Fig. 5 Metafluorophores with different photostability.

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Fig. 5 Metafluorophores with different photostability. Metafluorophores with different photostability. (A) Time-lapsed fluorescence micrographs of a sample composed of two spectrally indistinct metafluorophore species: one containing 44 Atto 647N dyes (more photostable) and one containing 44 Alexa 647 dyes (less photostable). Images were acquired at t1 = 0 s, t2 = 20 s, and t3 = 40 s, with an integration time of 10 s, whereas the sample was constantly illuminated during acquisition. The time-lapsed micrographs show two species where one bleaches faster than the other. The two species can be visually identified by superimposing the images taken at t1 (false color blue) and t3 (false color yellow). The metafluorophore containing more photostable dyes (that is, Atto 647N) appears green (blue + yellow), whereas the one with the less photostable dyes (that is, Alexa 647) appears blue. The fluorescence decay constant can be used as a parameter to quantitatively describe the photostability. The decay constant is obtained by fitting a single exponential decay to the intensity versus time trace. Scale bars, 5 μm. (B) Intensity versus decay constant histograms for three different metafluorophore samples containing Atto 647N dyes (green), Alexa 647 dyes (blue), and both dyes (red), respectively (note that only one species was present in each sample). (C) 1D histogram of the decay constants shows three distinguishable decay constant distributions (schematics in the legend show the dye arrangement on the metafluorophores). Johannes B. Woehrstein et al. Sci Adv 2017;3:e1602128 Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).