The PerkinElmer IVIS Spectrum is a State-of-the-art instrument for whole animan fluorescence and luminescence imaign.  This enables sensitive in vivo detection and quantification of optical signals from engineered reporter alleles engineered into whole animals or in implanted tumor cells. One of the two IVIS Spectrum instruments is located within an ABSL3 animal facility to support applications with mice infected with experimental pathogens or otherwise placed under quarantine. Spectral deconvolution that accounts for absorption of photons by tissues enables a pseudo-3D imaging technique that may also be combined with micro CT datasets acquired on the Quantum FX instrument, via an automated software process in Living Image 3.

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Specifications:

• in vivo imaging of fluorescence, bioluminescence, and Cherenkov Radiation

• throughput (5 mice at a time) with 23 cm field of view

• resolution (to 20 microns) with 3.9 cm field of view

• eight high efficiency filters spanning 430 – 850 nm

• spectral unmixing applications (enhanced fluorescent imaging ability)

• for distinguishing multiple bioluminescent and fluorescent reporters

• switch in the fluorescence illumination path allows reflection-mode or transmission-mode illumination

• 3Dforand

• import and automatically co-register CT or MRI images yielding a functional and anatomical context for your scientific data.

• traceable absolute calibrations

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Application: Bioluminescent Imaging

     Bioluminescence imaging measures light emission resulting from an enzymatic reaction catalyzed by one of several different luciferase enzymes. The luciferase gene may be incorporated into cells that are implanted in mice, or directly into mouse tissues through genetic engineering or viral transduction. Expression may be driven from general promoters as an indirect measure of tumor volume, or from gene- specific promoters to functionally visualize pathway activity.

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Three luciferase systems are in wide use:

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     Firefly luciferase requires D-luciferin to be injected into the subject prior to imaging. The peak emission wavelength is about 560 nm. Due to the attenuation of blue-green light in tissues, the red-shift of this emission makes detection of firefly luciferase much more sensitive in vivo (compared to the other systems).

     Renilla luciferase requires its substrate, coelenterazine, to be injected. As opposed to luciferin, coelenterazine has a lower bioavailability. Additionally, the peak emission wavelength is about 480 nm, a wavelength at which tissue attenuation is greater.

     Bacterial luciferase has an advantage in that the lux operon used to express it also encodes the enzymes required for substrate biosynthesis. Although originally believed to be functional only in prokaryotic organisms, where it is widely used for developing bioluminescent pathogens, it has been genetically engineered to work in mammalian expression systems as well. This luciferase reaction has a peak wavelength of about 490 nm.

     While the total amount of light emitted from bioluminescence is comparatively low (not detectable by the human eye), the fact that there is no background light emission makes it extremely specific. The ultra- sensitive CCD camera within the IVIS Spectrum can image bioluminescence with great sensitivity. Common applications of BLI include in vivo studies of infection (with bioluminescent pathogens), cancer progression (using a bioluminescent cancer cell line), and reconstitution kinetics (using bioluminescent stem cells).

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Application: Fluorescent Imaging

 

     The IVIS Spectrum can image and quantify all commonly used fluorophores, including fluorescent proteins, dyes and conjugates. Our IVIS Spectrum achieves superior spectral unmixing through a wide range of high resolution, short cut-off filters and advanced spectral unmixing algorithms.

 

     Spectral unmixing not only allows detection and separation of multiple reporters, but greatly reduces   the effects of tissue auto-fluorescence. The IVIS Spectrum is the most sensitive system to visualize these fluorescent agents in various in vivo research needs for Principal Investigators here at Columbia University Medical Center

 

The IVIS Spectrum optical imaging system:

• sensitivity and flexibility

• and epi-illumination imaging

• light from the excitation filter wheel feeds through a fiber optic bundle to illuminate the specimen from either the top, in epi-illumination (reflectance) mode, or from underneath the stage, by means of an automated bundle switch. Trans illuminating the subject from below at precise x, y-locations allows for transmission imaging, enabling more sensitive detection and accurate quantification of deep sources. Transmission fluorescence imaging also reduces the effects of auto fluorescence.

• designed to simplify advanced and complex biological models by intuitively guiding the user through experiential setup and analysis. The imaging wizard with the newly added probe library will help design imaging settings and select the right filter pair for fluorescence studies. The software also offers a step by step guide for spectrally unmixing multiple fluorescent signals from the same animal. Advanced spectral-unmixing algorithms and a broad range of high spectral resolution filter sets minimize auto fluorescence and provides the opportunity to image a wide variety of targeted and activatable fluorescent probes and reporters.

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