Source: http://aoot.osa.org/oe/abstract.cfm?uri=oe-27-6-8092
Timestamp: 2019-04-21 22:05:58+00:00

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Stimulated emission depletion (STED) fluorescence microscopy squeezes an excited spot well below the wavelength scale using a doughnut-shaped depletion beam. To generate a doughnut, a scale-free vortex phase modulation (2D-STED) is often used because it provides maximal transverse confinement and radial-aberration immunity (RAI) to the central dip. However, RAI also means blindness to a defocus term, making the axial origin of fluorescence photons uncertain within the wavelength scale provided by the confocal detection pinhole. Here, to reduce the uncertainty, we perturb the 2D-STED phase mask so as to change the sign of the axial concavity near focus, creating a dilated dip. By providing laser depletion power, the dip can be compressed back in three dimensions to retrieve lateral resolution, now at a significantly higher contrast. We test this coherent-hybrid STED (CH-STED) mode in x-y imaging of complex biological structures, such as the dividing cell. The proposed strategy creates an orthogonal direction in the STED parametric space that uniquely allows independent tuning of resolution and contrast using a single depletion beam in a conventional (circular polarization-based) STED setup.
S. T. Hess, T. P. Girirajan, and M. D. Mason, “Ultra-high resolution imaging by fluorescence photoactivation localization microscopy,” Biophys. J. 91(11), 4258–4272 (2006).
T. A. Klar, S. Jakobs, M. Dyba, A. Egner, and S. W. Hell, “Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission,” Proc. Natl. Acad. Sci. U.S.A. 97(15), 8206–8210 (2000).
M. Dyba and S. W. Hell, “Focal spots of size lambda/23 open up far-field fluorescence microscopy at 33 nm axial resolution,” Phys. Rev. Lett. 88(16), 163901 (2002).
M. Leutenegger, C. Eggeling, and S. W. Hell, “Analytical description of STED microscopy performance,” Opt. Express 18(25), 26417–26429 (2010).
T. Kaldewey, A. V. Kuhlmann, S. R. Valentin, A. Ludwig, A. D. Wieck, and R. J. Warburton, “Far-field nanoscopy on a semiconductor quantum dot via a rapid-adiabatic-passage-based switch,” Nat. Photonics 12(2), 68–72 (2018).
J. Keller, A. Schönle, and S. W. Hell, “Efficient fluorescence inhibition patterns for RESOLFT microscopy,” Opt. Express 15(6), 3361–3371 (2007).
E. Rittweger, K. Y. Han, S. E. Irvine, C. Eggeling, and S. W. Hell, “STED microscopy reveals crystal colour centres with nanometric resolution,” Nat. Photonics 3(3), 144–147 (2009).
M. Hofmann, C. Eggeling, S. Jakobs, and S. W. Hell, “Breaking the diffraction barrier in fluorescence microscopy at low light intensities by using reversibly photoswitchable proteins,” Proc. Natl. Acad. Sci. U.S.A. 102(49), 17565–17569 (2005).
J. Arlt and M. J. Padgett, “Generation of a beam with a dark focus surrounded by regions of higher intensity: the optical bottle beam,” Opt. Lett. 25(4), 191–193 (2000).
J. Heine, C. A. Wurm, J. Keller-Findeisen, A. Schönle, B. Harke, M. Reuss, F. R. Winter, and G. Donnert, “Three dimensional live-cell STED microscopy at increased depth using a water immersion objective,” Rev. Sci. Instrum. 89(5), 053701 (2018).
P. Török and P. Munro, “The use of Gauss-Laguerre vector beams in STED microscopy,” Opt. Express 12(15), 3605–3617 (2004).
S. Deng, L. Liu, Y. Cheng, R. Li, and Z. Xu, “Effects of primary aberrations on the fluorescence depletion patterns of STED microscopy,” Opt. Express 18(2), 1657–1666 (2010).
B. Harke, C. K. Ullal, J. Keller, and S. W. Hell, “Three-dimensional nanoscopy of colloidal crystals,” Nano Lett. 8(5), 1309–1313 (2008).
B. R. Patton, D. Burke, D. Owald, T. J. Gould, J. Bewersdorf, and M. J. Booth, “Three-dimensional STED microscopy of aberrating tissue using dual adaptive optics,” Opt. Express 24(8), 8862–8876 (2016).
K. Y. Han and T. Ha, “Dual-color three-dimensional STED microscopy with a single high-repetition-rate laser,” Opt. Lett. 40(11), 2653–2656 (2015).
Y. Xue, C. Kuang, X. Hao, Z. Gu, and X. Liu, “A method for generating a three-dimensional dark spot using a radially polarized beam,” J. Opt. 13(12), 125704 (2011).
