Single-molecule imaging and PICS analysis (S1 Fig Data).

<p><i>A</i>: Signal of individual eYFP-GR molecules on an emCCD camera. <i>B</i>: The signal of an individual molecule is fitted to a Gaussian yielding the position, the width and the strength of the signal. <i>C</i>: Distance calculation between molecules in subsequent frames. <i>D</i>: Cumulative distribution function (cdf) of distances of molecules in subsequent frames correlated by diffusion.</p

Calibration of the depth of field (DOF). eYFP was coated on a glass slide and the objective was moved by a piezo scanner (S1 Fig Data).

<p>The resulting peak-widths were fitted as previously described [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141080#pone.0141080.ref025" target="_blank">25</a>]. The data were subsequently fit to Eq (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141080#pone.0141080.e001" target="_blank">1</a>) yielding the signal width at focus, <i>σ</i>₀ = 263 nm and the DOF = 750 nm. All data characterized by a width larger than √2 × 263 nm = 372 nm (dashed line) were discarded from further analysis.</p

Result of Eq (6) for DOF = 750 nm and four different time lags, t (S1 Fig Data).

<p>For a diffusion constant of D = 2 μm²/s the probability to reside inside the DOF after t = 10 ms is 0.79, whereas for D = 0.1 mm2/s the probability is 0.97.</p

PICS analysis of glucocorticoid receptor at different time lags (S1 Fig Data).

<p>In blue the uncorrected result. A decrease of the fast fraction is observed. In green the result corrected by Eq (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141080#pone.0141080.e008" target="_blank">8</a>) taking into account the DOF. The fast fraction stays constant for time lags at least up to 150 ms. Dashed lines are linear fits to the data. Error-bars represent the standard deviation.</p

Depth-of-Focus Correction in Single-Molecule Data Allows Analysis of 3D Diffusion of the Glucocorticoid Receptor in the Nucleus

<div><p>Single-molecule imaging of proteins in a 2D environment like membranes has been frequently used to extract diffusive properties of multiple fractions of receptors. In a 3D environment the apparent fractions however change with observation time due to the movements of molecules out of the depth-of-field of the microscope. Here we developed a mathematical framework that allowed us to correct for the change in fraction size due to the limited detection volume in 3D single-molecule imaging. We applied our findings on the mobility of activated glucocorticoid receptors in the cell nucleus, and found a freely diffusing fraction of 0.49±0.02. Our analysis further showed that interchange between this mobile fraction and an immobile fraction does not occur on time scales shorter than 150 ms.</p></div

Imaging of diffusing fluorophores inside the nucleus.

<p>Since the depth of focus (DOF = 750 nm) is shallow, molecules can diffuse in and out of the observation volume. This will deplete the relative contribution of the fast diffusing fraction to the analysis.</p

Simulation result that shows depletion of the fast fraction for increasing time lags (S1 Fig Data).

<p>The time lag is given by the number of frames between detections. In blue the uncorrected result, in green the result after correction with Eq (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141080#pone.0141080.e008" target="_blank">8</a>).</p

Super-resolution images of internalized α-syn aggregates in endosomal vesicles in time.

<p>(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).</p

Characteristic properties of the optical setup.

<p>(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).</p

Size distribution of α-syn aggregates in endosomal vesicles in time.

<p>(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).</p