Coherent-hybrid STED: high contrast sub-diffraction imaging using a bi-vortex depletion beam

Abstract

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.

1. Introduction

The capacity to label proteins and other macromolecules with highly specific fluorescent reporters makes fluorescence microscopy an essential tool in the life sciences. Although spatial resolution is conventionally limited to a half-wavelength in the far-field, techniques have been developed that evade this limit by exploiting the fact that the fluorescence microscope is not governed exclusively by optics laws, but involves a (generally nonlinear) sample response [1–6].

In one seminal example, STED microscopy exploits the nano-second excited-state time window by delivering a second, red-shifted, depletion beam (the ‘STED’ beam) to silence significant portions of the excited (diffraction-limited) fluorophore spot [1,7]. Although the depletion beam can equally lead to re-excitation, this is minimized if fluorophores that spontaneously undergo a rapid post-depletion decay are used [8,9] (or by employing coherent population inversion techniques [10]). In the standard implementation, the depletion beam is a dark spot surrounded by steep intensity gradients [11] - a ‘doughnut’- that scans the sample along with the excitation beam. At each position, and if saturated depletion is reached, only a sub-diffraction-sized fluorophore ensemble survives the doughnut beam and fluoresces. Although the fluorescence signal is diffracted as it travels to the detector, its origin - the target coordinate - can be looked up at a scanner-defined (sub-diffraction) resolution. With a resolution limited by technical details only, the use of favorable samples has allowed STED to reach better than 10nm lateral resolution using excitation/depletion wavelengths above 500nm [12].

A problem in single-objective, single-beam, STED (or more generally, RESOLFT implementations [13]) is that the two doughnut beams used in the field, z-STED and 2D-STED, have geometries that are rigid, while displaying contrasting performances regarding lateral resolution, axial resolution, signal-to-background ratio (SBR) and aberrations resilience. The z-STED mode for example, where a π-shifted top-hat phase mask modulates the depletion beam, generates a very strong axial confinement [7,14] but a comparatively low lateral confinement (see Fig. 1(a)). z-STED is also affected by the spurious signal emitted by undepleted excitation lobes (‘ghosts’) and by simple aberrations, such as an imprecision in mask scale definition which, by unbalancing the π-shifted contributions, fills the central ‘zero’ and attenuates the signal of interest [11,15]. This sensitivity to scale and to the other radial aberrations (such as spherical) often demands mask readaptation to imaging conditions, some of which may vary in the course of a bio-imaging ‘session’ (e.g. the refractive index mismatch between samples and the objective immersion medium, the depth of the structure under observation and the objective lens [15]). Ultimately, z-STED is typically used for cross-section (x-z) imaging only, where its notable axial resolution may prove decisive.

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.

For the highest lateral resolution, a helical phase ramp (‘vortex’) phase mask [16] is used. This so-called 2D-STED mode (Fig. 1(b)) is regarded as a standard in STED microscopy, particularly when performing x-y imaging. 2D-STED maximizes lateral confinement for a given energy [11], suffers from low ghost noise and generates an aberration-resilient intensity-zero [16,17]. Crucially, the absence of a radial scale in the phase mask provides RAI, eliminating the scale mismatch-elicited noise that compromised z-STED. However, the feature that confers RAI to 2D-STED also determines that the vortex-generated dip is not a point, but a nodal line along the optical axis. Thus, in 2D-STED, it is still the detection pinhole that solely defines the axial response. The effective point-spread function (PSF) is thus a very thin but long (>λ) needle along the optical axis.

This rigid and complementary nature of the two STED modes prompted the use of beam superposition architectures. These two-beam (or many-beam) approaches allow a major improvement in depletion isotropy [18–20], which can be further improved by using radially or azimuthally-polarized depletion beams [21,22]. However, in addition to compound aberration effects [23,24] and alignment requirements at the nanometer scale, an incoherent superposition can never rectify a residual filling of the intensity-zero which may be present in either beam (usually the z-STED component). To provide an alternative to incoherent superpositions, the present work focuses on the definition of a single depletion beam to improve the 2D-STED axial response, aiming at increasing contrast in x-y STED imaging.

