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(Color) Schematic representation of the measurement system used. (a) Schematic representation of the experiment. (b) The schematic figure of the GFP molecule. An AFM probe partially applied either compression or extension force to GFPs in the contact area which resulted in the structure being disrupted by applied force.
Force curve and fluorescence spectra measured simultaneously. Panels (a) and (b) show the results measured with the reactive probe and the complex probe, respectively. The velocity of the AFM probe was during force curve measurement. Exposure time for the CCD camera was and the input power of excitation light was adjusted to . All fluorescence spectra were measured sequentially from 1. The spectra indicated by curves 1 and 4 are noncontact spectra and curves 2 and 3 are the compression and extension spectra, respectively. The vertical position where compression and extension spectra were measured is indicated by the same number in force scans. The errors in the position of the AFM probe that occurred due to the movement of the AFM probe during spectroscopic measurement are shown with bars. The approach and retract curves of force scans are indicated by the solid and dotted line, respectively.
Temporal changes in the fluorescence intensity. The vertical axis corresponds to the integrated fluorescence intensity and the horizontal axis corresponds to the sequential number of spectroscopic measurement starting from 1. The fluorescence intensity in the region was integrated. Closed and open circles represent data obtained when the AFM probe was noncontacted and when the compression force was applied to GFPs, respectively. Closed triangles indicate data acquired when extension force was applied to GFPs. Solid line in each graph is the calibration curve of photobleaching. It was created on the basis of the noncontact spectra. The net change in the fluorescence intensity can be calculated by subtracting the experimental value from the value on the calibration curve (Ref. 7). Panels (a) and (b) show typical results of the measurements using the reactive probe and the complex probe, respectively. Panel (c) shows the results obtained when the either spectroscopic measurement was performed without the force curve measurement.
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