Although ex vivo techniques, such as recently developed rapid sampling microscopy methods combined with the increased stability of fluorescent probes, now make it possible to routinely acquire three-dimensional microscopy data sets over time a method commonly referred to as four-dimensional microscopy , these types of experiments generate large data sets that often cannot be intuitively and quantitatively interpreted Phair and Misteli An alternative is the laborious and often crude three-dimensional reconstruction of information from many contiguous histological slides.
Instead, and with continued developments in this field, molecular imaging should be capable of generating four-dimensional information including temporal changes of biological phenomena much quicker and easier than by more conventional invasive means of investigation. A further incentive for use of molecular imaging strategies in basic biological research is the rising cost of laboratory mice and the relative scarcity of some genetically engineered mice.
This has provided a major impetus for establishing in vivo mouse imaging as an alternative to killing many animals for histological processing at different time points. The use of fewer animals in biological assays with molecular imaging would also be more appealing on ethical grounds. In theory, approval for research projects requiring large numbers or many separate cohorts of experimental animals could be obtained more easily. Biological discovery has moved at an accelerated pace in recent years, with considerable focus on the transition from in vitro to in vivo models.
As such, there has been a greater need to adapt clinical imaging methods for noninvasive assays of biochemical processes. Considerable efforts have been directed in recent years toward the development of noninvasive, high-resolution, small animal in vivo imaging technologies Fig. The widespread availability and use of miniaturized imaging systems for rodents are not the fanciful and futuristic concepts that many researchers might imagine; these systems are generally cheaper than their clinical counterparts and can be housed in shared resources of basic science laboratories.
In small animal research, the primary goal is to obtain as high a signal as possible and to localize the signal as accurately as possible with high temporal resolution and with minimal amount of molecular probe. The ultimate goal is to provide a single device that produces a final three-dimensional image of anatomical and biological information fused together, an objective that is likely to be achieved in the not too distant future. Multiple imaging modalities are available for small-animal molecular imaging.
Shown are views of typical instruments available, and illustrative examples of the variety of images that can be obtained with these modalities. A microPET whole-body coronal image of a rat injected with 18 FDG, showing uptake of tracer in tissues including muscles, heart, brain, and accumulation in bladder owing to renal clearance.
B microCT coronal image of a mouse abdomen after injection of intravenous iodinated contrast medium. C microSPECT coronal image of a mouse abdomen and pelvis regions after injection of 99m Tc methylene diphosphonate, showing spine, pelvis, tail vertebrae, femurs, and knee joints owing to accumulation of tracer in bone.
D Optical reflectance fluorescence image of a mouse showing GFP fluorescence from the liver, abdomen, spine, and brain. The mouse contains GFP-expressing tumor cells that have spread to various sites. Images are courtesy of Dr.
Hoffman, Anticancer Inc. E microMRI coronal T2-weighted image of a mouse brain. F Optical bioluminescence image of a mouse with a subcutaneous xenograft expressing Renilla luciferase in the left shoulder region, after tail-vein injection of the substrate coelenterazine. Images were obtained using a cooled CCD camera. The color image of visible light is superimposed on a photographic image of the mouse with a scale in photons per second per square centimeter per steradian sr. Collapsing the volume of an animal or tumor into a single image, known as planar imaging, is generally fast, the data sets generated are small, and imaging can be done in high throughput fashion, at the expense of internal resolution.
Volumetric image acquisition shows a volume of interest in all three dimensions and results in the highest spatial information content, although it can generate very large data sets. Further reviews of issues centered on molecular imaging techniques can be found elsewhere Cherry and Gambhir ; Weissleder , ; Weissleder and Mahmood ; Chatziioannou Moreover, a glossary of molecular imaging terminology has been published recently to enhance collaborative efforts between multiple disciplines Wagenaar et al.
Table 1 outlines some of the general characteristics of the imaging modalities available, and serves also as a simplified guide for biologists in choosing appropriate molecular imaging modalities and approaches. Please see additional useful links listed at the end of this article. Positron-emitting isotopes frequently used include 15 O, 13 N, 11 C, and 18 F, the latter used as a substitute for hydrogen.
Most of these isotopes are produced in a cyclotron Strijckmans , but some can be produced using a generator e. Labeled molecular probes see below or tracers can be introduced into the subject, and then PET imaging can follow the distribution and concentration of the injected molecules. Many of the positron-emitting isotopes used have relatively short half-lives e. PET radiopharmacies exist throughout the world and are capable of providing commonly used PET tracers on a daily basis Gambhir In contrast to SPECT, attenuation quantifiable reduction in events present at the face of the detector due to absorption or scatter through tissues of the emitted radiation in PET can be corrected precisely because the total length through the body determines the attenuation factor along a coincidence line.
By doing so, quantitative information about the tracer distribution can be obtained. The reconstruction software then takes the coincidence events measured at all angular and linear positions to reconstruct an image that depicts the localization and concentration of the positron-emitting radioisotope within a plane of the organ that was scanned. If single photon emitters are used, the direction of flight has to be determined by geometric collimation. Typically, several million cells accumulating reporter probe have to be in relative close proximity for a PET scanner to record them as a distinct entity relative to the background.
In SPECT, collimator design is always a compromise between spatial resolution and sensitivity: reducing the size of the holes or using longer septae improves spatial resolution but reduces sensitivity at the same time. For example, even a triple-head SPECT system designed to image 99m Tc-labeled tracers in the human brain is 15 times less sensitive than a PET if a 1-cm resolution is assumed in both systems Budinger Positron-emitting isotopes can usually be substituted readily for naturally occurring atoms, and therefore PET is a more robust technique than SPECT for imaging most molecular events.
