It is possible to image particular molecules, cells or organisms using optical imaging techniques. Bioluminescence imaging provides a relatively inexpensive means of imaging genetically-engineered luciferase-expressing cells. When luciferase-labelled cells or genes are exposed to substrate (e.g., luciferin, injected just prior to imaging), photons are emitted that can be detected and quantified by a charge-coupled device (CCD) camera. Relatively small numbers of cells (< 1,000) can be detected with this technique because background noise levels are inherently low. The availability of genetically-altered luciferases and natural luciferase variants, characterized by differing emission spectra, may allow simultaneous tracking of more than one molecular target.

Using fluorescently labelled antibodies or other molecules, or inducing expression of fluorescence proteins, fluorescence imaging can also be used for cellular and molecular imaging. Unlike bioluminescence imaging, the animal is typically illuminated with an external light source. The resulting autofluorescence reduces the signal-to-noise ratio, but background filtration can be used to improve sensitivity. Diverse fluorescent dyes are now commercially available, allowing imaging of several fluorophores simultaneously in a single animal. In addition, dual-function reporter genes, e.g, a luciferase-GFP fusion reporter, have been utilized to couple in vivo imaging with ex vivo assays, and triple-fusion genes have been developed to link optical and PET imaging.

In vivo optical imaging is limited by the absorption and scatter of light as it passes through living tissue. The development of optical probes that transmit in the near infra-red spectrum has improved resolution, but optical imaging will likely remain a lower resolution modality in comparison to PET, SPECT, CT and MRI. Nevertheless, it has widespread and important application in many fields of biological and biomedical research, including tumor growth and response to therapy, efficacy of gene transfer/gene expression, bacterial infection and immune cell trafficking, beta-amyloid plaque development, and drug efficacy.

IVIS 100

Ivis 100 Our IVIS 100 Imaging System (Xenogen Corp.) is equipped for inhalation (isoflurane) anesthesia and uses Windows-based data acquisition and analysis software. As many as 5 mice can be imaged simultaneously, and imaging times are typically 1-20 sec (fluorescence) and 1-5 min (bioluminescence). For fluoresence imaging, 4 filter sets are available to allow the use of a wide range of fluorescent proteins and dyes. Although the data from this system are 2-dimensional and have lower resolution than the other imaging modalities, optical imaging has the advantage of high throughput screening afforded by multi-animal scanning and short image acquisition times.

Maestro 2

Maestro 2 The Maestro 2 (CRi) is a high-performance system designed for in vivo measurement of fluorescence signals in small animals. It is distinguished by its multispectral imaging capability, which allows for imaging several fluorophore contrast agents simultaneously. Seven excitation and seven emission filters are available to allow myriad excitation/emission combinations for differentiating a wide range of fluorophores from blue to near-infra red wavelengths. Spectral curves for each fluorophore as well as for autofluorescence can be generated and saved into a spectral library. Using a spectral library, spectral decomposition of several fluorophores and autofluorescence is achieved. The autofluorescence component is thus removed and provides individual curves for each fluorophore. By using contrasting, artificial coloration for each spectral curve, a composite image is obtained with the locations of the fluorophores displayed in distinctly different colors. The enhanced signal-to-noise ratio allows imaging of smaller or fainter signals from molecular targets than is possible with systems that cannot eliminate autofluorescence. Regions of interest can be delineated manually or by threshold segmentation for quantitative analysis of images. The Maestro is equipped for inhalation (isoflurane) anesthesia and the chamber and animal stage are heated to maintain a stable body temperature while the animal(s) are in the chamber. Up to three animals can be imaged simultaneously and imaging times are typically on the order of a few seconds, thus providing a potentially high-throughput imaging system. Although intended as a planar imaging system, limited 3-D positional information can be acquired using a multiview platform with side mirrors to generate simultaneous lateral views in addition to the dorsal or ventral view. Finally, the system can also be used to acquire non-fluorescence, low light images, although our Xenogen IVIS system is better suited to this.

MODALITY	MODEL	MANUFACTURER	RESOLUTION
PET	Inveon DPET	Siemens	~ 1.5 mm
PET	Focus 120	Siemens	~ 1.5 mm
PET	Primate 4	Siemens	~ 2.0 mm
PET	microPET II	Custom-built	~ 1 mm
SPECT	Inveon	Siemens	~ 0.5 – 3 mm
CT	Inveon	Siemens	~ 50 – 150 µm
MRI	Biospec 7T	Bruker	~ 100 – 250 µm
Ultrasound	Sequoia	Siemens	~ 100 – 500 µm
Optical	Maestro 2	CRi (Caliper)	~ 1 – 5 mm
Optical	IVIS 100	Xenogen (Caliper)	~ 1 – 5 mm
Autoradiography	Storm 860	Amersham Biosciences	~ 50 – 100 µm

For additional information:

Contag CH, Bachmann MH. Advances in in vivo bioluminescence imaging of gene expression. Annu Rev Biomed Eng; 4:235-60, 2002.

Ntziachristos V, Bremer C, Weissleder R. Fluorescence imaging with near-infrared light: new technological advances that enable in vivo molecular imaging. Eur Radiol; 13:195-208, 2003.

Bremer C, Ntziachristos V, Weissleder R. Optical-based molecular imaging: contrast agents and potential medical applications. Eur Radiol; 13:231-43, 2003.

Cherry SR. In vivo molecular and genomic imaging: new challenges for imaging physics. Phys Med Biol; 49:R13-R48, 2004.

Contag CH. In vivo pathology: Seeing with molecular specificity and cellular resolution in the living body. Annu Rev Pathol Mech Dis; 2:277-305, 2007.