In Vivo VIS/NIR Fluorescent Imaging System
VIS/NIR Fluorescent Imaging System Design
Block diagram showing major system components.
Spectral response of a hypothetical bandpass excitation filter, and long-pass emission filter overlaid with the absorption and emission characteristics of Alexa Fluor 680 (Molecular Probes) fluorescent dye.
Details
The imaging system illustrated in the figure is designed for fluorescent optical imaging of small animals. The system employs epi-illumination via a 450 W Xe arc lamp (Oriel) attached to a condenser, heat-removal filter, electronic shutter and lens. Output light intensity from this block has a substantially flat spectral response from about 350 nm to 900 nm. Excitation wavelength selection is done by high performance, narrow band interference filters (Omega Optical, Alpha Vivid series, custom order) mounted in a filter wheel. Filtered excitation light is directed at a 10 cm square stage. Excitation illumination intensity measured at the stage is approximately 2 mW/cm2 for a typical configuration consisting of an excitation filter with bandwidth of 35 nm.
Emission filtration is achieved by either high performance bandpass or longpass optical interference filters (Omega Optical, Alpha Vivid series). Emission pass bands are chosen to maximize sensitivity to the particular fluorophore while minimizing sensitivity to scattered excitation light and fluorescence emission from endogenous fluorophores. The filtered emission light is collected by either a fixed focal length lens (Tamron, 25 mm, F/1.6) or a macro zoom lens (Optem 18-108 mm, F/2.5) mounted 320 mm above the stage. In plane resolution at the stage surface is approximately 100 mm FWHM for the system configured with the fixed focal length lens and 20 mm FWHM for the zoom lens set to 108 mm focal length. The zoom lens is employed for close-up imaging during in vivo experiments and for medium power epi-fluorescence video microscopy imaging of in vitro and histological preparations.
Images are digitized by a high-resolution, 1280 x 1024 x 12-bit pixel, cooled, 2/3" format CCD, low-noise, digital camera (ORCA-ER, Hamamatsu). The camera is capable of real-time acquisition of 8 frames per second in non binned mode or 30 frames per second using 4 x 4 binning. Longer integration times are possible for imaging in low light conditions by control of the frame integration duration (up to 2 seconds) and by summing successive frames under software control. System control and image acquisition hardware is interfaced (National Instruments) to a Pentium III-based personal computer running system integration and automation software (National Instruments LabVIEW 6). Imaging components are mounted on an optical bench that incorporates active vibration isolation (Kinetic Systems 9100 series).
For in vivo experiments, images are typically acquired and then processed and manipulated using custom software running on MATLAB Release 13 (Mathworks). Manual image manipulations such as contrast adjustments, format translations, scaling, compositions and annotations are performed with Photoshop 6 (Adobe). Because hair scatters light and degrades the emission image quality, experimental animals are usually either hairless mice such as athymic nu/nu or imuno-competent SKH1 (Charles River) or any other variety having undergone mechanical or chemical hair removal.
Animals are imaged while under anesthesia which is effected by either itraperitoneal (IP) injection of Ketamine and Xylazine for short (<10 min) imaging sessions, or by continuous inhalation of isoflurane for protracted imaging studies (10 min to several hours). ketamine and xylazine are administered at doses of 100 mg/kg and 10 mg/kg respectively mixed in sterile saline (0.85%). isofluorane at a concentration of 2% in air (4% for induction) is supplied by an anesthetic vaporizer connected to a pressurized air supply.
