Figure 7.
Linear unmixing was applied to flat-field corrected spectral images. (A) Total fluorescence intensity of a 10 μm-thick lung cryoslice containing perfused GFP-expressing pulmonary microvascular endothelial cells (PMVECs); (B) image of Nuclei (Hoechst 33342 staining) with 360/40 nm excitation and 450 nm emission; (C) brightfield image at 500 nm; unmixed images of autofluorescence (D), GFP (E), and background (F); the spectra used for unmixing (G) were acquired from GFP-expressing PMVECs and negative control tissue; (H) false-colored overlay of unmixed image showing autofluorescence (red), GFP (green), nuclei (blue), and background (orange). Control lung cryoslices showed minimal false-positive GFP signal with identical imaging and linear unmixing (data not shown).