Fig. 3: Colour unmixing of four fluorescent labels in a single spectral channel.
From: Electrochemical fluorescence modulation enables simultaneous multicolour imaging
a, Raw image of multitarget labelled HeLa cell before colour unmixing, where the mitochondria, actin, cell nucleus and transferrin receptor are labelled using Alexa 594, Alexa 555, mCherry and Alexa 568, respectively. b, Representative EC spectra for Alexa 568, mCherry, Alexa 594 and Alexa 555, as indicated. The potential was linearly scanned back and forth between −0.8 V and 0.15 V, at a scan rate of 500 mV s–1, and the time-lapse series consists of 19 frames of images at a frame rate of 200 ms. c, Unmixed images with different cellular structures as indicated. d, Overlaid image after colour unmixing with various structures colour-coded. Actin filaments appear in yellow; mitochondria, in green; transferrin, in magenta; and nucleus, in blue. e, Box plot (n = 6) showing the cross-talk factor for unmixed images. The boxes extend from the 25th to 75th percentiles, with the centre lines representing the median values. The whiskers reach out to the largest and smallest values, not exceeding 1.5 times the IQR from the upper- and lower-box hinges, respectively. Scale bar, 10 µm (a, c and d).
