Figure 4
Synchronized AFM and single molecule fluorescence microscopy experiments. (A) Representative force curve of a transfer experiment. AFM-tips were functionalized with fluorescent HDL and brought into contact with glass-supported DOPC bilayers (i), kept at constant force F < Fp for 500 ms (ii-iii), and finally retracted (iv). The small spikes in the trace and retrace curve are caused by the excitation laser, which is partially detected on the photodiode of the AFM. (B) Fluorescence images at the indicated time points are shown for HDL pre-loaded with C-Bodipy, DiI, or CE-Bodipy; for control, also the apoA-I-Alexa647 signal is shown. Upon contact, only C-Bodipy and DiI – but not CE-Bodipy and the covalently linked apoA-I-Alexa647 – moved away from the contact point and diffused freely in the bilayer after tip retraction. Diffusion analysis of transferred versus pre-inserted C-Bodipy (C, left) and DiI (C, right) in supported DOPC bilayers. The plot shows the time-dependent variance for transfer experiments (filled circles), and the mean-square displacements versus time for pre-incubated samples (open circles). For transfer experiments, the time-point zero specifies the first image, after the AFM-tip was retracted from the surface. Experiments were fit with Eq. 2 (pre-incubated samples) or Eq. 3 (transfer experiments). From the slopes of the C-Bodipy data (left), we calculated a diffusion constant of D = 2.9 ± 0.29 µm2/s and D = 3.3 ± 0.87 µm2/s for the transfer experiments and the pre-incubated sample, respectively. The same analysis was performed for DiI (right), yielding D = 1.36 ± 0.04 µm²/s for the transferred probe and D = 1.06 ± 0.02 µm²/s for the pre-incubated probe. Error bars indicate the standard error of the mean.
