Optical Imaging


The IVIS® Spectrum advanced preclinical optical imaging system from Perkin Elmer combines high throughput and full tomographic optical imaging in one platform. The system uses leading optical imaging technology to facilitate non-invasive longitudinal monitoring of disease progression, cell trafficking and gene expression patterns in living animals. It takes full advantage of bioluminescent and fluorescent reporters across the blue to near infrared wavelength region using optimized sets of high efficiency filters and spectral unmixing.

Field Of Applications:
  • • Quantitative longitudinal measurement of tumor development, metastasis, angiogenesis, and response to therapy.
  • • Trafficking of cellular and other biological therapies (e.g., immune cell, stem cell, or viral therapeutic platforms or antibodies, peptides, metabolites, etc.).
  • • Distribution, quantification, and kinetic analyses of gene expression or enzymatic activity in vivo


Bioluminescence Living Imaging (BLI) visualizes light produced by luciferase within a living subject. Luciferases are unique light-producing enzymes capable of using luciferin as a substrate. The most commonly used luciferase for imaging applications is firefly luciferase, which catalyzes luciferin oxidization in the presence of oxygen and ATP. The DNA coding sequence of firefly luciferase has been identified and further optimized for mammalian expression.
With the aid of molecular biology techniques, a luciferase-expressing cassette can be genetically introduced into viruses, cancer cells, or even the whole animal (transgenic) for BLI imaging research. One great advantage of luciferase-based bioluminescence imaging is the very low background signal. Since the substrate has uniquely evolved together with its partner enzyme, D-luciferin is not a substrate for any mammalian enzymes. Thus BLI imaging is extremely specific and highly sensitive; only luciferase-expressing cells are able to generate light signal.

Firefly bioluminescence


Fluorescence imaging requires an external light source for fluorophore excitation. As each fluorophore has its unique excitation and emission profile, the IVIS Spectrum is equipped with a full range of excitation and emission filters to capture light from the visible to near infrared spectrum, a feature that allows the use of a great variety of commercially-available and lab-made near infrared fluorescent agents.

The IVIS Spectrum excitation and emission filters:
  • • 10 narrow band excitation filters: 415 nm – 760 nm (30 nm bandwidth).
  • • 18 narrow band emission filters: 490 nm – 850 nm (20 nm bandwidth).
The IVIS Spectrum has the capability to use either trans-illumination (from the bottom) or epi-illumination (from the top) to illuminate in vivo fluorescent sources.

For a typical 2D epi-fluorescence (epi-FL) imaging session, a single pair of excitation and emission filters were used in alignment with the fluorescent agent’s optimal ex/em wavelengths.


Spectral Unmixing

The Living Image software is capable of spectral unmixing (SPUM) analysis on a series of 2D fluorescent surface radiance images. SPUM analysis require the series to be acquired with a range of excitation/emission filters to distinguish two or more fluorescent sources provided that the image series covers the fluorescent spectra of all responsible sources. A common application of SPUM is to separate the interfering tissue autofluorescence and food fluorescence from the true fluorescence signals contributed by the agent of interest. Typical SPUM analysis involves the use of multiple excitation lights for the background autofluorescence and the specific fluorescent agents. These filters along with spectral scanning filters and spectral unmixing algorithms significantly reduce autoflorescence. In addition, the spectral unmixing tools allow the researcher to separate signals from multiple fluorescent reporters within the same animal.

3D Reconstitution

The IVIS SpectrumCT features two advanced modes for acquiring optical 3D tomography in bioluminescence and fluorescence.
Diffuse Light Tomography (DLIT) is a 3D bioluminescence imaging module built into the Living Image software. In tissue, firefly luciferase has a broad emission range from 540 to 660 nm, and the maximal emission is about 620 nm. Lights with different wavelengths scatter and attenuate differently in tissue, with red-shifted wavelengths showing better tissue penetration, so source bioluminescence from deeper positions will show more loss of the bluer 540 signals than the redder 660 signals. DLIT generates 3D tomographic images by acquiring a series of 2D bioluminescence surface radiance images with different emission filters.
Diffuse Light Tomography

Fluorescence Imaging Tomography
Fluorescence Imaging Tomography (FLIT) uses a different approach for determining 3D localization of light signal. FLIT imaging uses a transillumination excitation light source placed on the opposite side of the camera and close to the animal’s body. As a result, the system can deliver more energy to compensate for possible light attenuation and scattering, ideal for deep tissue tomography. For optimal excitation and emission, FLIT uses distinct pairs of excitation/ emission filters aligned to each fluorescent agent used. In order to establish the distribution of fluorescent agent in the body, a series of 2D fluorescent surface radiance images are acquired with the light source at various transillumination positions throughout the region of interest.