J. Antonello, E. B. Kromann, D. Burke, J. Bewersdorf, and M. J. Booth, “Coma aberrations in combined two- and three-dimensional STED nanoscopy,” Opt. Lett. 41(15), 3631–3634 (2016).
J. Antonello, D. Burke, and M. J. Booth, “Aberrations in stimulated emission depletion (STED) microscopy,” Opt. Commun. 404, 203–209 (2017).
X. Weng, X. Gao, H. Guo, and S. Zhuang, “Creation of tunable multiple 3D dark spots with cylindrical vector beam,” Appl. Opt. 53(11), 2470–2476 (2014).
C. Alpmann, M. Esseling, P. Rose, and C. Denz, “Holographic optical bottle beams,” Appl. Phys. Lett. 100(11), 111101 (2012).
Y. Zhang, “Generation of three-dimensional dark spots with a perfect light shell with a radially polarized Laguerre-Gaussian beam,” Appl. Opt. 49(32), 6217–6223 (2010).
Z. Zhang, H. Fan, H.-F. Xu, J. Qu, and W. Huang, “Three-dimensional focus shaping of partially coherent circularly polarized vortex beams using a binary optic,” J. Opt. 17(6), 065611 (2015).
L. Gong, W. Liu, Q. Zhao, Y. Ren, X. Qiu, M. Zhong, and Y. Li, “Controllable light capsules employing modified Bessel-Gauss beams,” Sci. Rep. 6(1), 29001 (2016).
W. Condell, “Fraunhofer diffraction from a circular annular aperture with helical phase factor,” J. Opt. Soc. Am. A 2(2), 206–208 (1985).
Y. Kozawa and S. Sato, “Dark-spot formation by vector beams,” Opt. Lett. 33(20), 2326–2328 (2008).
A. M. Yao and M. J. Padgett, “Orbital angular momentum: origins, behavior and applications,” Adv. Opt. Photonics 3(2), 161–204 (2011).
B. Wang, J. Shi, T. Zhang, X. Xu, Y. Cao, and X. Li, “Improved lateral resolution with an annular vortex depletion beam in STED microscopy,” Opt. Lett. 42(23), 4885–4888 (2017).
C. Sheppard and Z. Hegedus, “Axial behavior of pupil-plane filters,” J. Opt. Soc. Am. A 5(5), 643–647 (1988).
B. Richards and E. Wolf, “Electromagnetic Diffraction in Optical Systems. 2. Structure of the Image Field in an Aplanatic System,” Proc R Soc Lon Ser-A253, 358–379 (1959).
B. Harke, J. Keller, C. K. Ullal, V. Westphal, A. Schönle, and S. W. Hell, “Resolution scaling in STED microscopy,” Opt. Express 16(6), 4154–4162 (2008).
V. Westphal and S. W. Hell, “Nanoscale resolution in the focal plane of an optical microscope,” Phys. Rev. Lett. 94(14), 143903 (2005).
A. Punge, S. O. Rizzoli, R. Jahn, J. D. Wildanger, L. Meyer, A. Schönle, L. Kastrup, and S. W. Hell, “3D reconstruction of high-resolution STED microscope images,” Microsc. Res. Tech. 71(9), 644–650 (2008).
T. J. Gould, J. R. Myers, and J. Bewersdorf, “Total internal reflection STED microscopy,” Opt. Express 19(14), 13351–13357 (2011).
L. Kastrup, H. Blom, C. Eggeling, and S. W. Hell, “Fluorescence fluctuation spectroscopy in subdiffraction focal volumes,” Phys. Rev. Lett. 94(17), 178104 (2005).
C. Eggeling, K. I. Willig, and F. J. Barrantes, “STED microscopy of living cells--new frontiers in membrane and neurobiology,” J. Neurochem. 126(2), 203–212 (2013).
C. Eggeling, C. Ringemann, R. Medda, G. Schwarzmann, K. Sandhoff, S. Polyakova, V. N. Belov, B. Hein, C. von Middendorff, A. Schönle, and S. W. Hell, “Direct observation of the nanoscale dynamics of membrane lipids in a living cell,” Nature 457(7233), 1159–1162 (2009).
K. Sozanski, E. Sisamakis, X. Zhang, and R. Holyst, “Quantitative fluorescence correlation spectroscopy in three-dimensional systems under stimulated emission depletion conditions,” Optica 4(8), 982–988 (2017).
R. Wollhofen, J. Katzmann, C. Hrelescu, J. Jacak, and T. A. Klar, “120 nm resolution and 55 nm structure size in STED-lithography,” Opt. Express 21(9), 10831–10840 (2013).
