effmann et al. [83], who used an implantable 18 mm diameter RF coil wrapped around the chick embryo, inductively coupled to an outside MR receiver [84]. With the resulting gain in signal-to-noise ratio (SNR), the PS 3D-FT could provide images with spatial resolutions of up to 50 x 50 x 600 µm 3 within 1-2 h scan times.
Assessment of metabolic developments by phosphorus 31 P MR spectroscopy (MRS) in correlation with anatomic developments were first reported by Moseley et al. in 1989 [54], who could clearly demonstrate the decrease of the observable 31 P volume by 80% indicating the respective tissue uptake. Further spectroscopy work was performed by Lirette et al. [85] for longitudinal quantification of the fat/water ratio ( 1 H MRS) and the phosphormono-/phosphordiester ratio ( 31 P MRS). Data were acquired with a 5 cm-diameter surface coil, in which the eggs were placed centrally. Acquisition times were below 1 h. Embryo motion was controlled by applying 1-2% halothane during scanning. Peebles et al. [86] applied 1 H MRS for monitoring of brain metabolites and diffusion MRI for assessment of changes in the apparent diffusion coefficient (ADC) in the brain during hypoxia. Immobilization of the chick embryo was achieved by Falen et al. [53] and Hutchison et al. [87] applied MRI with T1 and T2-weighted imaging techniques to the assessment of the yolk structure. Both reported the applicability of MRI for the assessment of the morphology of yolk, albumen, air space, and eggshell. The inner structure of the yolk, including concentric yolk rings, could be clearly visualized by this non-destructive imaging technique. In 2000, Donoghue et al. [88] reported the application of MRI for monitoring residue transfer into egg yolk. After injection of Gd-DTPA, the transfer of the drug into the yolk was monitored by scanning the egg applying a T1-weighted (MP-RAGE) sequence. It could be shown that Gd-DTPA residues were incorporated into the yolk ring structure.
Assessment of the chick embryo vasculature by MRI was reported by Smith et al. in 1992 [89]. They performed ex vivo high-resolution MRI of the embryonic vasculature after perfusion fixation of the vasculature structure with gadoliniumdoped gelatin. A similar approach was chosen by Hogers et al. [52] to demonstrate the benefit of ultra-highfield imaging by 1, albumen; 2, yolk; 3, air sac; 4, head; 5, limb; 6, rump; a, brain; b, eye; c, gizzard; d, heart; e, liver; f, pectoral muscles; g, intestine; h, umbilical vessels. The complete scans can be viewed at www.gla.ac.uk/7tmr/chickegg.htm. This research was originally published in JMRI: Noninvasive Monitoring of Chick Development In Ovo Using a 7T MRI System From Day 12 of Incubation Through to Hatching. Bain et al. [51].
comparing MRI in vitro microscopy between 7T and 17.6T field strength. A similar ex vivo technique was applied by Zhang et al. [90] and Yelbuz et al. [91,92], who used the combination of perfusion with immersion fixation and a small molecular gadolinium agent to improve image contrast between the myocardial wall and heart lumen. Isotropic three-dimensional images with up to 25 3 µm 3 spatial resolution were acquired with a T1-weighted spin warp technique in about 29 h scan time at 9.4T field strength. In ovo quantification of the cardiac function was reported by Holmes et al. [93,94] applying a self-gating technique for cardiac synchronization of the data. Even though adequate image quality could be obtained, the authors identified the bulk embryo motion at earlier stages as main limiting factor for reproducible image quality.
Noninvasive monitoring of chick embryo development was reported by Bain et al. [51] in 2007. For optimal image contrast a T2-weighted spin echo technique (RARE) was applied yielding 195 x 195 x 500 µm 3 spatial resolution within 25 minutes scan time at 7T (Figure 2). Rapid three-dimensional T1-weighted techniques provided higher spatial resolution, but organ contrast was not sufficient. For chick embryo immobilization, Bain et al. [51] used a precooling protocol, keeping the chicken eggs at 4 • C for 60 min prior to data acquisition with subsequent data acquisition at room temperature. No slowdown or arrest of the chick embryo development was observed by the cooling or the MRI examinations. As alternative cooling protocol, Li et al. [95] suggested data acquisition at 19 • C thus enabling scan times of up to 4 hours in quail embryos. More advanced imaging techniques including T1 and T2 mapping as well as magnetization transfer ratio (MTR) quantification were reported by Boss et al. [50]. Motion reduction was achieved by application of 0.5 mL Ketamine onto the CAM. Images were acquired with two-dimensional multi-slice techniques at a spatial resolution of 180 x 180 x 1000 µm 3 (T1), 260 x 260 x 1000 µm 3 (T2), and 230 x 230 x 1000 µm 3 (MTR). In 2015, Zhou et al. [49] translated developmental imaging of the brain to a 3T clinical scanner equipped with a dedicated small animal coil. T2 weighted RARE images as well as DTI data could be acquired at reasonable spatial resolution (T2w: 170 x 170 x 1000 µm 3 in 8 minutes, DTI: 1.25 x 1.25 x 1 mm 3 in 6 minutes), enabling noninvasive analysis of brain development including structural information. Imaging of the chick embryo brain development after Zika virus infection was reported by Goodfellow et al. [96], who could clearly demonstrate Zika virus induced microcephaly. Highresolution MRI at 7T were presented by Lindner et al. [48] for monitoring the chick embryo eye development. Immobilization of the embryo was achieved by bedding the egg on crushed ice ten minutes before scanning.
