- Monitoring gene therapy: By inserting the gene of a fluorescent or bioluminescent protein (e.g., GFP or luciferase) behind a therapeutic gene’s promoter, one can monitor the status of gene therapy in vivo by measurement of the intensity and localization of the optical signal.
- Measuring expression of transgenic/knock-in gene expression: As with gene therapy, coupling the genes of fluorescent or bioluminescent proteins with the knock-in genes of transgenic animals permits the in vivo monitoring of the expression of the genes of interest.
- Measuring proteolytic activity: With the recent design of targeted fluorescent optical beacons that remain dark until cleaved by specific proteases (e.g., cathepsin B or matrix metalloproteases), protease-related pathologies can be readily visualized in vivo and monitored over time.
- Protein-protein interactions: Exploiting the nature of Fluorescence Resonance Energy Transfer (FRET) – in which the fluorophores of two proteins of interest alter their respective fluorescence signals in a proximity/conformation-dependent manner – one can monitor protein-protein interactions in situ.
- Monitoring tumor growth and metastases: Using cancer cells specifically engineered to express fluorescent or bioluminescent proteins, tumor growth and metastasis – as well as the efficacy of therapeutic interventions – can be followed for extended periods in the same animal.
- Monitoring Infectious disease: Using bioluminescent (luciferase-expressing) pathogenic bacteria and fungi, the progression of a variety of infectious diseases and inflammatory conditions (as well as their responses to pharmacological therapy) can be monitored over time.
- Mapping Vascularization and Blood Pooling: Injection of fluorescent contrast agents (e.g., Indocyanine/Cardio Green or quantum dots) enables direct visualization of pathologic (genesis/necrosis) vasculature and blood pooling.
Located within the University of Chicago’s Carlson Animal Research Facility, the Optical Imaging Core Facility (OICF) provides quantitative fluorescence and bioluminescence imaging services for in vivo and in vitro studies of small animals and specimens using three different modalities.The faciliy's imaging instruments include the Xenogen IVIS 200 imaging system, the Olympus OV100 in vivo imaging system, and the VisEN Flurescence Molecular Tomography imaging system. All three in vivo optical imaging systems allow for multiple images to be acquired from the same animal so users can tract physiological changes occuring within animal over the course of seconds to minutes and days to weeks.
The Xenogen IVIS 200 is ideal for bioluminescence and fluorescence in vivo and in vitro imaging. It has the capacity to monitor and image up to 5 mice (or 3 rats) simultaneously, and in addition imaging cell culture plates. The instrument includes dual 12-position emission filter wheels and a 12-position excitation wheel; a variety of bandpass optical filters, tailored for specific fluorophore visualization (e.g., GFP, DsRed, Cy5.5, and ICG) and lumiphores (luciferase activity). Sequential imaging enables the approximation of depth of fluorophores and lumiphores in vivo. A CCD camera–cryogenically cooled to -105°C minimizes system noise while maximizing sensitivity. Combined with custom low-autofluorescence optics, the IVIS 200 offers 5 magnifications (FOVs of 26cm, 19.5cm, 13cm, 6.5cm, and 3.9cm) and is capable of detecting and quantifying the light emission from single cells in vitro at a maximum resolution of 60 microns. The IVIS 200 software allows for absolute quantification (photoms/sec/cmsq/sr) of bioluminescence and fluorescence signal, both in vivo and in vitro.
|Set||Name||Excitation (nm)||Emission (nm)||Background (nm)|
Spectral Imaging Filters
|Set||Name||Transmission Band (nm)|
|Sensor||Back-thinned, back-illuminated Grade 1 CCD|
|CCD Operating Temperature||Nominal -105°C|
|Imaging Pixels||2048 x 2048|
|Minimum Resolution||60 micron (at f/1)|
|Pixel Size||>13.5 micron square|
|CCD Size||26 x 26 mm|
|Quantum Efficiency||>85% 500-700 nm; >50% 350-950 nm|
|Read Noise||<5 electrons RMS|
|Dark Current||<100 electrons/s/cm²|
|Minimum Detectable Radiance||<70 photons/s/sr/cm²|
|Lens||f/1-f/8, 1.5x, 2.5x, 5x, 7.5x, 10x magnifications|
|Fields of View||3.9 x 3.9 cm to 26 x 26 cm (3.9, 6.5, 13, 19.5, 26 cm)|
|Scanning Laser||< 1 mW power at 532 nm|
|Stage temperature||Ambient to 40°C|
|Imaging Chamber Dimensions||51 cm X 51 cm x 66 cm (D x W x H)|
|Power Requirements||20 Amps for 120VAC or 10 Amps for 230VAC|
|Flourescence Imaging Components||Specifications|
|Excitation Filter||25 mm diameter 12-position (11-filter capacity)|
|Emission Filter||60 mm diameter 24-position (22-filter capacity)|
|Background (Autoflourescence and leakage)||<3 part in 106 typical|
|Lamp||150 W Quartz halogen 3250° Kelvin|
|Flourescence Field of View||6.5, 13, 19.5, 26 cm (flourescence not available at 3.9 cm field of view)|
|System Features: Heated stage up to 40°C; Gas anesthesia inlet and outlet ports; scanning laser for alignment and surface topography; High-performance acquisition computer; 20-inch, high resolution flat screen monitor; Software-controlled focus, f-stop, field of view; Excitation/Emission filter wheel; Xenogen's Living Image software provides image acquisition controls and image analysis.|
The Olympus OV100 allows for observations from the cellular level to the entire animal. In addition, it allows for time lapse observations of a variety of physiolgical events at the cellular level up to the whole-animal. With high light-collection efficiency optics, and a highly-sensitive Hamamatsu Electron Multiplying (EM) CCD camera, the OV100 is suited for weak in vitro and in vivo flurescence imaging. The OV100 software includes spectral unmixing algorithms that allow for tissue autofluorescence correction from visible to NIR wavelengths of up to 1000 nm.
The VisEn FMT allows for detection, monitoring, and measurement of disease progression and therapeutic response in various animal models of disease, including oncology, arthritis, and respiratory, cardiovascular, and bone disease. VisEn FMT molecular optical imaging enables true quantification and tomographic slicing of fluorochrome concentration and distribution at any depth within living small animals. The VisEn FMT system is optimized for probes with near-infrared (NIR) flurochromes in both the 680 nm and the 750 nm channels. The system includes a mouse positioning cartridge for rapid and easy alignment, an integrated index-matching fluid handling system, a heated imaging chamber with gas anethesia compatibility, and fully automated software that controls laser, camera, filter settings and fluid handling. A low-noise thermoelectrically-cooled CCD camera with low autofluorescence optics and image reconstruction algorithms, enable the VisEN FMT to image NIR flurorophores deep into the animal, with sub-millimeter spatial resolution and picomolar sensitvity. In addition, the software incorporates acquisition and archiving of images, as well as the analysis of flurochrome concentration, and distribution in user-specified three-dimensional regions of interest. VisEn Medical also provides a variety of pathology specific fluorescent molecular probes and a broad array of fluorochrome-based tags and reporters that allow for user specific labeling (e.g. antibody conjugation).
Other equipment within the OICF include a variety of bench-top apparatus (e.g., digital scale, slide warmer, post-op cages, etc.), a minor-surgery / imaging preparation area with gas anesthesia and instruments, and two high-performance workstations for data acquisition/reduction and post-acquisition image analysis.
In vivo optical imaging permits visualization of molecular processes within living subjects, thereby enabling the detection of molecular abnormalities long before they manifest themselves physiologically. Some applications of fluorescence and bioluminescence imaging of small animals include: