Fig 1.
Characteristic properties of the optical setup.
(a) Frame with the signal of several Alexa532 molecules. Scale bar = 2μm. (b) Histogram of the sigma of positional accuracy (Mean: 11 nm). (c) Zoom-in of the white square in Fig 1A showing the Gaussian intensity profile. (d) Histogram of the intensity of localizations (Mean: 447 photons).
Fig 2.
Super-resolution imaging of the in vitro prepared α-syn fibrils.
(a) AFM and (b) dSTORM images of intact wild-type α-syn fibrils covalently labeled with the NHS derivate of Alexa 532 fluorophore. (c) AFM and (d) dSTORM images of sonicated labeled α-syn fibrils.
Fig 3.
Characterization of different sized α-syn fibrils by dSTORM.
(a) Detailed view of an intact α-syn-Alexa532 fibril (the marked fibril from Fig 2A). (b) Histogram corresponding to the localization data (see S1 Fig for details on the method). A FWHM of 47 nm was calculated. (c) Histogram distribution of FWHM for intact α-syn fibrils. A mean diameter of 43 ± 12 nm was calculated from FWHM data (> 25 fibrils). (d) dSTORM images of two different sized sonicated fibrils (the marked sonicated fibrils from Fig 2B). (e) Histogram distribution of FWHM for sonicated α-syn fibrils. The mean diameter of α-syn sonicated fibrils was 42 ± 11 nm.
Fig 4.
Internalization of α-syn sonicated fibrils in human neuroblastoma cells.
Images show co-localization of Alexa 532 labeled α-syn aggregates (green) with LysoTracker Deep Red (red). SH-SY5Y cells were treated with 50 nM LysoTracker Deep Red, then washed, incubated further with Alexa532-labeled α-syn sonicated fibrils and imaged live on a confocal microscope.
Fig 5.
Super-resolution images of internalized α-syn aggregates in endosomal vesicles in time.
(a) dSTORM image of a cell treated for half an hour with α-syn -Alexa532 aggregates. A detailed view of the aggregates in the cell membrane is shown below a). (b) After 2 hours of incubation, α-syn aggregates are internalized in vesicles. Detailed view of the aggregates in a vesicle shown in the image below b). (c) Internalized α-syn aggregates after 24 hours of incubation, with two different sized clusters highlighted bellow image c).
Fig 6.
Size distribution of α-syn aggregates in endosomal vesicles in time.
(a)-(c) Histogram of FWHM of intracellular α-syn clusters in time. (ANOVA significance levels: (a)-(b): 10−4; (b)-(c):5×10−3; ((a)-(c):10−7). (d) A decrease in α-syn cluster size is observed in the mean average FWHM of α-syn clusters in time (median and 50% interval).