A first application of in ovo MRI for the assessment of the biodistribution of new compounds was reported by Dingman et al. [97] in 2003. They longitudinally investigated the distribution of a 19F-labeled L-6-heptafluorobutyryl-5hydroxytryptophan including uptake dynamics and crossing of the blood-brain barrier. In 2007, Oppitz et al. [98] suggested the use of chick embryo model for evaluation of the advantages and limitations of MRI to monitor the migration of superparamagnetic iron oxide (SPIO) labeled cells. A similar approach was presented by Pereira et al. [99] who showed in the chick embryo model an improved sensitivity for in vivo cell tracking after implantation by supplementing the culture medium with adequate iron sources as compared to the use of reporter genes. Faucher et al. [100] used the HET-CAM model for initial investigation of ultra-small gadolinium oxide nanoparticles for labeling of glioblastoma cells, seeded on the CAM. Taylor et al. [101] used the chick embryo model for initial evaluation of new nano-and micro-sized magnetic particles for cell tracking. All imaging was performed ex vivo. In 2017, Zuo et al. [65] reported the use of human cancer cell lines xenotransplanted onto the CAM for initial assessment of the biodistribution of MR labeled drugs. After injection of Gd-DOTA, the biodistribution of the compound in the chick embryo as well as in the xenotransplanted tumor was observed. By longitudinal imaging studies over 40 h, the accumulation and clearance of the contrast agent could be monitored. The technique was applied for demonstrating the feasibility of the HET-CAM tumor model for monitoring the fate of new MR labeled drugs by following the image contrast after intravenous administration of a gadolinium-labelled polymeric nanoparticle at high spatial resolution, applying an immobilization protocol as suggested earlier [61]. In direct comparison with conventional contrast agents, a significantly prolonged retention time of the polymeric nanoparticle in the tumor could be shown (Figure 3). For assessment of the intra-tumor distribution, 3D T1 weighted data were acquired at high spatial resolution of 100 x 100 x 560 µm 3 . Hafner et al. [102] used a similar approach for initial evaluation of the biodistribution of a multifunctional drug carrier.
Buschmann et al. [103] reported implantation of human osteoblasts-seeded scaffolds onto the CAM. One week after xenotransplantation, MRI (T1, T2 quantification) analysis was performed ex vivo, after intravenous injection or dripping of gadolinium onto the scaffold before sacrificing the embryo allowing for the analysis of the formation of new capillaries. The published ex vivo data clearly indicated the potential of the HET-CAM model as cheap reliable model for monitoring the angiogenesis in tissue-engineering. This work was further developed by Chesnick et al. [104] by initial testing of a new adrenolate labeled gadolinium complex for specific targeting to bone mineral. In 2015, Pfiffner et al. [105] presented in vivo MRI at 4.7T to noninvasively quantify and monitor the perfusion capacity in the HET-CAM model. After placing a biomaterial on the CAM, its perfusion capacity was quantified by relaxation rate changes after intravenous injection of the gadolinium-based contrast agent. Immobilization of the chick embryo was achieved by 5 drops of ketamine 1:100 (Ketasol-100, Graeub, Switzerland) dripped onto the CAM surface. Anatomical reference images were obtained applying a FLASH sequence at measured spatial resolution of roughly 500 2 µm 2 . T1 and T2 maps were derived applying RARE sequences with multiple repetition times (TR) and echo times (TE) at spatial resolution of 200 x 200 x 1000 µm 3 before and at different time points after intravenous injection of 100 µL Gd-DOTA (0.5M) MRI contrast agent. Relaxation rate changes over the scaffold could be clearly assessed indicating different vascular density, which was confirmed by histology.
