in addition, the contrast resolution of micro-ct is quite poor, and thus it is unsuitable for distinguishing between similar tissue types, such as normal vs. diseased tissues. cancer research: micro-ct is most often used as an anatomical imaging system in animal research because of the benefits that were mentioned earlier. contrast agents can also be injected to study blood flow. however, contrast agents for micro-ct, such as iodine, are difficult to conjugate molecular targets1 with, and thus it is rarely used in molecular imaging techniques. as such, micro-ct is often combined with micro-pet/spect for anatomical and molecular imaging in research. micro-pet principle: positron emission tomography (pet) images living systems by recording high-energy γ-rays emitted from within the subject. the source of the radiation comes from positron-emitting-bound biological molecules, such as 18f-fdg (fludeoxyglucose), which is injected into the test subject. as the radioisotopes decay, they emit positrons which annihilates with electrons found naturally in the body. this produces 2 γ-rays at ~180° apart, which are picked up by sensors on opposite ends of the pet machine. this allows individual emission events to be localized within the body, and the data set is reconstructed to produce images. strengths: the strength of micro-pet is that because the radiation source is within the animal, it has practically unlimited depth of imaging. the acquisition time is also reasonably fast, usually around minutes. since different tissues have different rates of uptake radiolabelled molecular probes, micro-pet is also extremely sensitive to molecular details, and thus only nanograms of molecular probes are needed for imaging. weaknesses: radioactive isotopes used in micro-pet have very short half-lives (110 min for 18f-fdg). in order to generate these isotopes, cyclotrons in radiochemistry laboratories are needed in close proximity of the micro-pet machines. 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. micro-pet also has poor spatial resolution of around 1 mm. in order to conduct a well rounded research that involves not only molecular imaging but also anatomical imaging, micro-pet needs to be used in conjunction with micro-mri or micro-ct, which further decreases accessibility to many researchers because of high cost and specialized facilities. cancer research: pet is usually widely used in clinical oncology, and thus results from small animal research are easily translated. because of the way 18f-fdg is metabolized by tissues, it results in intense radiolabelling in most cancers, such as brain and liver tumors. almost any biological compound can be traced by micro-pet, as long as it can be conjugated to a radioisotope, which makes it suitable towards studying novel pathways. micro-spect high resolution 99mtc-mdp mouse spect scan: animated image of rotating maximum intensity projections. principle: similar to pet, single photon emission computed tomography (spect) also images living systems through γ-rays emitted from within the subject. unlike pet, the radioisotopes used in spect (such as technetium-99m) emit γ-rays directly, instead of from annihilation events of a positron and electron. these rays are then captured by a γ-camera rotated around the subject and subsequently rendered into images. strengths: the benefit of this approach is that the nuclear isotopes are much more readily available, cheaper, and have longer half-lives as compared to micro-pet isotopes. like micro-pet, micro-spect also has very good sensitivity and only nanograms of molecular probes are needed. furthermore, by using different energy radioisotopes conjugated to different molecular targets, micro-spect has the advantage over micro-pet in being able to image several molecular events simultaneously. at the same time, unlike micro-pet, micro-spect can reach very high spatial resolution by exploring pinhole collimation principle (beekman et al.) in this approach, by placing the object (e.g. rodent) close to the aperture of the pinhole, one can reach high magnification of its projection on detector surface and effectively compensate for intrinsic resolution of the crystal. weaknesses: micro-spect still has considerable radiation which may affect physiological and immunological pathways in the small animals. 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. micro-spect can also be up to two orders of magnitude less sensitive than pet. furthermore, labeling compounds with micro-spect isotopes require chelating molarities which may alter their biochemical or physical properties. cancer research: micro-spect is often used in cancer research for molecular imaging of cancer-specific ligands. it can also be used to image the brain because of its penetration power. since newer radioisotopes involve nanoparticles such as 99mtc-labelled iron oxide nanoparticles, they could potentially be combined with drug delivery systems in the future. the following small-animal spect systems have been developed in different groups and are available commercially: reference brand system description radius of rotation (cm) resolution (mm) sensitivity (cps/mbq) sajedi et al., 2014, hirespect pixelated csi(tl) crystals, lehr parallel hole collimator, rat and mice imaging 25 1.7 36 magota et al., 2011, inveon nai(tl) crystals, 0.5mm single pinhole collimators 25 0.84 35.3 van der have et al., 2009, ivashchenko et al., 2015, u-spect ii three stationary nai(tl) crystals, 75 pinholes in 5 rings, no multiplexing na 0.25 best 340 (0.25mm) 13000 best del guerra et al., 2007, x-spect two rotating nai(tl) detectors, various apertures na 0.62 best 855 combined pet-mr the image shows a 3t preclinical mri multi-modality imaging system with a clip-on pet for sequential imaging. principle: the pet-mr technology for small animal imaging offers a major breakthrough in high performance functional imaging technology, particularly when combined with a cryogen-free mri system. a pet-mr system provides superior soft tissue contrast and molecular imaging capability for great visualisation, quantification and translational studies. a pet-mr preclinical system can be used for simultaneous multi-modality imaging. use of cryogen-free magnet technology also greatly reduces infrastructure requirements and dependency on the availability of increasingly hard to obtain cryogenic coolants. strengths: researchers can use standalone pet or mri operation, or use multi-modality imaging. pet and mri techniques can be carried out either independently (using either the pet or mri systems as standalone devices), or in sequence (with a clip-on pet) in front of the bore of the mri system, or simultaneously (with