preclinical imaging preclinical imaging preclinical imaging is the visualization of living animals for research purposes, such as drug development. imaging modalities have long been crucial to the researcher in observing changes, either at the organ, tissue, cell, or molecular level, in animals responding to physiological or environmental changes. imaging modalities that are non-invasive and in vivo have become especially important to study animal models longitudinally. broadly speaking, these imaging systems can be categorized into primarily morphological/anatomical and primarily molecular imaging techniques. techniques such as high-frequency micro-ultrasound, magnetic resonance imaging (mri) and computed tomography (ct) are usually used for anatomical imaging, while optical imaging (fluorescence and bioluminescence), positron emission tomography (pet), and single photon emission computed tomography (spect) are usually used for molecular visualizations. these days, many manufacturers provide multi-modal systems combining the advantages of anatomical modalities such as ct and mr with the functional imaging of pet and spect. as in the clinical market, common combinations are spect/ct, pet/ct and pet/mr. micro-ultrasound principle: high-frequency micro-ultrasound works through the generation of harmless sound waves from transducers into living systems. as the sound waves propagate through tissue, they are reflected back and picked up by the transducer, and can then be translated into 2d and 3d images. micro-ultrasound is specifically developed for small animal research, with frequencies ranging from 15 mhz to 80 mhz. strengths: micro-ultrasound is the only real-time imaging modality per se, capturing data at up to 1000 frames per second. this means that not only is it more than capable of visualizing blood flow in vivo, it can even be used to study high speed events such as blood flow and cardiac function in mice. micro-ultrasound systems are portable, do not require any dedicated facilities, and is extremely cost-effective compared to other systems. it also does not run the risk of confounding results through side-effects of radiation. currently, imaging of up to 30 µm is possible, allowing the visualization of tiny vasculature in cancer angiogenesis. to image capillaries, this resolution can be further increased to 3–5 µm with the injection of microbubble contrast agents. furthermore, microbubbles can be conjugated to markers such as activated glycoprotein iib/iiia (gpiib/iiia) receptors on platelets and clots, αvβ3 integrin, as well as vascular endothelial growth factor receptors (vegfr), in order to provide molecular visualization. thus, it is capable of a wide range of applications that can only be achieved through dual imaging modalities such as micro-mri/pet. micro-ultrasound devices have unique properties pertaining to an ultrasound research interface, where users of these devices get access to raw data typically unavailable on most commercial ultrasound (micro and non-micro) systems. weaknesses: unlike micro-mri, micro-ct, micro-pet, and micro-spect, micro-ultrasound has a limited depth of penetration. as frequency increases (and so does resolution), maximum imaging depth decreases. typically, micro-ultrasound can image tissue of around 3 cm below the skin, and this is more than sufficient for small animals such as mice. the performance of ultrasound imaging is often perceived as to be linked with the experience and skills of the operator. however, this is changing rapidly as systems are being designed into user-friendly devices that produce highly reproducible results. one other potential disadvantage of micro-ultrasound is that the targeted microbubble contrast agents cannot diffuse out of vasculature, even in tumors. however, this may actually be advantageous for applications such as tumor perfusion and angiogenesis imaging. cancer research: the advances in micro-ultrasound has been able to aid cancer research in a plethora of ways. for example, researchers can easily quantify tumor size in two and three dimensions. not only so, blood flow speed and direction can also be observed through ultrasound. furthermore, micro-ultrasound can be used to detect and quantify cardiotoxicity in response to anti-tumor therapy, since it is the only imaging modality that has instantaneous image acquisition. because of its real-time nature, micro-ultrasound can also guide micro-injections of drugs, stem cells, etc. into small animals without the need for surgical intervention. contrast agents can be injected into the animal to perform real-time tumor perfusion and targeted molecular imaging and quantification of biomarkers. recently, micro-ultrasound has even been shown to be an effective method of gene delivery. functional ultrasound brain imaging unlike conventional micro-ultrasound device with limited blood-flow sensitivity, dedicated real-time ultra fast ultrasound scanners with appropriate sequence and processing have been shown to be able to capture very subtle hemodynamic changes in the brain of small animals in real-time. this data can then be used to infer neuronal activity through the neurovascular coupling. the functional ultrasound imaging (fus) technique can be seen as an analogue to functional magnetic resonance imaging (fmri). fus can be used for brain angiography, brain functional activity mapping, brain functional connectivity from mice to primates including awake animals. micro-pat principle: photoacoustic tomography (pat) works on the natural phenomenon of tissues to thermalelastically expand when stimulated with externally applied electromagnetic waves, such as short laser pulses. this causes ultrasound waves to be emitted from these tissues, which can then be captured by an ultrasound transducer. the thermoelastic expansion and the resulting ultrasound wave is dependent on the wavelength of light used. pat allows for complete non-invasiveness when imaging the animal. this is especially important when working with brain tumor models, which are notoriously hard to study. strengths: micro-pat can be described as an imaging modality that is applicable in a wide variety of functions. it combines the high sensitivity of optical imaging with the high spatial resolution of ultrasound imaging. for this reason, it can not only image structure, but also separate between different tissue types, study hemodynamic responses, and even track molecular contrast agents conjugated to specific biological molecules. furthermore, it is non-invasive and can be quickly performed, making it ideal for longitudinal studies of the same animal. weaknesses: because micro-pat is still limited by the penetrating strength of light and sound, it does not have unlimited depth of penetration. however, it is sufficient to pass through rat skull and image up