Motion artifacts introduced by bulk motion of the chick embryo is a major limiting factor for high-resolution imaging of the CAM (Figure 4). In 2015, Waschkies et al. [106] investigated the use of different anesthesia drugs for immobilization, whereas Zuo et al. [61] evaluated an age-adjusted precooling protocol for high-resolution imaging of the CAM. In Waschkies et al, medetomidine at a dosage of 0.3 mg/kg, was compared to thiopental at 100 mg/kg and ketamine/midazolam at 50 and 1 mg/kg. The soluble anesthetics were applied by dropping a total volume of 0.3 mL onto the surface of the CAM. It was demonstrated that medetomidine performed best, enabling motion-free MRI for a period of about 30 min starting 10 min after application. Ketamine/midazolam yielded insufficient depth of anesthesia and thiopental anesthesia did not immobilize the chick embryo sufficiently long. In contrast, Zuo et al. investigated the use of an anesthesia-free immobilization approach. As extension to earlier published work [51], they suggested adaptation of the precooling time according to the age of the chick embryo, thereby achieving almost complete immobilization of the chick embryo for at least 60 min thus allowing multi-contrast high-resolution imaging of the chick embryo and xenotransplanted tumors on the CAM (Figures 5, 6). The suggested cooling protocol allowed in vivo imaging at high spatial resolution as 77 x 91 x 500 µm 3 (T2 weighted anatomic, 2D), 200 x 200 x 500 µm 3 (diffusion weighted, 2D), 104 x 98 x 500 µm 3 (T2 mapping, 2D), and 100 x 100 x 560 µm 3 (T1 weighted, 3D). Tumor volume and growth could be monitored longitudinally from day 4 to day 9 after xenotransplantation. The tumor progression could be monitored for each individual case (Figure 7) and the volumes derived from MRI at day 9 excellently correlated with the respective volumes derived after resection of the tumors. In 2018, Herrmann et al. [58] reported the application of MRI to measure primary neuroblastoma tumor size and metastasis in a chick embryo model. Human neuroblastoma cells labeled with green fluorescent protein (GFP) and micron-sized iron particles were xenotransplanted on the CAM at EDD 7. At EDD 14, T2 RARE and T2-weighted fast low angle shot (FLASH) data were acquired (Figure 8) using the cooling protocol as suggested by Zuo et al. [61]. Additionally, Herrmann et al. performed timeof-flight (ToF) MR angiography (MRA) and reported a reduced blood flow if using the cooling protocol, making successful ToF acquisition unfeasible. Instead, ketamine anesthesia (3.6 mM ketamine in 500 µL PBS) was applied resulting in MRI data free

POSITRON EMISSION TOMOGRAPHY (PET)
Most anatomic and functional imaging of the HET-CAM model has been performed by MRI so far. The main limitation of PET results from its rather low spatial resolution, which does not fit the high spatial resolution demands for imaging of the CAM. To gain from the outstanding sensitivity of PET, thus in almost all work published, PET (or SPECT) imaging was complimented by Computer Tomography (CT), x-ray, or MRI for providing additional anatomic details.
In 2012, Würbach et al. [71] introduced using 18 F-fluoride PET for assessment of bone metabolism. For radionuclide injection, a self-built catheter made of a 30G needle and a polythene tube with 0.28 mm inner diameter was introduced into one of the CAM vessels. Imaging was performed for a period of roughly 75 minutes yielding dynamic images (55 time frames) as well as high-quality static data. Applying an iterative ordinary Poisson maximum a posteriori reconstruction yield voxel sizes of roughly 400 x 400 x 800 µm 3 . The results proved the quantitative and reproducible assessment of bone metabolism in anesthetized chick embryos. Immobilization of the chick embryo during scanning was achieved by exposing the CAM to isoflurane at 1.5% concentration as suggested by Heidrich et al. [107], who investigated different anesthesia schemes for in vivo imaging of avian embryos, including isoflurane, 2,2,2-tribromoethanol (Avertin), and urethane/αchloralose (UC). UC and Avertin were directly applied as liquids onto the CAM. For isoflurane anesthesia, the egg was exposed to an isoflurane concentration of 5% in oxygen. For induction, the eggs were placed into a narcosis induction chamber. Toxic side effects and only poor correlation between narcosis depth and dose limited the application of UC and Avertin and the authors clearly favored the use of Isoflurane due to its high tolerability enabling repeated imaging of the avian embryos at a daily basis. In 2013, Gebhardt et al. [70] applied a similar approach as Würbach et al. [71] to initially   evaluate the dynamic behavior of new PET tracers in the chick embryo model as an in vivo assay. Various 18 F, 64 Cu, and 68 Ga-labeled compounds were investigated and the potential of the chick embryo model as efficient in vivo model could be shown.
