Figure 4.

Single-band analysis of GFP-expressing pulmonary microvascular endothelial cells in lung cryosections reveals poor sensitivity and specificity for GFP-expressing cells. Using single-band (500 nm, 50 nm bandwidth) fluorescence emission, both control lung (A & B) and lung perfused with GFP-expressing endothelial cells (D & E) show high levels of fluorescence. Thresholding may be used in an attempt to identify GFP emission (arrow indicates a possible GFP-expressing cell), but also results in a significant number of false-positive regions, as evident in the control. For quantitative analysis, single-band images (C) were analyzed using Cell Profiler software[34] and intensity-thresholding techniques. Nuclei and GFP regions (F) were identified, and the total number of nuclei and the number of nuclei within GFP regions were measured (G). Results show a poor sensitivity and high incidence of type I errors. This threshold corresponds to the medium threshold in the theoretical sensitivity analysis (Figure 3) and the threshold used for spectral analysis (Figure 6).