click to enlarge CRi’s Maestro system with Dynamic Contrast Enhancement.

A wide range of molecular imaging modalities have become common in the clinic—including MRI, CT, and PET—and each of these has a miniaturized version used for small animals. Optical imaging is used primarily for small animal research and is not commonly used in the clinic. Of the two optical techniques—bioluminescence and fluorescence imaging—only fluorescence imaging is potentially translatable to the clinic.

Designing a system for bioluminescence imaging requires only a very dark box and a high-quality CCD camera to obtain useful information. However, the simplest fluorescence imaging methodology using monochrome filters for single excitation and emission wavelengths, is not as useful due to the strong interference from tissue autofluorescence, which limits the sensitivity of in vivo fluorescence measurements regardless of the wavelengths of excitation. Obtaining results that are in the same order of magnitude of sensitivity of bioluminescence requires careful use of spectral imaging technologies to separate the fluorophore-of-interest signal from the interfering autofluorescence.

The CRi Maestro system works by using liquid-crystal tunable filter (LCTF) spectral-imaging technology combined with a patented software approach for separating the signals-of-interest from autofluorescence. An LCTF can be thought of as no-moving-parts filter wheel equipped with ~200 bandpass filters. The LCTF enables the rapid collection (often under 10 seconds, total) of spectral imaging data. An important aspect of achieving quantitative results is the means by which the spectral library, or spectral signatures of the fluorophores in the sample, is obtained. It is critical that the correct spectral signatures are used. It is relatively easy to obtain the spectral signature of tissue autofluorescence, only requiring that the animal has no additional fluorophores. However, obtaining the spectral signature of the fluorophore-of-interest is more difficult, as there can often be no location on an animal that contains the pure signature of that fluorophore. CRi’s Compute Pure Spectrum (CPS) algorithm utilizes the negative control autofluorescence signature to calculate the purified spectral signature. Subsequent spectral unmixing using the correct spectral signatures yields a quantitative separation of the fluorophores, and, hence, a quantitative measure of the fluorophores in the animal.

The Maestro system provides rapid spectral imaging from up to three adult mice simultaneously down to a high-resolution image (25 microns/pixel resolution). It is equipped to image any fluorophore or combination of fluorophores, which emits beyond 500 nm. When combined with CRi’s Dynamic Contrast Enhancement (DyCE) kinetic imaging package, the Maestro provides rapid kinetic imaging data, down to near-video rate (15 images per second).

References
1. Weissleder R, Mahmood, U. Radiolog. 2001;219(2):316.
2. Frangioni J. Cur Opin Chem Bio. 2003; 7(5):626.
3. Mansfield JR, Gossage KW, Levenson RW. Autofluorescence removal, multiplexing, and automated analysis methods for in-vivo fluorescence imaging. J Biomed Optics. 2005; 10(4):41207.

This article was published in Drug Discovery & Development magazine: Vol. 13, No. 2, March 2010, p. 19.