Warnock et al. [69] demonstrated the use of the CAM for screening of novel PET tracers. At EDD 11, the eggs were opened and 5 x 10 6 human U87 glioblastoma cells in 20 µL of culture medium xenotransplanted onto the CAM. At EDD 18 PET/CT imaging of the tumors was performed. During scanning the egg was reproducible positioned in both systems in a small animal imaging cell (Minerve equipment veterenaire) allowing temperature control and isoflurane anesthesia (2% in air). Uptake of the radiotracer was clearly demonstrated by timeactivity curves and in the PET images (Figure 9). Contrast-agent enhanced µCT data provided accurate anatomic correlation, Haller et al. [19] investigated the tissue distribution and stability of different 18 F, 125 I, 99m Tc, and 177 Lu-labeled radiopharmaceuticals. For imaging purposes the chick embryos were euthanized by shock-freezing in liquid nitrogen at different time points after administration of the radioactivity. In comparison with the established mouse models, they concluded a similar tissue distribution and stability of radiopharmaceuticals in the chick embryo. The very similar behavior in the two in vivo models indicate the potential of using the chick embryo as an inexpensive and simple test model for preclinical screening of novel radiopharmaceuticals. To overcome the limitations of the small anatomies with respective requirement of dedicated highresolution imaging equipment, Freesmeyer et al. [108] suggested the translation of the work into ostrich eggs and demonstrated the potential use for different radiotracers even on conventional PET/CT systems. Even though overcoming the issues with small anatomy and high-spatial resolution requirements, ostrich eggs cannot be seen as a real alternative to chick embryos, since they are not readily available, hardly established in science, expensive and often do not fit into conventional small animal imaging equipment.
In a recent work of Zlatopolskiy et al. [68] the HET-CAM model was used for evaluation of radiotracers addressing the tryptophan metabolic pathway. An efficient method for the synthesis of fluorotryptophans, labeled in different positions with 18  Even though CT represents an important imaging tool providing relevant anatomic information and has proven clinical success in combination with PET, its only limited soft tissue contrast is often insufficient. To that respect in 2019 Steinemann et al. [109] and Winter et al. [110] combined in ovo PET with MRI to make full advantage from the high sensitivity of PET and the excellent soft tissue contrast in MRI. Steinemann et al. used the imaging approach for monitoring the effectiveness of a new chimeric inhibitor (animacroxam, which combines histone deacetylase (HDAC) inhibitory and cytoskeleton-interfering pharmacophores) to the clinical approved HDAC in testicular germ cell tumor. Tumor plaques were grown from 10 × 10 6 2102EP cells mixed with 150 µl matrigel and transplanted onto the CAM. Imaging was performed three days after xenotransplantation to allow angiogenic connection of the xenograft to the CAM. Injection cells) mixed in Matrigel (40%, v/v) were grafted on the CAM in two silicon rings. MR and PET imaging was performed starting on EDD 12. Anatomical information was provided by high-resolution imaging using a small animal MR based on the protocol of Zuo et al. [61,65]. For PET, 150 µl of [ 68 Ga]-PSMA-11 solution was injected into a chorioallantoic membrane vessel followed by a dynamic 60 minutes PET scan. Registration between both imaging modalities was achieved by a self-built animal holder with PET and MRI visible fiducial markers. Tumor growth could be quantified by MR imaging. In addition to PET imaging the tumor entities were excised from the membrane after measurement and the accumulated activity was separately quantified by γ-counter (COBRA II, Perkin Elmer) detection. As expected, in comparison to the PC-3 tumors, higher accumulation of [ 68 Ga]-PSMA-11 was observed in the LNCaP C4-2 tumors, indicating the applicability of the HET-CAM model for initial testing of binding specificity of targeted compounds.

DISCUSSION
The HET-CAM model has been exerted to numerous applications. It represents a simple, quick, and low-cost model, not rising any regulatory concerns in many countries if sacrificed before hatching. However, there is a consensus in the scientific community that it is illogical to conclude that the neural capacity to experience pain is not fully developed prior to hatching and that beyond a critical point in development avian embryos are capable of experiencing pain. The exact stage of development at which this capacity is sufficiently developed to warrant concern has not yet been determined. Society recognizes that a critical period in chick embryo development occurs 72 h prior to hatching and from an ethical point of view embryos should be sacrificed prior to EDD 18.