In this context, it is instructive to visualize a ‘signal rescue’ scenario as it is encountered in actual 2D-STED experiments. If the depletion laser power is gradually increased, the needle-shaped fluorescence source is eventually made so thin that a vanishingly small number of molecules remain in the ‘on’ state. This condition of ‘excess resolution’ is eventually reached even if an ideal beam with a perfect nodal line is used, or otherwise sooner. The recourse is to reduce back the laser power, which widens the parabolic dip, so that neighboring molecules are allowed to fluoresce. By doing this, the needle-shaped source dilates in all planes, recovering focal signal but also the unwanted background. Thus, the usual power-modulation protocol does not permit an independent tuning of lateral resolution and contrast. In principle, addition of a doughnut-geometry degree of freedom to the ‘power-only’ modulation could decouple control over these metrics.

Here, applying a RAI-assisted modulation to the vortex mask, we generate a depletion doughnut that dilates the dip primarily at the focal plane, providing focus-specific signal selection, thus fundamentally changing the axial response. Dip dilation does not reflect a fundamental loss in lateral resolution. Instead, the transformed axial response sets the stage for a non-linear process (e.g., STED) to compress the PSF in a more isotropic manner. With both beam geometry and depletion non-linearity at hand, one can expect to achieve independent control over lateral resolution and depth sectioning.

2. Coherent-hybrid depletion beam

To test the concept, we aimed at defining a phase-only mask that generates a hollow depletion beam featuring a dilation at the focal plane. A comprehensive resource for hollow beam engineering [25–29] is the field of optical trapping, where these beams find application in manipulating low-refractive index particles. However, in optical trapping the absolute intensity-zero is less important than sophistication of the dielectric maps (e.g. for dynamic, multiple, tunable trap generation). In contrast, the major concern in STED bio-imaging is signal preservation, which demands one sharp dark spot. Thus, to create a dilated dip, we restrict ourselves to phase mask typologies providing a robust intensity-zero.

A sufficient condition for aberration resilience (RAI) is a mask constructed as a radial tile of concentric annular regions, each one filled with a vortex phase of integer, equal-sign but possibly different, topological charge. That is so because each annular vortex produces an intensity-zero [30] even in the vectorial regime (e.g. using equal-handedness circular polarization; see Appendix A). Trivially, the full complement of annular vortices still yields an intensity-zero, irrespective of the annulus mutual phase (rotation) and amplitude (Fig. 1(c), Appendix A).

This annular tile is equivalent to the addition of a vortex to a radial-only function, obeying a ‘radial vortex’ condition, ϕ(r,θ) = f(r)+θ, where f(r) is an arbitrary function, generally complex, of the distance to the optical axis r, and θ is the azimuthal angle. While the vortex component θ, provides RAI, f(r) can represent, for example, a lens phase function (paraxial or non-paraxial defocus term), an imprecision in setting the mask scale, spherical aberration, a radial amplitude profile of the depletion beam or their arbitrary combination.

For our purposes, f(r) provides the degree of freedom permitting a shift in the sign of the isophotes’ concavity near the focal point (Fig. 2(a)), even if, by symmetry, the concavity vanishes at the optical axis. To achieve concavity inversion we choose a step function, justified by the fact that only an off-axis phase gradient (absent in the pure vortex mask, Δ = 0 in Fig. 1(b)) can generate axially varying interference conditions, as in z-STED. However, in contrast to z-STED, where the step needs to be precisely located at a radius r₀ for scale matching ($r_0=R/\sqrt{2}$ for a uniformly illuminated pupil of radius R), the vortex presence eliminates the constraint.

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.

