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. 2012 Jul 6;2(7):655–667. doi: 10.7150/thno.3890

Figure 4.

Figure 4

Single QD tracking of Av-GPI by total internal reflection fluorescence (TIRF) microscopy. (A) First frame from a dual-color TIRF movie of a HeLa cell. Av-GPIs in the ventral plasma membrane are labeled with QDs (red) and GM1 molecules are labeled with Alexa-488 CT×B (green). (B) Selected frames from a region of interest (white square in (A)) in which diffusing Av-GPIs are tracked. Diffusion trajectories are determined by the series of fitted positions, connected by a straight line. Notice that Alexa-488 CT×B bleaches fast compared to QDs and the signal was nearly completely lost after 10 s. To facilitate visualization, the QD point-spread-function size was intentionally expanded. Tracking was performed on raw images. (C) Overlay of Av-GPI trajectories with the mean intensity projection image (ΣImean) for the Alexa-488 CT×B channel. This approach allows colocalization studies of Av-GPIs within fixed/slow diffusing GM1-rich domains despite the fast photobleaching of Alexa-488 CT×B. Reprinted with permission from Fabien Pinaud, Xavier Michalet, Gopal Iyer, Emmanuel Margeat, Hsiao-Ping Moore, and Shimon Weiss, Fig.2, Traffic Vol 10, 691-712 (2009). Copyright (2009) John Wiley and Sons.