Since the HET-CAM model represents a naturally immunodeficient host, xenotransplantation of many kinds of tumors without species-specific limitations are possible. Even considering the fact that not all organs are fully developed, the established circulation and the highly vascularized CAM, which is connected to the embryo through a continuous circulatory system make it a natural candidate in-between cell culture and animal experiments, especially for initial testing of new compounds. Even though not finally established, it will likely play an important role in early drug development as pointed out in Figure 12. Compounds without detectable toxicity in cell and eventually zebra fish assays, may be initially evaluated regarding efficacy and biodistribution in the HET-CAM model, thereby reinforcing the potential of this convenient, 3R compliant, in vivo model for cancer research. Only compounds showing promising properties will then further be evaluated in animal models, thus reducing the need for animal studies and related costs.
Non-invasive imaging will likely play an increasingly important role for direct visualization of the biodistribution of respectively labeled compounds and longitudinal monitoring of surrogate markers such as tumor progression and metabolism. MRI appears as an attractive imaging approach providing flexible image contrast and assessment to different tissue-specific parameters like MR relaxation times, diffusion and perfusion. Even though it offers the possibility of tailoring image contrast to the specific application, its intrinsic low sensitivity often limits its application in identification of small amounts or only traces of compounds. This limitation raises the increasing interest in using PET in the context of HET-CAM imaging. The excellent sensitivity of PET combined with the excellent anatomic detail of MRI appears as an ideal combination for anatomic, metabolic, and molecular imaging in the HET-CAM model.
Even though in PET motion compensation of the embryo may not be of paramount importance due to its only limited spatial resolution, in MRI coping with embryo motion is one of the major challenges to finally achieve the required highspatial resolution. Over the last years efficient immobilization approaches of the chick embryo have successfully been evaluated in ovo ( Table 1). Promising approaches include the precooling of the egg prior to scanning, the application of halothane and isoflurane, and the use of anesthesia agents dropped directly onto the CAM. Good to excellent immobilization could be achieved with almost all approaches. Major differences were reported regarding immobilization duration and resulting possible image acquisition times, tolerability by the chick embryo, side effects, and easiness to use. For high-resolution anatomic imaging, precooling appears as an excellent approach, allowing long scan times of up to one hour and being easy-to-use. However, the related slowdown in metabolism [113] and blood flow [58] may limit its application in cases where physiological properties are under investigation. Here, in contrast to halothane, isoflurane was reported to not impact aortic blood flow and cardiac performance [114] and may be a good alternative with the limitation of a quite complex imaging setup. Both approaches are well tolerated by the chick embryo allowing for repeated measurements in longitudinal studies. Liquid anesthetics applied by directly dropping onto the CAM are easy to use and a variety of agents have been reported with widely varying anesthesia efficiency and often toxic side effects [58,106,107]. Even though, ketamine was reported to reduce cardiac contraction force in isolated chick embryo heart at EDD 4 and EDD 7 [115], Herrmann et al. reported the advantage of ketamine over precooling for time-of-flight MRI [58] and showed its possible use in applications being sensitive compromised circulation or metabolism in cases of rather short acquisition times.
This review specifically addresses MRI/PET imaging techniques for HET-CAM applications. Even though highly attractive for high-quality multi-contrast morphological and functional imaging in combination with high-sensitive imaging of radio-labeled compounds, both represent high-cost imaging techniques requiring long acquisition times with only limited applicability to high-throughput applications. A wide range of alternative somehow competing techniques have been proposed over recent years. Most prominent to mention at this point are  Frontiers in Physics | www.frontiersin.org optical methods [116,117] including bioluminescence [118], fluorescence [119], and tomographic [120,121] techniques. Furthermore, ultrasonographic imaging [122], x-ray based tomographic [45] and even photoacoustic techniques [123] have been introduced, the latter of which with the potential for label-free imaging. In combination with single photon emission tomography (SPECT), tomographic x-ray techniques have been applied to initial evaluation of radiopharmaceuticals in chick embryos [19].
In conclusion, the expected increasing interest in the HET-CAM model as an intermediate step between cell culture and animal model for initial testing of new compounds makes in ovo imaging an important tool for monitoring the fate of compounds after systemic injection or surrogate markers. The combination of MRI and PET appears promising by combining the sensitivity of PET with detailed anatomic and functional information provided by MRI.

AUTHOR CONTRIBUTIONS
GW, AK, JL, AA, AB, HL, and ZZ performed measurements in the context of the presented work. FJ and ML provided in-depth knowledge on nano-particles. GW and VR wrote the manuscript, and are responsible for the in ovo experiments.