(a) Absorption spectra of the different carbocyanine dyes used for three-color single-molecule imaging. The vertical lines indicate the different excitation wavelengths. (b) A composite overlay of the three individual channels to render the multicolor single-molecule image is shown. Arrows highlight discrete photobleaching events on three spectrally different molecules. (c–e) Individual channels for DiO (c), DiI (d), and DiD (e) are shown. Intensity scales correspond to the number of photons detected within 5-ms pixel integration time. Scale bars, 2 μm. (f–h) Histograms of the average counts/pixel measured for the individual molecules shown in (c)–(e), respectively, are displayed. To see this figure in color, go online.