the pet inserted inside the mri magnet). this provides a much more accurate picture far more quickly. by operating the pet and mri systems simultaneously workflow within a laboratory can be increased. the mr-pet system from mr solutions incorporates the latest technology in silicon photomultipliers (sipm), which significantly reduces the size of the system and avoids the problems of using photomultipliers or other legacy detector types within the magnetic field of the mri. the performance characteristics of sipm are similar to a conventional pmt, but with the practical advantages of solid-state technology. weaknesses: as this is a combination of imaging systems the weaknesses associated with each imaging modality are largely compensated for by the other. in sequential pet-mr, the operator needs to allow a little time to transfer the subject between the pet and mr acquisition positions. this is negated in simultaneous pet-mr. however, in sequential pet-mr systems, the pet ring itself is easy to clip-on or off and transfer between rooms for independent use. the researcher requires sufficient knowledge to interpret images and data from the two different systems and would require training for this. cancer research: the combination of mr and pet imaging is far more time efficient than using one technique at a time. images from the two modalities may also be registered far more precisely, since the time delay between modalities is limited for sequential pet-mr systems, and effectively non-existent for simultaneous systems. this means that there is little to no opportunity for gross movement of the subject between acquisitions. combined spect-mr a preclinical imaging system with clip-on spect principle: the new spect-mr for small animal imaging is based on multi-pinhole technology, allowing high resolution and high sensitivity. when coupled with cryogen-free mri the combined spect-mr technology dramatically increases the workflow in research laboratories whilst reducing laboratory infrastructure requirements and vulnerability to cryogen supply. strengths: research facilities no longer need to purchase multiple systems and may choose between different system imaging configurations. the spect or mri equipment can each be used as a standalone device on a bench, or sequential imaging can be carried out by clipping the spect module on to the mri system. the animal translates automatically from one modality to the other along the same axis. by inserting a spect module inside the mri magnet simultaneous acquisition of spect and mri data is possible. the workflow of the laboratory can be increased by acquiring multiple modalities of the same subject in one session or by operating the spect and mri systems separately, imaging different subjects at the same time. spect-mr is available in different configurations with different trans-axial field of views, allowing imaging from mice to rats. weaknesses: as this is a combination of imaging systems the weaknesses associated with one or other imaging modality are no longer applicable. in sequential spect-mr, the operator needs to allow a little time to transfer the subject between the spect and mr acquisition positions. this is negated in simultaneous spect-mr. however, for sequential spect-mr, when the spect module is clipped on it is easy to clip-on or off and transfer between rooms. the researcher has to have sufficient knowledge to interpret two different system outputs and would require training for this. cancer research: the combination of mri, which is used as a non-invasive imaging technique, and spect provide results far more quickly when compared to using one technique at a time. images from the two modalities may also be registered far more precisely, since the time delay between modalities is limited for sequential spect-mr systems, and effectively non-existent for simultaneous systems. this means that there is little to no opportunity for gross movement of the subject between acquisitions. with separate, independent operation of the mri and spect systems workflow can easily be increased. optical imaging main article: optical imaging principle: optical imaging is divided into fluorescence and bioluminescence. fluorescence imaging works on the basis of fluorochromes inside the subject that are excited by an external light source, and which emit light of a different wavelength in response. traditional fluorochromes include gfp, rfp, and their many mutants. however significant challenges emerge in vivo due to the autofluorescence of tissue at wavelengths below 700 nm. this has led to a transition to near-infrared dyes and infrared fluorescent proteins (700 nm–800 nm) which have demonstrated much more feasibility for in vivo imaging due to the much lower autofluorescence of tissue and deeper tissue penetration at these wavelengths. bioluminescence imaging, on the other hand, is based on light generated by chemiluminescent enzymatic reactions. in both fluorescence and bioluminescence imaging, the light signals are captured by charged coupled device (ccd) cameras cooled up to −150 °c, making them extremely light-sensitive. in events where more light is produced, less sensitive cameras or even the naked eye can be used to visualize the image. strengths: optical imaging is fast and easy to perform, and is relatively inexpensive compared to many of the other imaging modalities. furthermore, it is extremely sensitive, being able to detect molecular events in the 10–15 m range. in addition, since bioluminescence imaging does not require excitation of the reporter, but rather the catalysis reaction itself, it is indicative of the biological / molecular process and has almost no background noise. weaknesses: a major weakness of optical imaging has been the depth of penetration, which, in the case of visible dyes is only a few millimeters. near-infrared fluorescence has allowed depths of several centimeters to be feasible. since light in the infrared region has the best penetration depth, numerous fluorochromes have been specifically designed to be optimally excited in this area. optical imaging, fluorescence has a resolution limited to the diffraction of light of ~270 nm and bioluminescence has a resolution of ~1–10 mm, depending on time of acquisition, compared to mri at 100 µm, and micro-ultrasound at 30 µm. cancer research: because of poor depth of penetration, optical imaging is typically only used for molecular purposes, and not anatomical imaging. due to poor depth of penetration in visible wavelengths, it is used for subcutaneous models of cancer, however near-infrared fluorescence has enabled orthotopic models to now be feasible. often, investigation of specific protein