to a few centimeters down, which is more than sufficient for most animal research. one other drawback of micro-pat is that it relies on optical absorbance of tissue to receive feedback, and thus poorly vascularized tissue such as the prostate is difficult to visualize. to date, 3 commercially available systems are on the market, namely by visualsonics, ithera and endra, the last one being the only machine doing real 3d image acquisition. cancer research: the study of brain cancers has been significantly hampered by the lack of an easy imaging modality to study animals in vivo. to do so, a craniotomy is often needed, in addition to hours of anesthesia, mechanical ventilation, etc. which significantly alters experimental parameters. for this reason, many researchers have been content to sacrifice animals at different time points and study brain tissue with traditional histological methods. compared to an in vivo longitudinal study, many more animals are needed to obtain significant results, and the sensitivity of the entire experiment is cast in doubt. as stated earlier, the problem is not reluctance by researchers to use in vivo imaging modalities, but rather a lack of suitable ones. for example, although optical imaging provides fast functional data and oxy- and deoxyhemoglobin analysis, it requires a craniotomy and only provides a few hundred micrometres of penetration depth. furthermore, it is focused on one area of the brain, while research has made it apparently clear that brain function is interrelated as a whole. on the other hand, micro-fmri is extremely expensive, and offers dismal resolution and image acquisition times when scanning the entire brain. it also provides little vasculature information. micro-pat has been demonstrated to be a significant enhancement over existing in vivo neuro-imaging devices. it is fast, non-invasive, and provides a plethora of data output. micro-pat can image the brain with high spatial resolution, detect molecular targeted contrast agents, simultaneously quantify functional parameters such as so2 and hbt, and provide complementary information from functional and molecular imaging which would be extremely useful in tumor quantification and cell-centered therapeutic analysis. micro-mri principle: magnetic resonance imaging (mri) exploits the nuclear magnetic alignments of different atoms inside a magnetic field to generate images. mri machines consist of large magnets that generate magnetic fields around the target of analysis. these magnetic fields cause atoms with non-zero spin quantum number such as hydrogen, gadolinium, and manganese to align themselves with the magnetic dipole along the magnetic field. a radio frequency (rf) signal is applied closely matching the larmor precession frequency of the target nuclei, perturbing the nuclei's alignment with the magnetic field. after the rf pulse the nuclei relax and emit a characteristic rf signal, which is captured by the machine. with this data a computer will generate an image of the subject based on the resonance characteristics of different tissue types. 7t cryogen free preclinical mri imaging system – this shows the mrs 7000 series since 2012, the use of cryogen-free magnet technology has greatly reduced infrastructure requirements and dependency on the availability of increasingly hard to obtain cryogenic coolants. strengths: the advantage of micro-mri is that it has good spatial resolution, up to 100 µm and even 25 µm in very high strength magnetic fields. it also has excellent contrast resolution to distinguish between normal and pathological tissue. micro-mri can be used in a wide variety of applications, including anatomical, functional, and molecular imaging. furthermore, since micro-mri's mechanism is based on a magnetic field, it is much safer compared to radiation based imaging modalities such as micro-ct and micro-pet. weaknesses: one of the biggest drawbacks of micro-mri is its cost. depending on the magnetic strength (which determines resolution), systems used for animal imaging between 1.5 and 14 teslas in magnetic flux density range from $1 million to over $6 million, with most systems costing around $2 million. furthermore, the image acquisition time is extremely long, spanning into minutes and even hours. this may negatively affect animals that are anesthetized for long periods of time. in addition, micro-mri typically captures a snapshot of the subject in time, and thus it is unable to study blood flow and other real-time processes well. even with recent advances in high strength functional micro-mri, there is still around a 10–15 second lag time to reach peak signal intensity, making important information such as blood flow velocity quantification difficult to access. cancer research: micro-mri is often used to image the brain because of its ability to non-invasively penetrate the skull. because of its high resolution, micro-mri can also detect early small-sized tumors. antibody-bound paramagnetic nanoparticles can also be used to increase resolution and to visualize molecular expression in the system. stroke and traumatic brain injury research: micro-mri is often used for anatomical imaging in stroke and traumatic brain injury research. molecular imaging is a new area of research. micro-ct micro-ct system volume rendering of reconstructed ct of a mouse skull principle: computed tomography (ct) imaging works through x-rays that are emitted from a focused radiation source that is rotated around the test subject placed in the middle of the ct scanner. the x-ray is attenuated at different rates depending on the density of tissue it is passing through, and is then picked up by sensors on the opposite end of the ct scanner from the emission source. in contrast to traditional 2d x-ray, since the emission source in a ct scanner is rotated around the animal, a series of 2d images can then be combined into 3d structures by the computer. strengths: micro-ct can have excellent spatial resolution, which can be up to 6 µm when combined with contrast agents. however, the radiation dose needed to achieve this resolution is lethal to small animals, and a 50 µm spatial resolution is a better representation of the limits of micro-ct. it is also decent in terms of image acquisition times, which can be in the range of minutes for small animals. in addition, micro-ct is excellent for bone imaging. weaknesses: one of the major drawbacks of micro-ct is the radiation dosage placed on test animals. although this is generally not lethal, the radiation is high enough to affect the immune system and other biological pathways, which may ultimately change experimental outcomes. also, radiation may affect tumor size in cancer models as it mimics radiotherapy, and thus extra control groups might be needed to account for this potential confounding variable.