Preclinical imaging

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Preclinical imaging is the visualization of living animals for research purposes,

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

]

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.[3]

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,

, 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

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:

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.[7] 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

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.^[8] 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.

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.^[9]

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,^[10] 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.^[2]

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.^[11][12]

Micro-CT

Principle:

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.[8] In addition, micro-CT is excellent for bone imaging.

Weaknesses: One of the major drawbacks of micro-CT is the

. 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.^[14]

Micro-PET

Principle:

(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.^[15]

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

Principle: Similar to PET,

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.^[15] 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.)^[16] 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

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

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,[17]	HiReSPECT[18]	Pixelated CsI(Tl) crystals, LEHR parallel hole collimator, Rat and Mice imaging	25	1.7	36
Magota et al., 2011,[19]	Inveon	NaI(Tl) crystals, 0.5mm single pinhole collimators	25	0.84	35.3
van der Have et al., 2009, Ivashchenko et al., 2015,[20] [21]	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,[22]	X-SPECT	two rotating NaI(Tl) detectors, various apertures	NA	0.62 best	855

Combined PET-MR

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

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.^[23]

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

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.^{[24][25][26][27]}
• 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.^[2]
  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.^[8]

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.

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.[28] Often, investigation of specific protein expression in cancer and drug effects on these expressions are studied in vivo with genetically engineered light-emitting reporter genes.^[2] This also allows the identification of mechanisms for tissue-selective gene targeting in cancer and beyond.^[29]

Combined PET-optical imaging, fluorescence

Principle: Dioxaborolane chemistry enables radioactive

Strengths: Combines the strengths of

Weaknesses: Combining

with subcellular resolution (see Figure of multicolor HeLa cells). Multiple instruments are required to image PET, whole organ fluorescence, and single cell fluorescence with sub-cellular resolution.

References