Year 2

The project entitled “A novel SPECT microscopy system for 3D imaging of single stem cells in vivo” achieved significant progress toward the creation of an imaging tool that will ultimately find use in monitoring clinical stem cell therapies in humans. The imaging technique employs cutting edge technologies both in the preparation of the stem cells to be imaged and in the microscope system that we developed to visualize the stem cells in vivo. Certain radioactive atoms emit low-energy x-rays and gamma-rays when they undergo decay. The straight-line trajectories of these photons are unperturbed through a few millimeters of tissue, enabling high resolution image reconstruction when special lens-like, image-formation devices known as “coded apertures” are employed. The components of the microscope are: 1) the pixellated detector; 2) the coded aperture foil; 3) the motion control system; and 4) a radioactive calibration source. The design of each component and their relative geometrical configuration result in a system that is designed to produce cellular resolutions (on the order of 20 micrometers). The detector is the state-of-the-art TimePix detector developed as part of the international high-energy physics collaboration at CERN (Geneva, Switzerland). This detector has a special thickness of silicon that is responsive to the low-energy photon emission but relatively transparent to higher energy photons from the same radionuclide decay. The TimePix also has the necessary spatial resolution to achieve the targeted image resolution. Acceptance tests of the TimePix show that the low energy photon emission from the decay of 57Co can be detected and their energy value is discerned. The coded aperture foil has a special hole pattern called a modified uniformly redundant array (MURA) – this pattern has two properties: high acceptance of photon flux (because there are many holes), and ability to form an image of individual cells as if they are “stars in the sky” – that is, high contrast bright spots in a blank background. Our coded aperture is formed by thin layers of gold that have the hole patterns aligned to form a conical acceptance angle. For this initial aperture foil, we have selected a 30 micrometer hole size, with finer resolution planned once the performance of this prototype is understood. Precision motion control is enabled for both the coded aperture foil (for focus) and the specimen (for field-of-view). The microscope is designed to image an area of roughly 5 mm x 5 mm (variable depending upon magnification). The specimens that can be imaged range from microscope slides with radiolabeled cell populations to calibration phantoms that simulate cells of known radioactivity and location. The microscope was also designed to image radiolabeled cells injected into the spine or lower limbs of living laboratory mice. As the microscope is designed for low energies, other imaging devices are available for seeing the cells in larger volumes of interest and within their anatomical context. In particular, the translation from microscope to in vivo small animal imaging is done with a new microSPECT/MRI multi-modality instrument. The microSPECT has the ability to image the higher energy emissions from the same labeling radionuclides (e.g., 111In, 99mTc). The ability to image stem cells with hinges upon the technique used to incorporate the radioactive atoms to the cells. This project has made an exciting advance in the imaging technology needed to see the cells in vivo by incorporating “reporter genes” into the mesenchymal stem cells before they are injected into the laboratory animal. The reporter gene technique fundamentally transforms the use of nuclear imaging of cells. In conventional nuclear labeling, cells are visible only until the radioactivity that was originally attached ex vivo has decayed. With the reporter gene technique, however, we have demonstrated in laboratory animals the ability to visualize the cells in vivo in mice in three powerful and unique ways: 1) as the stem cells undergo mitosis in vivo, their progeny express the reporter imaging receptor that absorbs radioactivity injected at any time point after the genetically-engineered cells were inserted; 2) the expression of imaging receptors can be made to be promoted by differentation of the stem cells, generating an imaging signal when the stem cells have differentiated; and 3) non-radioactive fluorescent contrast agents can target the imaging receptors for optical microscopy of the stem cells and their differentiated progeny. Another feature of nuclear imaging of cells is the ability to image two photon energies – one can be from the stem cells and the other from a radiopharmaceutical that is targeted for the desired stem cell reparative function. This dual-energy imaging can monitor the location and action of stem cells in clinical trials of stem cell therapies.