FIGURE 7.

TIRFM analyses of HTT oligomers. A, photobleaching of Alexa Fluor 488-HTTQ53 molecule. B, distribution of photobleaching unitary step intensity of Alexa Fluor 488 dye (n = 209). The peak fluorescence intensity was estimated by Gaussian fitting and shown as mean ± S.D. C and D, fluorescence intensities of single particles of Alexa Fluor 488-GST-HTTQ53 (C) and Alexa Fluor 488-GST-HTTQ23 (D) were measured before (white column) and after (black column) incubation of proteins with PreScission protease in the presence of prefoldin for 13 h. Samples were diluted 3000-fold and illuminated by a blue laser (488 nm, 11.8 milliwatts). The gain level of the image intensifier was set at 4.2. To correctly measure the intensity of Alexa Fluor 488-GST-HTTQ53 samples that had been incubated with prefoldin for 13 h, an ND40 filter (5.2 milliwatts) was used to avoid saturation of fluorescence intensities, and their fluorescence intensities were multiplied by 2.4, which was calculated from the linear correlation between laser power and fluorescent intensity shown in E. E, fluorescent intensities of FluoSpheres (0.1 μm diameter and yellow-green, Molecular Probes) were excited with a blue laser using different ND filters. Each laser beam power was measured by a power meter. Fluorescence intensity was decreased to 41.5 ± 1.9% (1/2.4-fold) by the ND40 filter compared with that in the absence of an ND filter (ND100). a.u., arbitrary unit.