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Schematic model of the TOCCSL method. The top row represents images of the sample, the bottom row the respective timing protocol for illumination. (a) Initially, the biomembrane contains a high surface density of mobile fluorescent biomolecules (full circles), which form small clusters. Individual clusters are too close to being resolved as individual spots. The initial situation is probed by a short illumination with illumination time . (b) By using a field stop for excitation, a clear-cut region with adjustable diameter of several micrometers is illuminated by an extended laser pulse for . This leads to complete photobleaching of the fluorescence label for all exposed molecules. The molecules outside the illuminated region remain unaffected. (c) Immediately after photobleaching, the illuminated area contains only nonfluorescent biomolecules (open circles). (d) The recovery process starts, as fluorescent clusters move from the outside into the bleached region. After the recovery time , only a few clusters have populated the bleached region. These clusters can now be resolved as individual diffraction limited spots, and their intensity allows an estimation of the stoichiometry of labeling.
Proof of principle of TOCCSL on a supported lipid bilayer. Anti-DNP antibodies labeled with multiple fluorescein molecules were used to mimic stable clusters. A fluid supported lipid bilayer containing a fraction of DNP-labeled lipid provided the matrix for the experiment. (a) displays representative images recorded during the TOCCSL experiment. On the left, the initial equilibrium situation is shown: a surface density of clusters per makes direct observation of individual clusters impossible. Upon photobleaching for , clusters were allowed to diffuse into the bleached area. To the right, three images recorded after distinct recovery times are shown: after , no fluorescence signal can be observed within the illuminated part of the membrane; this image serves as control for complete photobleaching. After , individual clusters were clearly resolvable in the central part of the image, indicated by the dashed white circle; such single cluster signals were used for subsequent stoichiometric analysis. Using a much longer recovery time of , the system has nearly reached equilibrium again; 82% total recovery is achieved, and a homogenous fluorescence distribution can be observed. (b) pdf of the fluorescence signal of 2913 single clusters observed with a recovery time of . An average signal of counts was obtained. (c) Signal distribution of directly observed single antibody molecules. For this, the surface density of DNP-DPPE in the bilayer was reduced to 0.15 clusters per . The pdf (1875 clusters, mean signal counts) is in perfect agreement with the respective distribution obtained in TOCCSL mode.
Quantification of the cluster load. The probability distribution for the fluorescence signal of single fluorescein molecules has been determined on immobilized fluorescein-labeled antibodies upon prolonged excitation (a), and was used for calculation of . In (b), the signal distribution for fluorescein-labeled antibodies measured via TOCCSL (solid line) was fitted using Eq. (2) (dashed line); the residuals do not indicate any significant deviation over the full fitting range. The weighted contributions of the individual -mers, , are plotted as thin lines. The inset shows the distribution of cluster load , ; the dominant species was found to be dimers, however, a long tail towards high shifts the average load to .
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