Fig 1.
Setup realizing a STED upgrade.
Components integrated into the MicroTime 200 confocal microscope (PicoQuant, Germany) in order to reach diffraction-unlimited imaging are underlined. The laser beam inducing stimulated emission (766 nm) is coupled into the same polarization-maintaining (PM) fiber as the excitation light from the ps-pulsed laser diode at 635 nm. The polarizer (Glan-Thompson prism from Artifex, Germany) is not mandatory, as the linear polarization of the lasers used is preserved mostly by the fiber. Still, as the quality of the STED doughnut created by the EASYDOnut phase plate (Abberior, Germany) depends on the polarization state of the 766 nm light, further polarization control may be beneficial. Arrows in inset (D) indicate the slow axis’ direction of each of the phase plate’s segments. The achromatic quarter wave plate (λ/4) changes the polarization state to circular, the state the phase plate works best with. Fluorescence was recorded with a 100 Plan Apo Lambda oil immersion objective from Nikon. Time-correlated single-photon counting (TCSPC) electronics and analysis software were provided by PicoQuant. For single-photon detection, a single-photon avalanche diode (SPAD, PerkinElmer) was used. Inset (A) shows the point spread functions of the excitation (yellow) and STED (red) light as measured by monitoring the backscattered light of an 80 nm gold bead. Inset (B) shows the corresponding profile plots along the white line in (A). In inset (C), the absorption and emission spectra of the dye Abberior STAR635 are given together with the wavelengths of the chosen excitation and STED lasers. The detection window, realized by a 690/70 nm bandpass filter in front of the detector, is indicated as a yellow box.
Fig 2.
TCSPC approach for time-gated STED imaging.
(A) Double-logarithmic TCSPC histogram of the fluorescence signal of the F-actin marker Abberior STAR635-phalloidin under ps-pulsed excitation and ps-pulsed stimulated emission. Three time gates are indicated: gate i) contains the fluorescence emitted by the labeling dye before application of the STED pulse, the gate ii) intermediate gate contains residual fluorescence emitted during the stimulated emission process, and the gate iii) STED gate contains the fluorescence from the very center of the STED doughnut after the completion of the stimulated emission process. (B) Fluorescence images of STAR635-phalloidin labeled in vitro F-actin obtained from the different time gates i), ii), iii), and the sum of all three time gates, as indicated in (A). Gate i) comprises photons with the confocal spatial information of the structure, gate iii) photons with the diffraction-unlimited STED information, and gate ii) contains a mixture of fluorescence photons that differ in the spatial information they carry. (C) Profile plots along the yellow line in the confocal and the STED image in (B), indicating the same F-actin structure with different spatial resolutions.
Fig 3.
Diffraction-unlimited imaging of crimson beads.
(A) Confocal and STED image of the same preparation of 20 nm crimson beads on a glass substrate, embedded in Mowiol/DABCO. STED power 14.5 mW (at the objective’s back aperture) at a repetition rate of 2.5 MHz. (B) Profile plots along the dashed white line in (A). (C) Full width at half maximum (FWHM) values determined by fitting 2D-Gaussian single peaks to STED images taken at different STED powers. Each data point represents the mean FWHM value of 10–20 peaks with the corresponding standard deviation. The solid lines show the result of a simulation with a function proportional to one over the square root of the STED intensity (Eq 3).
Fig 4.
Joint deconvolution of STED images.
Left: confocal and STED contribution, derived from offline time gating of the emission from one pulsed STED image acquisition run (STED power of 5 mW at a 2.5 MHz repetition rate) of STAR635-phalloidin labeled in vitro F-actin. Right: result of a joint deconvolution of both contributions (joint dec.) and a simple deconvolution of only the STED image (STED dec.); both deconvolutions applied the Richardson-Lucy algorithm. For the joint approach, one PSF of Lorentzian shape with a FWHM of 70 nm and one Gaussian with a FWHM of 300 nm were applied. 108 iterations were performed, and the weighting was 4:1 in favor of the confocal image. For the simple STED-only deconvolution, the same Lorentzian PSF was used as in the joint case, but only 11 iterations were calculated. Insets show the enlarged ROIs at an adapted dynamic range to emphasize tiny differences in the results of the two deconvolution approaches.
Fig 5.
Measurements of in vitro F-actin filaments stained with Atto647N-phalloidin.
(A) Diffraction-limited and-unlimited images of a gated STED acquisition of a filament structure attached to the glass cover slip and labeled with the dye Atto647N-phalloidin. STED power of 5 mW at a 2.5 MHz repetition rate. (B) The marked region of the STED image in (A) is shown enlarged, and regions of interest (ROI) are marked by lines (black and green) and a red box. (C) Profile plots of the ROIs simulated by single peak (Lorentzian) or double peak functions. The specified FWHM indicates that a single filament is localized to an accuracy of 60 nm. Crossing filamentous structures at a distance of ~80 nm appear distinguishable.
Fig 6.
Immunofluorescence localization of microtubules in blowfly salivary glands.
Gland cryosections (10 μm thick) were treated with antibody YL1/2 against α-tubulin and fluorescence of secondary antibody coupled to STAR635P was detected. (A-C) A confocal overview image as well as enlarged views of regions indicated as yellow rectangles are shown. The gland lumen (L) and the nuclei (asterisk) are indicated. (D) STED image of the region shown in (C) acquired at a STED power of 5 mW (2.5 MHz). (E) Image with enhanced SBR as a result of a joint deconvolution of both images (C) and (D). Deconvolution parameter: 131 iterations, no scaling of input-images, FWHM values of 70 nm (Lorentzian) and 300 nm (Gaussian). (F) Profile Plots along the yellow lines in (C), (D) and (E), indicating the same microtubule structure with a different spatial resolution.