Determination of the spatial resolution.

<p>(A) point-spread function of a single fluorophore, (B) localization pattern of one single fluorophore that was localized multiple times through reversible photoswitching, (C) histogram of the standard deviation of localizations of 66 single-molecule point-spread functions (average standard deviation 12 nm) and (D) histogram of the full-width half-maximum (FWHM) of 66 single-molecule point-spread functions (average FWHM 28 nm).</p

Schematic drawing of the experimental approach (see main text for further details).

<p>Schematic drawing of the experimental approach (see main text for further details).</p

Dual color <i>d</i>STORM images of mitochondria and their localization within the calyx of Held.

<p>(A) Anti-cytochrome c oxidase stain. Nucleus spared. The soma of the principal neuron is densely populated with mitochodria. (B) Anti-mGFP stain of a single section through the calyx of Held. (C) Overlay of (A) and (B). (D) 3D rendering of a mitochondrion (blue) and surrounding membrane (yellow). Scale bars are 250 nm.</p

Distribution of HIV-1 Env at the plasma membrane of HeLa cells visualized by super-resolution TIRF microscopy.

<p>HeLa cells were transfected with equimolar amounts of pCHIV and pCHIV^mEosFP. Cells were fixed 24 hpt, stained by indirect immunofluorescence using MAb 2G12 and goat anti-human Alexa Fluor 647 and imaged by dSTORM as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003198#s4" target="_blank">Materials and Methods</a>. Summed intensity TIRF (<b>A</b>) and high resolution dSTORM (<b>B</b>) microscopy images of a representative cell are displayed. Scale bars correspond to 2 µm. (<b>C</b>) Region from the plasma membrane of a representative cell, showing the superposition of a PALM image for Gag.mEosFP (green) and the corresponding dSTORM image of Env stained with Alexa Fluor 647 (red), respectively. HeLa cells transfected with equimolar amounts of pCHIV and pCHIV^mEosFP were fixed and stained by indirect immunofluorescence using MAb 2G12 and goat anti-human Alexa Fluor 647. Cells were subjected to dual-color super-resolution microscopy as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003198#s4" target="_blank">Materials and Methods</a>. Scale bar corresponds to 1 µm. (<b>D</b>) Individual HIV-1 assembly sites from the boxed regions are shown. Scale bar corresponds to 100 nm. (<b>E</b>) Env surface distribution on extracellular immature HIV-1 particles. Particles were purified from the supernatant of 293T cells transfected with pCHIV carrying a point mutation in the HIV-1 protease active site <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003198#ppat.1003198-Chojnacki1" target="_blank">[39]</a>. An eGFP-tagged version of the accessory protein Vpr <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003198#ppat.1003198-McDonald1" target="_blank">[63]</a> was included as a marker to localize the position of individual virions in diffraction limited images. Virions were adhered to fibronectin-coated coverslips, fixed and immunostained with MAb 2G12 and goat anti-human Alexa Fluor 647. The panel shows images of four individual particles with the eGFP signal (green) recorded in diffraction limited mode and the Alexa Fluor 647 (red) signal recorded by dSTORM. Scale bars correspond to 100 nm. (<b>F</b>) Comparison of the Env signal intensities on single extracellular immature particles with the Env signal intensities colocalizing with HIV-1 assembly sites defined by the dSTORM signal for Gag.mEosFP. Fluorescence intensities of the Alexa Fluor 647 signals were determined for the super-resolution images of ten individual particles each. The histogram shows the average values and SD of the fluorescence intensity.</p

Comparative analysis of Env(wt) distribution expressed in the viral context or alone.

<p>(<b>A, B</b>) Distribution of HIV-1 Gag and Env at the plasma membrane of HeLa cells transfected with equimolar amounts of pCHIV and pCHIV^mEosFP. Unfixed cells were stained by indirect immunofluorescence using Fab 2G12 and Fab goat anti-human Alexa Fluor 647, fixed, and subjected to dual-color super-resolution microscopy as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003198#s4" target="_blank">Materials and Methods</a>. (<b>A</b>) Region from the plasma membrane of a representative cell, showing the superposition of a PALM image for Gag.mEosFP (green) and the corresponding dSTORM image of Env stained with Alexa Fluor 647 (red), respectively. Scale bar corresponds to 1 µm. (<b>B</b>) Enlargement of three individual assembly sites from the boxed regions indicated in (A). The figure shows merged super-resolution images (left panels), the dSTORM Env Alexa Fluor 647 image (middle panels) and individual Alexa Fluor 647 localizations from all images recorded in the defined area as black dots, with a black circle representing the rims of the Gag cluster (right panels), respectively. Scale bar corresponds to 100 nm. (<b>C, D</b>) Env distribution patterns in the presence (C) and absence (D) of other HIV-1 derived proteins. HeLa cells were transfected with equimolar amounts of pCHIV and pCHIV^mEosFP (<b>C</b>) or with pEnv(wt) (<b>D</b>), respectively. Unfixed cells were stained with 2G12 Fab and Alexa Fluor 647 Fab, fixed, and visualized by dual-color super-resolution microscopy as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003198#s4" target="_blank">Materials and Methods</a>. Scale bars represent 1 µm.</p

Distribution of Env(wt) or Env(ΔCT) in the context of an Env-interaction deficient Gag variant.

