Now you can image a mouse like a man

In Vivo animal imaging finally goes nano thanks to a new tomographic nano-imaging device

Mice and biologists everywhere can breathe a collective sign of relief. Far fewer small laboratory animals will need to be sacrificed and dissected to study the origins of disease, or the effectiveness of experimental therapies. Instead, thanks to a new tomographic imaging device, called NanoSPECT, investigators can study living biology in mice with nanolitre precision. By using a new multiplexed, multi-pinhole image projection technique for single photon emission tomography (SPECT), researchers can now investigate biology in living mice with the same exquisite detail as obtained in men with clinical PET and SPECT scans. This enables to track the development of disease and to evaluate new therapies in intact living animals for extended periods of time, thus obtaining a fuller, more accurate picture of what’s going on and why. In this article, we describe the technology and instrument that makes it possible to image subjects such as mice which are thousand times smaller than humans, with the same image detail as can be obtained from scanning humans. In addition, with a few examples, we illustrate the potential power of this method in modern biological research.

[caption id="" align="alignleft" width="232" caption="Figure 1: Despite the very small size of a mouse as compared to a human, it is possible with the new NanoSPECT/CT imaging device to obtain images of living mice with the same intricate detail that can be obtained from scanning humans. This is accomplished by exclusive multi-pinhole image magnification"][/caption]

 Mouse modelling Approximately 95% of all lab animals are mice and rats. Easily housed and bred, short lived (2-3 years) with a short reproductive cycle, small and relatively inexpensive, rodents have become the animal model of choice for modern biomedical researchers. Their physiology and genetic make up closely resemble that of humans. The mouse genome is believed to contain essentially the same complement of genes found in the human genome so studying how the genes work in mice is an effective way of discovering the role of a gene in human health and disease.

To date, mouse models of human disease are mostly studied using invasive techniques, destructive to the tissue, such as biopsy followed by tissue counting or autoradiography. Although well established, there are several disadvantages to these techniques. These include: large numbers of animals are required, all of which have to be sacrificed; follow-up studies are generally not possible in the same animal nor can multiple samples be obtained from the same animal; and drugs administered as prodrugs, requiring metabolism for efficacy, are difficult to study with destructive methods. Moreover stringent ethical regulations and economic demands (research time and cost of genetically manipulated animals) urge researchers to use other methods that restrict the numbers of animals involved1.

Over the last couple of years, in vivo imaging techniques such as X-ray micro CT (computed tomography), MRI (magnetic resonance imaging) microscopy, micro PET (positron emission tomography), micro SPECT (single photon emission computed tomography) and optical planar imaging have become available. Of these technologies, optical imaging doesn’t translate to the clinic. Indeed, light from optical tracers doesn’t penetrate far through tissue making the technology incompatible with both small animal and human imaging. Micro CT and MRI on the other hand are intended primarily for scanning structural or anatomical features. Some limited functional information can be obtained with modern MRI methods, although the sensitivity levels are typically in the milli - to micromolar range, insufficient for studying molecular interactions. Nuclear imaging methods on the other hand, such as PET and SPECT methods, can provide sensitivities beyond the nanomolar range.

The exquisite sensitivity of PET and SPECT imaging over other modalities and their applicability to both small animals and humans makes these techniques attractive for in vivo imaging. Results obtained in experimental small animals can be translated to a clinical setting in humans. In order to carry out such translational studies successfully, however, imaging systems must be available that meet the demanding spatial resolution requirements imposed when imaging subjects such as mice, thousand of times less massive than humans (Figure 1).

Meeting the challenge of imaging small animals with PET or SPECT The challenge to developing the technology for imaging small laboratory animals, and in particular the mouse, is that of achieving the necessary spatial resolution, which must be comparable with that realised in man. In round numbers, the linear dimensions in mice are ten times smaller in each dimension than in humans. Therefore, if imaging studies in small animals are to be “equivalent” to human studies, the spatial resolution of an animal scanner must be about ten times better than a human scanner2. “Equivalent” in this context means that the animal organ is visualised on the animal scale with the same relative acuity as the human organ is imaged on the human scale. Taking 6mm as an achievable PET and SPECT resolution in humans, the scaled up resolution to achieve anatomical parody for a mouse is 0.6 to 0mm (or sub-µL in volumetric terms). This is clearly beyond the realms of physical possibility with PET imaging. This follows from the finite positron range of PET tracers. Rather than detecting the location of the emission of a positron tracer, PET detects the location where gamma rays are being emitted as a result of the annihilation of a positron with an electron. This distance, the “positron range”, represents a built-in “blur” present in all PET images. While this effect is small compared to other effects in human imaging, this range extends from one to a couple of millimeters depending on the energy of the used tracer, making it quite impossible to perform small animal PET imaging with sub-mm resolution.