Therefore, to investigate multiple molecular events, molecular probes are usually injected separately, allowing for the decay of one isotope prior to administration of the other. In recent years, small animal micro-PET scanners have been developed. Development of molecular imaging assays with PET is particularly advantageous because of the ability to validate them in cell culture and small animal models prior to using the same reporter probe in established clinical PET centers around the world.
The ability to perform translational research from a cell culture setting to preclinical animal models to clinical applications is one of the most unique and powerful features of PET technology. Optical imaging techniques have already been developed for in vitro and ex vivo applications in molecular and cellular biology e.
An extension of this concept toward noninvasive in vivo imaging with light photons represents an interesting avenue for extracting relevant biological information from living subjects. Progress in optical molecular imaging strategies has come from the recent development of targeted bioluminescence probes, near-infrared fluorochromes, activatable near-infrared fluorochromes, and red-shifted fluorescent proteins Weissleder A notable theoretical advantage of optical techniques is the fact that multiple probes with different spectral characteristics could potentially be used for multichannel imaging, similar to in vivo karyotyping Weissleder Optical imaging also allows for a relatively low-cost alternative to studying reporter gene expression in small animal models see below.
A fundamental issue in optical imaging of living subjects is how to detect light emitted from the body, this being relevant to both bioluminescence and fluorescence imaging. In this regard, several technical advances for imaging very low levels of visible light have now emerged, allowing the use of highly sensitive detectors in living subjects, and not just restricted to cell cultures and small transparent animals.
Charged coupled device CCD detectors are made of silicon crystals sliced into thin sheets for fabrication into integrated circuits using similar technologies to those used in making computer silicon chips. For a detailed overview of CCD technology, please refer to Spibey et al. One of the properties of silicon-based detectors is their high sensitivity to light, allowing them to detect light in the visible to near-infrared range.
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A CCD contains semiconductors that are connected so that the output of one serves as the input of the next. In this way, an electrical charge pattern, corresponding to the intensity of incoming photons, is read out of the CCD into an output register and amplifier at the edge of the CCD for digitization. A camera controller, linked to a computer system, is used for data acquisition and analysis. A bioluminescence image is often shown as a color image that is superimposed on a gray-scale photographic image of the small animal using overlay and image analysis software.
Usually a region of interest is manually selected over an area of signal intensity, and the maximum or average intensity is recorded as photons per second per centimeter squared per steradian a steradian is a unit of solid angle; Wu et al. The main advantage of optical bioluminescence imaging is that it can be used to detect very low levels of signal because the light emitted is virtually background-free see below.
It is quick and easy to perform and allows rapid testing of biological hypotheses and proofs of principle in living experimental models. It is also uniquely suited for high-throughput imaging because of its ease of operation, short acquisition times typically 10—60 sec , and the possibility of simultaneous measurement of six or more anesthetized living mice Vooijs et al.
However, the cooled CCD camera has three main drawbacks Wu et al. Skin and muscle have the highest transmission and are fairly wavelength-dependent, whereas organs with a high vascular content such as liver and spleen have the lowest transmission because of absorption of light by oxyhemoglobin and deoxyhemoglobin.
Secondly, images obtained from the cooled CCD camera are two-dimensional and lack depth information. However, it is expected that future bioluminescence image acquisition using rotating CCD cameras or multiple views of the same animal with a single CCD camera may allow volumetric imaging, especially when combined with novel red-shifted luciferases that have better tissue penetration.
A third limitation is the lack of an equivalent imaging modality applicable for human studies, thus preventing direct translation of developed methods for clinical use. In fluorescence imaging, an excitation light of one wavelength in the visible light range of — nm illuminates the living subject, and a CCD camera usually a less-sensitive version than the cooled CCD required in bioluminescence detection, for technical reasons discussed in Golden and Ligler collects an emission light of shifted wavelength.
Cells tagged with fluorescently labeled antibodies or those in which expression of the green fluorescent protein GFP gene or its variants; Lippincott-Schwartz et al. GFP is a protein from the jellyfish Aequorea victoria that has become very popular over the last decade as a reporter in fixed and cultured cells and tissues. Wild-type GFP emits green nm light when excited by violet nm light. The variant EGFP has a shifted excitation spectrum to longer wavelengths and has increased fold brightness.
Between and 10, fluorescently-labeled cells in the peritoneal cavity of a mouse can be imaged on its external surface Kaneko et al. It may be necessary to expose internal organs surgically prior to their imaging Bouvet et al. This simple, reflectance type of fluorescence imaging has been used extensively in studies of feasibility and development of these approaches Kamiyama et al.
Li et al. However, these systems are not quantitative, and the image information is surface-weighted anything closer to the surface will appear brighter compared with deeper structures; Weissleder Direct comparisons of bioluminescence and fluorescence imaging have not been published to date, although these are presently underway in our laboratory. One clear difference between the two modalities is the observation of significantly more background signal owing to autofluorescence of tissues in fluorescence imaging as compared with bioluminescence imaging.
In contrast to fluorescence imaging in the visible light range, the use of the near-infrared NIR spectrum in the —nm range maximizes tissue penetration and minimizes autofluorescence from nontarget tissue Weissleder This is because hemoglobin and water, the major absorbers of visible and infrared light, respectively, have their lowest absorption coefficients in the NIR region. Several NIR fluorochromes have recently become available Lin et al. This type of NIR fluorescence reflectance imaging is still limited to targets that are fairly near the illuminated surface.