Calculated time evolution of the observation volume of STED nanoscopy (tE-PSF).

<p>Lateral (<i>r</i>) intensity distribution <i>h</i>(<i>t</i>,<i>r</i>) (A,B), the full-width-at-half-maximum (FWHM(<i>t</i>)) (C,D; black, left axis) and the peak signal <i>h</i>(<i>t</i>,0) (C,D; red, right axis) of the tE-PSF as a function of the time of the STED beam action (excitation at time 0) for the CW-STED (A,C) and P-STED (B,D) modality (A,B; left panel: original, un-normalized data; right panel: normalization to 1 for each time). The excitation intensity profile <i>h</i>_exc(<i>r</i>), the detection efficiency profile <i>h</i>_em(<i>r</i>) and the STED intensity profile <i>I</i>_sted(<i>r</i>) are computed using Fourier theory <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054421#pone.0054421-Leutenegger2" target="_blank">[39]</a>. Given <i>I</i>_sted(<i>r</i>), <i>h</i>_exc(<i>r</i>) and <i>h</i>_em(<i>r</i>) the time evolution of the observation volume <i>h</i>(<i>t</i>,<i>r</i>) is calculated using <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054421#pone.0054421.e004" target="_blank">Equation (4</a>). We assumed an oil immersion objective of 1.4 numerical aperture, <i>τ</i>₌3.4 ns, <i>T</i> = 1/80 MHz, <i>T</i>_STED = 300 ps, <i>λ</i>(excitation)₌635 nm, <i>λ</i>(STED)₌760 nm and <i>λ</i>(emission)₌670 nm, the same average powers for both modalities, i.e. ς^* = 4.8 and 200 for CW and pulsed mode, respectively, and a detection pinhole with a projected diameter of 500 nm in the sample space (0.9 × the Airy disc diameter).</p

Scanning gP-STED nanoscopy images.

<p>Scanning images of microtubule of fixed PtK2 cells immunolabeled with the dye KK114 (A) and ATTO647N (B): confocal (left), time-gated gP-STED (middle left) and non-gated P-STED (middle right) images, and normalized intensity profiles along the arrows marked in the P-STED images. Excitation: 635 nm, <i>f</i> = 76 Mhz, <i>P_exc</i>₌5 µW; STED: 760 nm, <i>f</i> = 76 Mhz, <i>P_STED</i>₌45 mW (a), 70 mW (b); gated detection: <i>T_g</i> = 500 ps. Pixel size: 20 nm. Scale bars: 1 µm.</p

Dependence of the confinement of the E-PSF of CW-STED and gCW-STED recordings on the fluorescence lifetime.

<p>Correlative plot of values pairs of intensity-weighted average lifetime <<i>τ</i>> (<i>i</i> = 3) and FWHM of the intensity profiles through images of individual FNDs. The solid lines show linear regression fits to the experimental data with slopes of −1.99, −0.70 and −0.41 for <i>T_g</i> = 0 ns, 5 ns and 10 ns, respectively. Imaging conditions as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054421#pone-0054421-g003" target="_blank">Figure 3B</a>.</p

Observation volume under gated detection (E-PSF).

<p>Calculated lateral line profiles of the E-PSF (A,B) and OTF (C,D) for confocal (grey line), pulsed STED (P-STED: solid blue line without gating, gP-STED: dotted blue line with <i>T</i>_g = <i>T</i>_STED) and CW-STED (CW-STED: solid red line without gating, gCW-STED: dotted red lines with different <i>T</i>_g as denoted) recordings (left panels un-normalized, right panels normalized data). The excitation intensity profile <i>h</i>_exc(<i>r</i>), the detection efficiency profile <i>h</i>_em(<i>r</i>) and the STED intensity profile <i>I</i>_sted(<i>r</i>) are computed using Fourier theory <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054421#pone.0054421-Leutenegger2" target="_blank">[39]</a>. Given <i>I</i>_sted(<i>r</i>), <i>h</i>_exc(<i>r</i>) and <i>h</i>_em(<i>r</i>) the E-PSF <i>h</i>(<i>r</i>) is calculated using the time integration of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054421#pone.0054421.e004" target="_blank">Equation (4</a>). Same parameters as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054421#pone-0054421-g001" target="_blank">Figure 1</a>. Inset (B): FWHM of the E-PSF of the gCW-STED nanoscope as a function of the time-delay <i>T</i>_g; Red dots depict the simulation values (see above); Red solid line depict the model values: Calculation are based on <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054421#pone.0054421.e012" target="_blank">Equation (12</a>) with <i>d</i>_c =232 nm, <i>a</i> = 3.63·10⁻³ nm⁻¹, τ = 3.4 ns and ς^* = 4.8. The dotted horizontal line represents the FWHM of the simulated E-PSF of the gP-STED nanoscope with <i>T</i>_g = <i>T</i>_STED and the same parameters as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054421#pone-0054421-g001" target="_blank">Figure 1</a>.</p