Purpose: To present a new multichannel optical imaging system for fluorescence reflectance imaging (FRI) of small animals covering the visible to infrared light spectrum.
Methods and Materials: Excitation in the near-infrared (NIR) and infrared (IR) spectrum is implemented by six light emitting diode arrays while photons in the blue and green range are delivered by a high-pressure mercury-vapor lamp. Four different emission filters allow for spectral separation of emitted photons. Images are acquired by a CCD-camera and processed on a PC-based system. The sensitivity, spatial resolution and depth penetration were tested in-vitro for each imaging channel using different fluorochromes and fluorescent proteins. Wavelength selectivity and spectral separation were assessed in phantoms and in-vivo using GFP- expressing tumors and Cy 5.5 labelled optical contrast agents.
Results: The imaging system allowed for detection of commonly used fluorochromes and fluorescent proteins such as Cy 5.5, Cy 7, green - and red fluorescent protein. Dilution series and cell cultures studies showed detection of fluorochromes even in the picomolar range. As little as 12 pmol Cy 7 and 30 pmol Cy 5.5 labeled contrast agent could be traced through 8 mm thick muscle tissue. The current set up of emission filters shows complete spectral separation in green, red and infrared channel and minor cross-excitation (30%) between the NIR and IR channel. The spatial resolution of the system in surface weighted images ranges between 0.1-0.2 mm. In-vivo experiments showed sensitive differentiation between a Cy 5.5 labelled cathepsin-B specific contrast agent and tumoral GFP-expression within the same animal
Conclusion: The multichannel fluorescence reflectance imaging system allows rapid and sensitive imaging of most commonly used fluorochromes or fluorescent proteins at four different wavelengths. Rapid data acquisition and versatile wavelength handling are ideal for in-vivo and ex-vivo applications to test novel optical contrast media.
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