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. 2016 Jul 26;111(2):395–408. doi: 10.1016/j.bpj.2016.06.007

Figure 2.

Figure 2

TIRFM visualization of GFP in vitro and in living cells versus GFP-CaMKII. (A) i: Antibody-immobilized GFP molecules (10-frame averaged image)). ii: Line intensity profiles of the four spots in field center, top and right (± standard error (SE), thin line), and of the brighter spot (arrow) show the diffraction-limited size. (B) Intensity-versus-time records of spots shown in A(i), illustrating single-step photobleaching (i–iii), blinking behavior (iv), and double-step photobleaching of the brighter spot (v).The single-step modal value was 27.5 ± 2.5 counts/pixel (doubling and tripling occurs when fluorophore PSFs overlap). (C) Single video frame (50 ms exposure) of a HUVEC expressing GFP alone, showing that motion blurring prevents single fluorophore observation. (D) Single video frame of a HUVEC expressing GFP-CaMKIIβ, showing that discrete fluorescent spots are now visible.