Living Image 3.1

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G. Planar Spectral Imaging 228 NOTE When using the 560 nm and 580 nm band pass filters, tissue optics result in a larger attenuation of light (due mainly to hemoglobin absorption). A longer exposure time is recommended at these wavelengths. Figure G.2 shows the metastasis sites. The signals from the lungs and right kidney are well defined in both animals. However, in the lower back area of the left mouse, the signals are in close proximity, causing an artifact in the planar spectral analysis. Figure G.2 Metastatic sites in nude mice. Mice were imaged 13 days after a tail vein injection of 5x10 5 B16F10 melanoma cells. Imaging parameters: high sensitivity binning, f/stop=1, FOV = C (13 cm), exposure time = 120 seconds at 560 and 580 nm, exposure time = 60 seconds at all other wavelengths. This resulted in signals of ~2000 counts on each image. To perform the planar spectral analysis, draw a measurement ROI that captures the entire signal of each site of interest without including a neighboring metastasis (Figure G.3). After the ROI is defined, start the planar spectral analysis (for more details, see page 107). The software: • Measures the total flux inside the ROI on each filtered image. • Normalizes the data to the luciferase spectrum (Plot of Intensity vs. Lambda, Figure G.4). • Fits the normalized data to the analytical expression in Equation 1, page 226 where S = absolute total photon flux emitted by the bioluminescence source and d = source depth (Plot of Linear Fit Results, Figure G.4)
<strong>Living</strong> <strong>Image</strong> ® Software User’s Manual After the analysis is completed, click a button to display graphical results. Figure G.4 Planar spectral analysis results Figure G.3 Metastatic site ROI includes the signal of the right kidney and separates it from other metastatic sites. The signal coming from the lower back area is spread out due to the presence of two bright spots. The dimmer signal in the lower bottom right of the image likely originates from the femoral bone of the animal. 229
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Living Image 3.1

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