Imaging Instrumentation For Living Subjects

A. Basic Principles of Optical Charged Couple Device (CCD) Imaging (Fluorescence and Bioluminescence)

Although photons emitted in the visible light range of the electromagnetic spectrum face considerable obstacles traveling through layers of tissues, notable advances in light sensor technology have permitted the use of optical reporter genes in intact organisms. There are fundamentally 2 different types of optically based imaging systems: a) fluorescence imaging, which uses emitters such as green fluorescent protein (GFP), wavelength-shifted GFP mutants, red fluorescent protein (RFP), ''smart'' near-infrared fluorescent (NIRF) probes, and b) bioluminescence imaging, which utilizes systems such as Firefly luciferase-D-Luciferin or Renilla luciferase-coelen-terazine (15,19,20). Each of these systems will be discussed in further detail in a later section. Emission of light from fluorescent markers requires external light excitation while bioluminescence systems generate light de novo after an injectable substrate is introduced and the appropriate conditions are met (Fig. 1). In both cases, light emitted from either system can be detected with a thermoelectrically cooled charge-couple device camera (CCD) since they emit light in the visible light range (400 nm to 750 nm) to near-infrared range (~800 nm). Cooled to - 120 to - 150°C, these cameras can detect weakly luminescent sources within a light-tight chamber. Being exquisitely sensitive to light, these desktop camera systems allow for quantitative analysis of the data. The method of imaging bioluminescence sources in living subjects with a CCD camera is relatively straightforward: the animal is anesthetized, subsequently injected intravenously or intraperito-neally with the substrate and placed in the light-tight chamber for a few seconds to minutes. A standard light photographic image of the animal is obtained, followed by a bioluminescence image captured by the cooled CCD camera positioned above the subject within the confines of the dark chamber.

A computer subsequently superimposes the 2 images on one another, and relative location of luciferase activity is inferred from the composite image. An adjacent color scale quantitates relative or absolute number of photons detected. This scale does not reflect the color (wavelength) of the emitted photons, but only the number of such photons, measured in relative light units per minute (RLU/min). Differences between fluorescence and bioluminescence systems are discussed in a later section of this chapter.

Comparison of optical-based imaging systems with the other imaging modalities, such as the radionuclide-based or MRI-based systems, reveals important differences. Advantages of optically-based reporter systems is that they are at least an order of magnitude more sensitive than the radionu-clide-based techniques at limited depths (21). Furthermore, the direct and indirect costs are generally less than radionu-clide-based techniques or MRI. However, there is significantly less spatial information obtained from optical imaging, and signal obtained from light-emitting reporter systems is limited by the tissue depth from which it arises. Furthermore, while significant progress has been made to localize fluorescent signals tomographically to obtain distribution of fluorochromes in deep tissues (22), there is currently no commercially available technique to obtain 3-dimensional localization of the targeted optical probes.

B. Basic Principles of PET

With recent breakthroughs in molecular/cell biology and target discovery, it is now possible to design specific markers to image events noninvasively in small animals and humans with positron emission tomography (PET) (23,24). Natural biological molecules such as glucose, peptides, proteins, and a variety of other structures can either be labeled with a radioisotope or slightly modified to accommodate a radioisotope. In the jargon of molecular imagers, these radiolabeled molecules are referred to as molecular probes, reporter probes, markers, or tracers. In PET imaging, molecules are labeled with isotopes that emit positrons from their nucleus. This is in contrast to gamma camera or SPECT imaging (discussed below), where molecules are ''tagged'' with radioisotopes that emit gamma rays. When a molecular probe is injected into a living subject and then placed into a PET scanner, the image we acquire is a snapshot of the physiological distribution and concentration of that tracer.

Let's take the example of 2-deoxy-2-[18F] fluoro-D-deoxy-glucose (FDG), a glucose analog labeled with the positron-emitting isotope, 18F. In the synthesis of this tracer, PET radio-chemists have devised a method to replace one of the hydrogens with the 18F radioisotope. Following intravenous injection, this particular probe distributes readily from the intravascular compartments to the extravascular and intracel-lular compartments. FDG becomes sequestered in cellular populations and tissues that have a predilection for glucose, i.e., cells that possess larger number of glucose transporters and/or hexokinase II activity. When FDG encounters hexokinase II, it becomes phosphorylated and subsequently trapped

Molecular Imaging Technologies Table 1 Summary of Reporter Gene Systems Used in Living Subjects

Method Imaging

Reporter gene


Imaging agent/substrate


In vitro Chromatography and



Radioactive acetyl group


(selected autoradiography


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