SPECT imaging on the other hand uses gamma emitters rather than positron emitting tracers. As a result, SPECT detects the localisation of the emission so that the blurring effect caused by the positron range is eliminated. However, in order to determine where the gamma rays come from, a SPECT device needs a collimator in front of its detectors to define the direction of the gamma rays. For high resolution imaging, small pinhole collimators are used which have the advantage that the gamma rays can be projected through the small pinhole on a large detector, thus magnifying the image of the subject. By using small pinholes, high resolution SPECT images of mice can be obtained3. The disadvantage of this approach is that gamma rays have to pass through a single small pinhole in order to be detected. This reduces the number of detectable events and the sensitivity of such single-pinhole micro SPECT devices drops by roughly one to two orders of magnitude below the sensitivity achievable with micro PET systems4.

Solving the resolution-sensitivity trade-off for imaging small animals Although micro PET and micro SPECT devices are being used for imaging of small animals, they each suffer from critical shortcomings to make them an in vivo alternative to the established ex vivo techniques such as autoradiography and tissue counting. For mouse imaging, the positron range of PET tracers blurs the images so much that micro PET systems are only capable of measuring tracer distributions and kinetics at the whole organ or large region-of-interest level5. Conventional single-pinhole micro SPECT devices on the other hand suffer from low detection sensitivity. To address this problem, manufacturers of micro SPECT system offer pinhole collimators with different size pinholes. Indeed, the detection sensitivity can be improved by enlarging the pinhole; however, such sensitivity improvements come at the detriment of lower image resolutions.

[caption id="" align="alignleft" width="425" caption="Figure 2: Bioscan’s ultra-high resolution NanoSPECT/CT system enables researchers to image molecular interactions in living small animals. The sedated animals are carefully positioned on an animal bed before imaging."][/caption]

To address this sensitivity versus resolution trade-off problem, Bioscan has recently introduced a new SPECT technology, called NanoSPECT, which uses a patented multiplexed, multi-pinhole SPECT technology (Figure 2). Instead of acquiring images through a single pinhole, NanoSPECT acquires images through thirty-six or more small pinholes (Figure 3). The use of small pinholes results in image magnification for high spatial resolution, while the projection of images through multiple pinholes

simultaneously ensures high detection sensitivities. With this unique approach, NanoSPECT addresses the sensitivity-resolution trade-off problem and is able to produce images with sub-mm resolution (less than 500nL volumetric resolution), and with a detection sensitivity approaching the sensitivity of micro PET systems. Compared to micro PET systems, NanoSPECT’s images are approximately ten times sharper, and when compared to micro SPECT systems, the sensitivity is increased by at least one order of magnitude6. This enables measuring tracer distribution and kinetics in mice at the sub-organ or micro-region level and establishes NanoSPECT as a real in vivo alternative to labor-intensive and animal-consuming ex vivo autoradiography and tissue counting techniques.

[caption id="" align="alignleft" width="293" caption="Figure 3: After administering a radiopharmaceutical or biological tracer, the single photon emissions are projected through thirty-six pinholes on four detectors surrounding the animal (only three projections are shown for simplicity). This simultaneous acquisition of multiple magnified images solves the traditional resolution-sensitivity trade-off problem for in vivo imaging of small animals."][/caption]

Studying biology at the molecular level in living mice By removing the fogginess from nuclear tomographic images, NanoSPECT provides a way to track the development of diseases and to evaluate new therapeutic approaches in intact, living small animals such as mice and rats. Moreover, the use of SPECT rather than PET tracers has the advantage that they are widely available from a mature isotope distribution network. For a wide range of applications, researchers can often use the so-called “shake and bake” kits to make a range of useful compounds in-house. This eliminates the need for an expensive on-site cyclotron/radiochemistry production facility typically required for the use of PET tracers. The use of SPECT tracers is therefore relatively cheap, and the longer half-lives as compared to PET tracers make SPECT well suited, if not required, when biologically active radiopharmaceuticals have slow kinetics.

Moreover, for drug discovery, development and delivery research at the pre-clinical level, it is not necessary to develop imaging analogues of every drug candidate. In vivo competition or displacement assays of unlabeled compounds with labelled probes for agents with known pharmacological characteristics and efficacy can be used instead.

As illustrated in Figure 4, NanoSPECT acts as a window into living biology, tracking a range of biological processes from metabolism to receptors, gene expression, cell trafficking, drug activity and others - all with unprecedented detail, at the nanolitre precision level.

Author: Staf C. Van Cauter, M.Sc., Washington, D.C. USA

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