Living Image 3.1

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G. Planar Spectral Imaging 230 To estimate the cell count, divide the absolute photon flux by the flux per cell. This is best determined by making independent in vitro measurements of the cell line used in the experiment. The Plot of Linear Fit Results is weighted by the uncertainty of the measured images and takes into account the uncertainty in the determination of the optical properties. The precision of the method is largely determined by the known precision of the optical properties. In most cases, the relative uncertainty in the depth determination is equal to the relative uncertainty in the optical properties. An analysis of the dorsal and ventral views of the mouse left lung in Figure G.5 results in total flux values that are very similar. The measured depth values are also close, indicating that the cells are distributed about the same distance from the front and back of the animal. Figure G.5 Planar spectral analysis results Top: Dorsal view of the left lung, bottom: ventral view of the left lung
<strong>Living</strong> <strong>Image</strong> ® Software User’s Manual G.5 Optimizing the Precision of Planar Spectral Analysis The accuracy of the planar spectral analysis is highly dependent on the quality of the: • Measured data for the firefly luciferase spectrum and the tissue optical properties. • Fit of the experimentally measured total flux at each wavelength to μ eff (effective attentuation coefficient). In general, more experimental values produce a better fit of the data. It is particularly important to be able to extract signals at all wavelengths to optimize the quality of the fit. If the software detects no signal above the animal background level at 560 nm and 580 nm (the wavelengths that absorb the most light), the dynamic range of the optical properties is reduced and with it, the precision of the fit. If a bioluminescent signal is dim or buried deep in the tissue, it may barely exceed the tissue autoluminescence at the shorter, more absorbing wavelengths (560 and 580 nm). In this case, it is recommended that you subtract the tissue autoluminescence from the image data. (For more details on subtracting tissue autoluminescence, see Appendix E, page 206). It is also recommended that you inspect all images in the sequence to confirm that the bioluminescent signal is greater than the tissue autoluminescence. If the bioluminescent signal does not exceed the tissue autoluminescence at a particular wavelength, do not include that wavelength in the analysis. 231
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Living Image 3.1

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