Types of Optical Imaging Devices
The two principal optical imaging options for whole-body small animal molecular imaging are continuous wave (CW) and time domain (TD). The ART Optix system is a TD-based instrument. Both CW and TD systems collect optical signals that can be used to determine the concentration and location of fluorophores within an animal. In order to effectively achieve this goal, CW devices necessitate multiple angular projections of the same subject, while TD systems operating in reflection mode can evaluate those directly from the temporal response of the subject’s tissue to ultra-short laser pulses. In similar acquisition settings, the added information content of TD systems allows for more accurate recovery of depth and concentration than CW systems.
A variant of fluorescence imaging, bioluminescence, is also used in molecular imaging. In this modality, the excitation energy is supplied by a chemical reaction rather than from an external light source. Typically, two agents are required for bioluminescence — a light producer such as luciferin, and a catalyzing agent, luciferase. The luciferase catalyzes the oxidation of luciferin, resulting in light emission. In order to fuel this reaction, luciferin must be brought into the system, either through diet or by internal synthesis. In bioluminescence, luciferase becomes a biomarker, tagged to specific proteins or genes in the organism.
The capabilities of bioluminescence, as well as CW and TD fluorescence technologies, are illustrated in the following table.
| Type of Data | Bioluminescence | CW Fluorescence | TD Fluorescence |
|---|---|---|---|
| Fluorescence intensity |  |
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| Bioluminescence intensity | |
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| Fluorescence lifetime | |
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| Fluorophore depth from a single projection scan | |
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| Fluorophore concentration from a single projection scan | |
Illumination Geometries
There are essentially two methods of illuminating exogenous fluorophores — wide-area and point-wise raster-scanning. The main disadvantage of wide-area illumination is the spatial cross-talk between the illuminated source points, which can make tomographic reconstruction difficult. Also, wide-area illumination does not ensure a uniform illumination density, especially in subjects with irregular surfaces. Non-uniformity is also an issue for longitudinal studies in which subjects may not always be positioned identically throughout consecutive imaging sessions. Raster-scanning, in which a quasi-punctual source such as a laser is directed at various locations on the subject’s surface in a consecutive manner, enhances light density uniformity across the subject’s surface and between scans within a longitudinal study, and is the method of choice for the Optix system.
Detection Geometries
Regardless of the type of illumination geometry used, two main categories of detection geometries exist in optical imaging systems — reflection mode and transmission mode. Reflection mode refers to a geometry in which photon detection occurs on the same surface as illumination, while transmission mode refers to detection on a different surface from that of the illumination. Each type of geometry has its advantages and disadvantages.
The main advantage of reflection mode is that it offers more sensitivity than transmission. In reflection mode, photons only need to travel from the surface of the medium to the fluorophore’s location and then back to the surface. For most applications, this represents only a few millimeters over which light is attenuated. In transmission mode, light is attenuated over the entire thickness of the sample and measured signal intensities are much lower. Reflection mode also offers a high level of precision when determining fluorophore depth since signal delay can be directly linked to fluorophore depth. In transmission mode, little to no delay occurs when fluorophore depth changes.