Figure 3.

NMF is capable of separating four simultaneously present labels when the spectrum of one label is known. A HeLa SS6 cell labeled with FITC phalloidin F-actin, A514 tubulin, A555 transferrin, and EtBr DNA was excited at 488 nm and imaged over 16 spectral channels ranging from 503 to 663 nm (width 10.7 nm, image size 49 × 49 μm). A555 transferrin is the weakest label and its spectrum was fixed to the literature spectrum. Running NMF with a slight segregation bias yielded the label distributions and the spectra from blue to red according to FITC, A514, A555, and EtBr (dotted lines, bare NMF; solid lines, NMF with segregation bias; dashed lines, reference spectra from singly labeled specimens). Both the label distributions and spectra are estimated to high accuracy. The RGB panels show the false color overlay of F-actin (blue), tubulin (green), and nucleic acids (red) (RGB₁₂₄), and of F-actin (blue), transferrin (green), and nucleic acids (red) (RGB₁₃₄). The NMF run was initialized with Gaussian spectra with FWHM of 75 nm and 524-, 558-, and 617-nm center positions, which represent the FWHM centers of the literature spectra.