Living Image 3.1

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F. Fluorescent Imaging 210 combination provides high emission throughout the 400-950 nm wavelength range (Figure F.2). The dichroic reflector reduces infrared coupling (>700 nm) to prevent overheating of the fiber-optic bundles, but allows sufficient infrared light throughput to enable imaging at these wavelengths. The Living Image software controls the illumination intensity level (off, low, or high). The illumination intensity at the low setting is approximately 18% that of the high setting. Figure F.2 Relative spectral radiance output for the quartz halogen lamp with dichroic reflector. The lamp output is delivered to the excitation filter wheel assembly located at the back of the IVIS ® Imaging System (Figure F.3). Light from the input fiber-optic bundle passes through a collimating lens followed by a 25 mm diameter excitation filter. The IVIS Imaging System provides a 12-position excitation filter wheel, allowing you to select from up to 11 fluorescent filters (five filters on older systems). A light block is provided in one filter slot for use during bioluminescent imaging to prevent external light from entering the imaging chamber. The Living Image software manages the motor control of the excitation filter wheel. Figure F.3 Excitation filter wheel cross section. Following the excitation filter, a second lens focuses light into a 0.25 inch fused silica fiber-optic bundle inside the imaging chamber. Fused silica fibers (core and clad), unlike ordinary glass fibers, prevent the generation of autofluorescence. The fused silica fiber bundle splits into four separate bundles that deliver filtered light to four reflectors in the ceiling of the imaging chamber (Figure F.1). The reflectors provide a
Living Image ® Software User’s Manual F.2 Filter Spectra diffuse and relatively uniform illumination of the sample stage. Analyzing image data in terms of efficiency corrects for nonuniformity in the illumination profile. When the efficiency mode is selected, the measured fluorescent image is normalized to a reference illumination image. (For more details on efficiency, see page 201.) The emission filter wheel at the top of the imaging chamber collects the fluorescent emission from the target fluorophore and focuses it into the CCD camera. All IVIS ® Imaging Systems require that one filter position on each wheel always be open for bioluminescent imaging. IVIS Imaging System Number of Emission Filter Wheel Positions 200 Series 24 (two levels, each with 12 positions) Number of Available Fluorescence Filters 22 (60 mm diameter) Lumina 8 7 100 or 50 6 5 (75 mm diameter) High quality filters are essential for obtaining good signal-to-background levels (contrast) in fluorescence measurements, particularly in highly sensitive instruments such as the IVIS ® Imaging Systems. Figure F.4 shows typical excitation and emission fluorophore spectra, along with idealized excitation and emission filter transmission curves. The excitation and emission filters are called bandpass filters. Ideally, bandpass filters transmit all of the wavelengths within the bandpass region and block (absorb or reflect) all wavelengths outside the bandpass region. This spectral band is like a window, characterized by its central wavelength and its width at 50% peak transmission, or full width half maximum. Figure F.5 shows filter transmission curves of a more realistic nature. Because the filters are not ideal, some leakage (undesirable light not blocked by the filter but detected by the camera) may occur outside the bandpass region. The materials used in filter construction may also cause the filters to autofluoresce. 211
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Living Image 3.1

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