Figure 1.
A. Overview of the dendrimer based labeling strategy. Anti-ErbB1 antibodies tethered to dendrimers functionalized with multiple biotin moieties are targeted to the receptors on the cell surface in a first labeling step. In a second labeling step, anti-biotin antibody functionalized NPs bind to the created binding sites on the cell surface. B. Synthesis of biotin-dendrimer-antibody construct. C. Functionalization of NP surface with anti-biotin antibodies.
Figure 2.
Stability of anti-biotin functionalized NPs.
A. UV-Vis spectra of 60 nm NPs functionalized with anti-biotin before and after incubation for 10 min with A431 cells in Hanks buffer with 10 mM HEPES pH 7.2. B. Size distribution as determined by dynamic light scattering. C. SEM image of surface immobilized NPs (on a BSA-biotin functionalized glass substrate) after incubation with cells. The inset shows the Hopkins statistics for the field of view. D. Histogram of the NP association levels after incubation with the cells.
Figure 3.
Clustering of NP immunolabels targeted at ErbB1.
A. Comparison of immunolabel densities obtained with different labeling strategies: dendrimer enhanced labeling (red), secondary antibody assisted labeling (olive), direct labeling (blue). Controls (dendrimer enhanced labeling in the presence of excess antibodies, see text) are included in magenta. B. Part of an SEM image of a labeled cell surface (dendrimer enhanced strategy). The Hopkins statistics for the full image in the inset shows that the NP distribution is not random but that the NPs show clustering. C. Histogram of the NP cluster sizes on the cell surface.
Figure 4.
Monitoring the configurational dynamics of laterally diffusing NP clusters through PCM.
A. Diffusion trajectory of a NP cluster on the cell surface. B. Reduced polarization dichroism (P, red) and total intensity (Itot, blue) of the light scattered off the diffusing cluster as function of time. We indicated high (pink) and low (olive) intensity levels in the Itot trajectory. The large fluctuations in Itot and P are characteristic of a rich configurational dynamics in which the NPs of the cluster change their separation and geometric arrangement.
Figure 5.
Correlation of |P| with high and low intensity (Itot) configurations.
The |P| values for the high Itot configuration for the cluster from Figure 4 are plotted in pink, the |P| values for the low Itot configuration are plotted in olive.
Figure 6.
Frequency domain analysis of the reduced polarization dichroism of NP clusters.
A. Power spectral density (PSD) of P for the NP cluster from Figure 4. B. PSD of P for a cluster of similar intensity immobilized on a glass substrate.
Figure 7.
Mapping the spatiotemporal history of NP cluster configurations through PCM.
P as function of time and location during the lateral translation of a NP cluster comprising three to four 60 nm diameter Au NPs. At t = 8.1 s the P value shows a systematic shift indicative of a change in the cluster configuration.
Figure 8.
Comparison of diffusion coefficient (D) distributions for individual NPs and NP clusters.
The graphs show the cumulative distributions of the D values of individual NP labels (blue) and of NP clusters (red). The D value distribution of the clusters is systematically shifted to lower values when compared with that of individual NPs.