Fig. 1. FCS measurements of a synthetically prepared Aβ (3.3 μM total Aβ concentration) aggregate suspension (right column) and 3.3 μM Aβ, prevented from aggregating by 0.2% SDS as control (left column). Samples were measured with an FCS Olympus IX 50 (Evotec OAI, Hamburg, Germany) equipped with a beam scanner unit in 24-well assay chips (Evotec OAI, Hamburg, Germany). Using the beam scanner the detection volume was moved through the sample in horizontal and vertical dimensions. Two different Aβ antibodies (Alexa-488 labeled 6E10 and Alexa-633 labeled 19H11) were used as detection probes in the samples. (A) Diffusion of an Aβ monomer bound to fluorescence labeled antibody through the confocal volume (schematically shown as a dotted circle in the upper row) results in relatively small fluctuations at low fluorescence intensities. Binding of several labeled antibodies to Aβ aggregates results in fluorescence bursts. (B) To increase the specificity of the assay, two antibodies with different fluorescence labels and a two-dimensional fluorescence intensity distribution analysis (2D-FIDA) was applied. Thus, simultaneous binding of both antibodies specifically indicates presence of an Aβ oligomer. Histograms of the fluorescence intensities of both antibodies are presented as points of single peaks with two intensities. A cut off (dotted lines) was set in both dimensions and signals above were summed up.

Fig. 2. Detection of synthetically prepared Aβ aggregates in solution and sensitivity of the assay. The sensitivity of the assay was determined by dilution of the aggregates. Data points show mean values (four measurements, ±standard deviation) of fluorescence bursts detected by 2D-FIDA during 0.5 min. Fluorescence labeled antibodies 6E10 and 19H11 were used as detection probes.

Fig. 3. (A) 2D-surface-FIDA of synthetically prepared Aβ aggregates (concentration 3.3 μM, dark-grey columns) in PBS. PBS without Aβ aggregates was used as control (light-grey columns). Measurements were done at different distances (5, 10, and 15 μm) to the slide surface. (B) 2D-surface-FIDA of synthetically prepared Aβ aggregates (concentration 3.3 μM) diluted in CSF. CSF without additional Aβ aggregates was used as control. Aβ preparations prevented from aggregation by 0.2% SDS did not yield more fluorescence bursts as seen in the control. (C) The sensitivity of the assay was determined by dilution of the aggregates in CSF. For all measurements, mean values (three measurements, ±standard deviation) of burst number detected by 2D-FIDA during 0.5 min are shown. Fluorescence labeled antibodies 6E10 and 19H11 were used as detection probes.

Fig. 4. 2D-surface-FIDA was applied to 20 μl crude CSF of AD patients and control patients not affected by AD. Number of bursts (mean values of three measurements, ±standard deviation) obtained by 2D-FIDA during 0.5 min are shown.