<p>HeLa cells were transfected with proviral constructs carrying both wt Gag and wt Env (<b>A</b>), wt Gag and Env(ΔCT) (<b>B</b>), Gag carrying the MA mutation and wt Env (<b>C</b>), or comprising both mutated MA and Env(ΔCT) (<b>D</b>), respectively. Fixed cells were stained by indirect immunofluorescence using MAb APR342 and goat anti-mouse Alexa Fluor 532 and MAb 2G12 and goat anti-human Alexa Fluor 647, and subjected to dual-color dSTORM. Scale bar corresponds to 1 µm. An overview of respective cells is presented in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003198#ppat.1003198.s007" target="_blank">Figure S7</a>.</p

Averaged super-resolution intensity distribution of Env at HIV-1 assembly sites.

<p>Image-based, averaged assembly site analysis performed on super-imposed high-resolution images of individual Gag.mEosFP clusters (green) from cells transfected with pCHIV/pCHIV^mEosFP (<b>A</b>) or pCHIV.Env(ΔCT)/pCHIV^mEosFPEnv(ΔCT) (<b>B</b>), respectively (n = 7 assembly sites per condition) and the averaged Env Alexa Fluor 647 signal in the respective area (red). Graphs show the profile of intensity through the center of the averaged, overlaid intensity images: green line, Gag.mEosFP; red line, Env. Scale bars represent 50 nm.</p

Computational cluster analysis of HIV-1 Env membrane distribution.

<p>Global cluster analysis of Env distribution at the plasma membrane was performed based on super-resolution images of HeLa cells transfected with pCHIV/pCHIV^mEos.FP, pCHIV. Env(ΔCT)/pCHIV^mEosFPEnv(ΔCT), pEnv(wt) or pEnv(ΔCT), or of A3.01 T-cells nucleofected with pCHIV/pCHIV^mEos.FP or pCHIV.Env(ΔCT)/pCHIV^mEosFPEnv(ΔCT), respectively. (<b>A</b>) Image-based, morphological cluster analysis of entire cells. (<b>B</b>) Differential distribution of cluster size for Env(wt) on the surface of HeLa cells in the presence and absence of other viral proteins. The curve was derived by subtraction of the normalized distribution of cluster size obtained by image-based cluster analysis for Env(wt) expressed in the HIV-1 context from that obtained upon expression of Env(wt) alone. (<b>C</b>) Coordinate based all-distance distribution analysis by Ripley's H-function. Statistical evaluation of the maximal H-values [nm] was performed as a correlate of the average cluster diameter. (<b>D</b>) Statistical evaluation of the amplitude of the H-function [a.u.] as a measure of the average degree of clustering. Box plots display 5^th percentile, 25^th percentile, median (straight line), mean (square), 75^th percentile and 95^th percentile.</p

Implementation details of WaveTracer software.

<p>(<b>A</b>) 2D real-time localization steps: 1) During the acquisition process, images are temporarily transferred to the CCD camera buffer. 2) The current image is transferred to the GPU memory for processing. 3) The image is split into 16×16 overlapping sub-images and sent to different processors of the GPU. 4) Wavelet filtering is performed in parallel on each sub-image. 5) Sub-images are stitched back to reconstruct the filtered image. 6) The filtered image is transferred to the CPU for thresholding, watershed processing and centroid extraction. 7) The super-resolution image is then reconstructed and the localized molecule coordinates are saved into the memory for later 3D analysis. (<b>B</b>) 3D post-acquisition localization steps: 1) Images are split into 7×7 sub-images centered on localized molecule coordinates. 2) Anisotropic Gaussian fitting is performed on each sub-image in parallel on GPU. 3) Axial coordinate retrieval of each localized molecule is performed in parallel on GPU. 4) 3D reconstruction is made from the (X,Y,Z) coordinates of all the localized molecules.</p

Real-time super-resolution imaging.

<p>(<b>A</b>) Diffraction-limited epifluorescence image of microtubules labeled with Alexa Fluor 647. (<b>B</b>) 2D super-resolved image of the cell in figure (<b>A</b>), reconstructed in real-time from 20,000 frames and 1.2 million single-molecule localizations. (<b>C</b>) 3D super-resolved image of the microtubules of figure (<b>A</b>) obtained only 15 seconds just after the end of the acquisition. Colors encode for the axial position, in µm. (<b>E</b>) A selected region of interest (ROI) from figure (<b>A</b>). (<b>F</b>) A selected ROI from figure (<b>B</b>). (<b>G</b>) Corresponding ROI from figure (<b>C</b>). (<b>D</b>), (<b>H</b>) Intermediate real-time visualization obtained after 1,000 and 4,000 frames respectively. (<b>I</b>) Diffraction limited epifluorescence image of microtubules labeled with Alexa Fluor 647. (<b>J</b>), (<b>K</b>) 2D super-resolved images of the cell in figure (<b>I</b>), reconstructed in real-time from 3,000 frames, without (78,341 localizations) and with (96,298 localizations) feedback control respectively. (<b>L</b>) Graph of the number of localizations over the number of images, without (red) and with (green) feedback loop control. Solid and dashed black lines represent their respective trends. For better clarity, only one point over 25 points is displayed.</p