Figure 1.
Schematic of the CW STED system for far-field super-resolution optical nanoscopy.
The green line represents the excitation 635 nm laser, red line is the CW Ti: Sapphire STED beam, and the yellow line represents the fluorescence signal. SMF: single mode fiber; MMF: multi-mode fiber; DC1 and DC2 are dichotic filters. VPP: vortex 0–2π phase plate; QWP: quarter waveplate. The excitation PSF (a), doughnut depletion PSF overlapped with excitation PSF (b), and STED PSF (c) clearly shows the process of achieving super-resolution. Scale bar: 500 nm. The modulation efficiency versus the depletion intensity was measured with crimson beads, DyLight 650 and ATTO 647N solution (d). The STED wavelengths are 763 nm for crimson beads and ATTO 647N, and 783 nm for DyLight 650.
Figure 2.
Confocal and corresponding CW-STED images of fluorescent 20-nm-diameter nanospheres.
(a) Confocal; (b) CW-STED; (c) The profile along the dashed line in (a) and (b) exhibits a spot size of 63 nm, indicating an effective resolution of ∼59 nm after subtraction of the bead size. Scale bar: 500 nm.
Figure 3.
Comparison of diffraction-limited confocal microscopy and STED nanoscopy for fluorescent 200-nm-diameter nanospheres.
(a) Confocal and (b) corresponding CW-STED images; (c) and (d) are the deconvolved results of (a) and (b). Scale bar: 500 nm.
Figure 4.
Confocal, corresponding CW-STED, and deconvolved STED images of microtubules in a Vero cell stained with Dylight 650.
(a) Confocal; (b) CW-STED; (c) R-L deconvolution of (b). The intensity at the cross-section marked in (a), (b), and (c) inlets are plotted in (d). The wavelength and power density for the STED beam is 783 nm and ∼200 MW/cm2. Scale bar: 1 µm.
Figure 5.
Confocal, corresponding CW-STED, and deconvolved STED images of keratin intermediate filaments in a PtK2 cell stained with ATTO 647N.
(a) Confocal; (b) CW-STED; (c) R-L deconvolution of (b). The intensity at the cross-section marked in (a), (b), and (c) boxed regions are plotted in (d). The wavelength and power density for STED beam is 763 nm and ∼200 MW/cm2. Scale bar: 1 µm.
Figure 6.
Confocal and corresponding CW-STED images of actin filaments in a HeLa cell.
(a) Confocal; (b) CW-STED. The boxed regions in (a) and (b) are magnified in (c) and (d), respectively. The intensities at the cross-section marked in (c) and (d) are plotted in (e). The wavelength and power density for the STED beam is 763 nm and ∼127 MW/cm2. Scale bar: 500 nm.
Figure 7.
Confocal and corresponding CW-STED images of hRSV genomic RNA granules in a HEp2 cell.
Confocal (a) and corresponding CW-STED (b) results of isolated individual RNA, as well as RNA granules images by confocal (c) and CW-STED (d) are shown. The intensity at the cross-section marked in (d) and (e) are plotted in (c, f). The wavelength and power intensity for STED beam is 783 nm and ∼162 MW/cm2. Scale bar: 1 µm.