Facilities > Imaging

We believe that molecular imaging will emerge as an objective standard for validating biomarkers and targets as well as assessing the tissue-targeting specificity and endothelial processing in vivo of probes such as intravenously injected antibodies. Over the last few years, a number of instruments and methodologies, such as gamma scintigraphy imaging, small animal single photon emission computed tomography coupled with x-ray computed tomography (SPECT-CT), and intravital microscopy (IVM), have been developed and/or optimized by us and others to allow increasingly higher resolution imaging in both humans and small animals. Molecular imaging can provide a non-invasive, highly objective, and stringent test of targeting that is not possible with other techniques. It allows dynamic, live imaging of the whole body at once so that minor accumulations can be detected in real time in all parts of the body, including minor tissues that are either not normally considered or are not amenable to dissection for biodistribution analysis. It is invaluable to be able to perform longitudinal studies monitoring the distribution of the targeting probes or the tissue response to our vascular targeting agents with time within the same animal. Importantly, the technology also allows the targeting, uptake and accumulation of probe conjugates to be readily quantified. It is vital to use two or more validation methods in order to confidently assess the binding specificity of targeting probes. At a minimum, we always couple dynamic imaging techniques with static imaging, including confocal and electron microscopy, to verify our findings and provide additional detailed analysis. The imaging facility has two main aims: 1) to determine the in vivo targeting and endothelial cell processing of intravenously injected probes in vivo and 2) to create a topological map of protein expression across multiple organs and disease states. This facility has more than 2 TB of digital images stored from intravital, confocal, fluorescent, electron, and light microscopy studies.

Intravital Microscopy (IVM):
IVM permits live, dynamic video microscopy of endothelial cell surface binding as well as transport across the endothelial cell barrier for accumulation in the tissue parenchyma. Individual tissue cells can easily be discerned by standard light and fluorescence microscopy so that many cellular events such as immune cell migration, mitosis, pyknosis and apoptosis, and the growth and connectivity of blood vessels can be readily quantified.  Used in conjunction with higher magnification static imaging methods, one can obtain a relatively complete picture of endothelial cell targeting and processing of candidate targeting probes.
To image the vasculature and endothelial cells in vivo, we implant mice with a dorsal skin fold window chamber. Small pieces of tissue are engrafted into this window chamber. We have shown that this implanted tissue maintains both tissue- and species-specificity, even expressing key organ-specific endothelial cell markers and that the blood vessels from the donor tissue connected to the vessels of the underlying host skin tissue to revascularize with ample blood flow within one week. Once vasculature is readily apparent throughout the tissue, targeting and processing of fluorescent dyes, antibodies, drugs, gene vectors, and nanoparticles can be imaged dynamically through the transparent chamber.

Gamma scintigraphy and SPECT-CT:
Optical imaging and gamma scintigraphy allows us to view and quantify signal accumulation in different regions of the body in two dimensions. Dynamic planar imaging assesses targeting and accumulation at the whole body level in real time. SPECT-CT provides a means to analyze and quantify signal accumulation at high resolution in three dimensions. This allows the radiosignal from a probe to be correlated with distinct anatomical structures in the body non-invasively. Radiolabeled antibodies can be readily imaged following intravenous injection with our Gamma Medica SPECT-CT imaging system. This system has a resolution down to 200 – 400 mm; can detect radioisotopes 125I, 123I, 99mTc, and 111In; and can construct 3D images of the radiosignal and skeletal structure (through x-ray CT scanning). With proper acquisition and processing of the radionuclide image data, actual quantitative rates of the compound's processing may be obtained. Image data analysis consists of visual detection of a focal area of increased radiotracer. Included software permits region-of-interest analysis correlated with the CT to quantify probe accumulation in each tissue.

Static Imaging:
The static imaging used in this facility includes confocal microscopy, immunohistochemistry, and electron microscopy. This imaging serves two purposes: to localize proteins to specific tissues, cells, or subcellular fractions, and to provide greater detail for the live imaging described above. Though these methods are not dynamic, they provide far greater detail and resolution than is possible with mass spectrometry or live imaging.

Mass spectrometry-based proteomics and static imaging are complementary approaches to define a topological map of protein expression. Proteomics approaches aim to identify the proteins that are present in a particular subcellular fraction in a high-throughput manner. The final results are dependant on the purity of the isolated fraction. Light and fluorescence microscopy can reveal probe location in tissues and cells while electron microscopy can reveal probe location at the subcellular level. Additionally, it is straightforward to analyze many tissues from both healthy and diseased states. Electron microscopy is especially important to validate mass spectrometry results. We have analyzed 100’s of antibodies to validate 50 proteins concentrated in caveolae. The small size of caveolae (approx. 70 nm in diameter) demands that electron microscopy, not light microscopy, be used to accomplish the task. The technique is referred to as immunocytochemistry. However, static imaging analyzes proteins one at a time, or at best, a few at a time and is simply too tedious to screen large number of proteins.

Static imaging can also be used to provide detailed “snap shots” of endothelial cell targeting and processing of intravenously injected probes. For these types of studies, tissues is excised at set times following probe injection, and the probe is localized with immunohistochemistry, immunofluorescence, and/or electron microscopy. This can provide valuable confirmation of the ability of antibody to target the endothelial cell surface, to be transcytosed across the endothelial cell barrier, and to penetrate deep into the tissue.