P. Gao, B. Prunsche, L. Zhou, K. Nienhaus, and G. U. Nienhaus, “Background suppression in fluorescence nanoscopy with stimulated emission double depletion,” Nat. Photonics 11(3), 163–169 (2017).
G. Vicidomini, G. Moneron, K. Y. Han, V. Westphal, H. Ta, M. Reuss, J. Engelhardt, C. Eggeling, and S. W. Hell, “Sharper low-power STED nanoscopy by time gating,” Nat. Methods 8(7), 571–573 (2011).
L. Lanzanò, I. Coto Hernández, M. Castello, E. Gratton, A. Diaspro, and G. Vicidomini, “Encoding and decoding spatio-temporal information for super-resolution microscopy,” Nat. Commun. 6(1), 6701 (2015).
J. Heine, M. Reuss, B. Harke, E. D’Este, S. J. Sahl, and S. W. Hell, “Adaptive-illumination STED nanoscopy,” Proc. Natl. Acad. Sci. U.S.A. 114(37), 9797–9802 (2017).
F. Göttfert, T. Pleiner, J. Heine, V. Westphal, D. Görlich, S. J. Sahl, and S. W. Hell, “Strong signal increase in STED fluorescence microscopy by imaging regions of subdiffraction extent,” Proc. Natl. Acad. Sci. U.S.A. 114(9), 2125–2130 (2017).
L. Wang, B. Chen, W. Yan, Z. Yang, X. Peng, D. Lin, X. Weng, T. Ye, and J. Qu, “Resolution improvement in STED super-resolution microscopy at low power using a phasor plot approach,” Nanoscale 10(34), 16252–16260 (2018).
T. Grotjohann, I. Testa, M. Reuss, T. Brakemann, C. Eggeling, S. W. Hell, and S. Jakobs, “rsEGFP2 enables fast RESOLFT nanoscopy of living cells,” eLife 1, e00248 (2012).
F. Balzarotti, Y. Eilers, K. C. Gwosch, A. H. Gynnå, V. Westphal, F. D. Stefani, J. Elf, and S. W. Hell, “Nanometer resolution imaging and tracking of fluorescent molecules with minimal photon fluxes,” Science 355(6325), 606–612 (2017).
M. Abramowitz and I. A. Stegun, “Handbook of mathematical function: with formulas, graphs and mathematical tables,” in Handbook of mathematical function: with formulas, graphs and mathematical tables (Dover Publications, 1965).
» Visualization 1 Raw data (z-stack acquisition) used to generate the chromo-projection in Fig. 4c.
» Visualization 2 Large field-of-view raw data (z-stack acquisition) of tubulin-labeled Indian muntjac cells. Left: 2D-STED (30mW), Center: 2D-STED (80mW); Right: CH-STED (80mW, rho=0.86). Slice distance is 200nm.
Fig. 1 Phase masks for the standard STED modes and for a ‘radial vortex’. a) z-STED mask for mostly-axial confinement. b) 2D-STED mask for transverse confinement. c) An intensity-zero is warranted whenever a vortex-phase is added to an arbitrary radial-only function, f(r) (Appendix A). Off-axis radial phase gradients (Δ=0) can be exploited to generate an axial gradient for STED confinement. The bottom rows show experimental cross-sections and focal profiles of z-STED and 2D-STED using gold-bead scattering (775nm wavelength, 1.4 NA objective) with corresponding x-y imaging of microtubule filaments.
Fig. 2 (a) Simplified layout of the modified STED setup. (b) Interpretation and observation by gold bead scattering of the tunable dip generation. The scattering images are for an xz-scanned gold bead displayed with a linear and a saturated look-up table (LUT), the latter providing a heuristic preview of the effective fluorescence source at high saturation. (c) Data-points and paraxial theory (solid lines) for the depletion beam focal plane profile. In the inset, beam’s geometrical confinement metric (second-order derivative of intensity) with experimental data and theory as a function of the bi-vortex radius, ρ. The single adjusted parameter (both in the main graph and in the inset) is a global vertical normalization factor.
Fig. 3 STED modes PSFs. (a) Nano-bead fluorescence xz-scans in different STED modes (same LUT display). The inset highlights the origin of the excited ghosts that are poorly depleted by the z-STED beam. (b) Left: gold bead scattering cross-section in 2D-STED and CH-STED with isophote lines defining saturation contours. White-to-red represent signal rescue in 2D-STED (depletion power change) and in CH-STED (ρ change). Red and white isophotes in the CH-STED panel are for the same intensity value and are therefore representative of an actual 2D- to CH-STED transition. Right: effective PSF in the two STED regimes. Red-blue images (same LUT) are log-intensity versions that highlight background noise.
