Living Image 3.1

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F. Fluorescent Imaging Specifying Signal Levels and f/stop Settings F.4 Image Data Display 214 Figure F.6 Illustration of the in vivo fluorescence process. At 600-900 nm, light transmission through tissue is highest and the generation of autofluorescence is lower. Therefore it is important to select fluorophores that are active in the 600-900 nm range. Fluorophores such as GFP that are active in the 450-600 nm range will still work, but the depth of detection may be limited to within several millimeters of the surface. Fluorescent signals are usually brighter than bioluminescent signals, so imaging times are shorter, typically from one to 30 seconds. The bright signal enables a lower binning level that produces better spatial resolution. Further, the f/stop can often be set to higher values; f/2 or f/4 is recommended for fluorescence imaging. A higher f/stop improves the depth of field, yielding a sharper image. For more details on the f/stop, see Lens Aperture, page 191. Fluorescent image data can be displayed in units of counts or photons (absolute, calibrated), or in terms of efficiency (calibrated, normalized). For more details, see Quantifying Image Data, page 199. If the image is displayed in photons, you can compare images with different exposure times, f/stop setting, or binning level. When an image is displayed in terms of efficiency, the fluorescent image is normalized against a stored reference image of the excitation light intensity. Efficiency image data is without units and represents the ratio of emitted light to incident light. For more details on efficiency, see page 201. Fluorescent Efficiency The detected fluorescent signal depends on the amount of fluorophore present in the sample and the intensity of the incident excitation light. At the sample stage, the incident excitation light is not uniform over the FOV. It peaks at the center of the FOV and drops of slowly toward the edges (Figure F.7). To eliminate the excitation light as a variable from the measurement, the data can be displayed in terms of efficiency (Figure F.8).
Living Image ® Software User’s Manual When efficiency is selected, the fluorescent image data is normalized (divided) by a stored, calibrated reference image of the excitation light intensity incident on a highly reflective white plate. The resulting image data is without units, typically in the range of 10 -2 to 10 -9 . NOTE F.5 Fluorescent Background Figure F.7 Illumination profiles for different FOVs on an IVIS Imaging System 100 Series measured from the center of the FOV. To enable a more quantitative comparison of fluorescent signals, choose Efficiency. Figure F.8 Fluorescent image data displayed in terms of efficiency On every IVIS ® Imaging System, a reference image of the excitation light intensity is measured for each excitation filter at every FOV and lamp power. The reference images are measured and stored in the Living Image folder prior to instrument delivery. Autofluorescence Autofluorescence is a fluorescent signal that originates from substances other than the fluorophore of interest and is a source of background. Almost every substance emits some 215
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Living Image 3.1

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