Fig. 4 2D-STED to CH-STED transition in constant-power mode with all acquisition parameters (apart from the phase mask) kept constant. (a) Single-slice and projections from a z-stack acquisition of a tubulin-labeled Indian muntjac dividing cell in metaphase, with a kinetochore marker shown in the right panel (red). Top- and bottom-halves are independent, vertically adjacent, acquisitions. (b) Low-signal Indian muntjac dividing cell in anaphase (chromosome staining in the inset) and zoomed ROI at the 2D to CH transition zone. Photon standard deviation, average and their ratio for the top and bottom halves are displayed. (c) Chromo-projection from a z-stack (Visualization 1) of a tubulin-labeled U2OS interphase cell. Color definition is a readout for optical sectioning. (d) Indian muntjac mitotic spindle in all single-beam and incoherent superposition modes. In (c) and (d), where a single object is consecutively imaged, a left-right acquisition sequence is followed.
Fig. 5 CH-STED vs. 2D-STED axial confinement and background suppression using 40-nm fluorescent beads. (a) PSF lateral dimension using the full-width half-maximum (FWHM) criteria (mean ± s.d., n = 10 beads per datapoint) at and away from the focal plane in 2D-STED as a function of STED laser average power (at back focal plane). In the inset, metrics for assessing performance, where the defocused plane chosen for measuring confinement is at a Rayleigh range distance (zR = 260nm) from the focal plane. (b) PSF lateral dimension (mean ± s.d., n = 10 beads per datapoint) at and away from the focal plane in CH-STED as a function of ρ, using an intermediate-range STED power (60mW). (c) Scatter plot comparison of 2D-STED and CH-STED (at 60mW and 200mW) using a common parameter (D0) as the independent variable. Each datapoint represents one bead. Axes were cropped at 400nm, leaving three 2D-STED (red) datapoints not displayed (used and accounted for in quantifications in (a). (d) Relative background suppression estimate using the focal plane Gaussian curve fit amplitude relative to an average background value at a one wavelength distance from the focal plane (as displayed in (a) inset).
Fig. 6 (a) Left: STED beam cross-sections with contour lines showing signal rescue (path A) followed by resolution rescue (path B); Right: Surface displaying theoretical resolution under the parabolic approximation (Eq. (2)). Example trajectories for the three base modes are outlined: constant-power (A or A’), constant-geometry (B or the 2D-STED case B’) and constant-resolution (green line). (b) Projection of an anaphase spindle in a tubulin filament-labeled U2OS cell. Blue-red insets are pictorial examples of the PSF cross-section in each condition (taken from Fig. 3(b)) (c) Single slice image of astral microtubules in a prometaphase U2OS cell showing suppression of defocused portions in CH-STED. (d) Central region of a tubulin-labeled Indian muntjac mitotic spindle. Photocount profiles (right) correspond to the yellow dashed lines. (e) Tubulin-labeled Indian muntjac mitotic spindle along alternative paths in the theoretical surface. Independent LUTs are used. All panels in this figure follow a left-right acquisition sequence, with all unstated settings kept constant.
Fig. 7 Intensity variation of the bi-vortex focal plane profile near the point at which the on-axis curvature vanishes, together with the leading term in the Taylor expansion (parabolic approximation).
Fig. 8 Predicted variation of the focal plane fluorescence spot size as the CH-STED parameter ρ is varied. The parabolic approximation deviates significantly from the experimental values for values of ρ below 0.9.
Fig. 9 (a) Actin-labeled axon. Transition to CH-STED displays mostly a loss in lateral resolution. In these thin structures (<λ) SBR improvement is less significant. (b) Standard and CH-STED modes compared to confocal in a cross-section acquisition of a spectrin-labeled neuron cell body. (c) Transition between STED modes during scanning (vertical slow axis) following the paths outlined in Fig. 6(a). The object is the peripheral area of an actin-labeled neuron cell body. (d) Confocal and dual-channel STED imaging of nuclear pore components in HeLa cells.
(10) M bV ( ρ,R;s,ϕ )= M sV ( R;s,ϕ )−2 M sV ( ρR;s,ϕ ).
(11) lim s→0 I bi−vortex ( ρ,R;s )→ I 0 ( 2πNA 3 s ) 2 ( 2 ρ 3 −1 ) 2 .
(12) ( FWHM ) 2 = ( FWH M 0 ) 2 1+P/ P Sat + B 2 .
(14) I bV ( λ STED , ρ, R; s )= I 0 (2π R 2 ) 2 | M bV ( λ STED , ρ, R; s ) | 2 .

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