A general expression for the phase disturbance imparted by the phase mask can be written as M(r,θ) = exp(inθ + aiπH(r–ρ)) with H the Heaviside step function. This includes, as limiting cases, the z-STED (n = 0, a = 1, ρ ≅ 0.71R) and the 2D-STED single vortex (n = 1, ρ > R) modes. In the particular case we focus on in this work (n = 1, ρ < R), the resultant is a ‘bi-vortex’ parameterized by ρ, the radius at which the vortex switches sign (Fig. 2(b)). It can be noted that at a particular scale and amplitude modulation, the beam created by a bi-vortex phase would degenerate to the standard Laguerre-Gauss (LG₁₁) beam [31,32].

To gain insight on the diffraction pattern created by the bi-vortex, it can be seen that the contracted (inner) vortex alone would generate a correspondingly wider doughnut at the Fourier plane. The exposed peripheral vortex, which has the same handedness but is out-of-phase, would by its own generate an elongated and narrow dip [33], reminiscent of an inverted annular-shaped aperture PSF [34,35]. The resultant is a long narrow dip, dilated specifically at the focal plane region. This tunable dark spot can be seen as arising from the destructive interference of the beam crests generated by the individual vortices (Fig. 2(b)).

We call the beam created by the bi-vortex mask a ‘coherent-hybrid (CH-) STED’ beam, as it amounts to the addition of a 2D-STED mask to a rescaled z-STED mask. Clearly, instrumental imprecisions in setting the desired ρ (or even the phase step magnitude) do not compromise the radial vortex condition for an intensity-zero (Appendix A), anticipating operational ruggedness regarding central fluorescence preservation.

Although vectorial diffraction is required for a complete analysis [36], assumption of sufficient polarization symmetry conditions (as imparted by circular polarization), allows the paraxial approximation to deliver a quantitative insight into the effect of the bi-vortex on lateral resolution. The intensity profile at the focal plane (Fig. 2(c)) in the neighborhood of the optical axis is, in the parabolic approximation, given by (Appendix B)

$$\underset{x\to 0}{\text{lim}}{I}_{bi-vortex}\to I_0{\left(\frac{2\pi NA}{3\lambda }x\right)}^2{\left(2{\rho }^3-1\right)}^2,$$ (1)

where I₀ represents the on-axis focal plane intensity created by the circular pupil without a phase mask, NA is the (beam width-dependent) effective numerical aperture of the focusing system, λ is the STED beam wavelength in vacuum and x is the distance to the optical axis. At this expansion level (parabolic approximation), the only difference relative to the single vortex (2D-STED) mask is the factor (2ρ³ – 1)². In contrast, the z-STED profile structure starts at fourth order, providing low (and fixed) geometrical confinement. Experimental data for the parabola concavity near the optical axis fits well this paraxial approximation (inset in Fig. 2(c)), with the slight left-shift being likely caused by the finite width of the STED beam incident on the objective.

3. Experimental results

To create a true intensity-zero using a high-NA objective, 2D-STED setups typically use a circularly polarized depletion beam matching the vortex handedness (or charge). This configuration (as well as other rotationally symmetric polarization states) warrants cancellation of the axial component of the electric field at the optical axis. The same applies for each annular vortex of a CH-STED mask, making existing STED setups adequate for CH-STED microscopy. Here, we used a confocal gated-STED (Abberior Instruments ‘Expert Line’) featuring 40 MHz-modulated excitation (560 and 640nm) and depletion (775nm) beams, coupled to a Nikon Ti microscope. The bi-vortex phase pattern was imprinted on a phase-only spatial light modulator (LCOS, Hamamatsu) on top of a factory-set flat-field correction phase map, with an additional grating being used to diffract the beam off zero-order. A 4f-system is used to image the SLM plane at the back focal plane of an oil-immersion 1.4NA 60x plan-apochromatic objective (Nikon, Lambda Series). All acquisitions were made with a confocal pinhole size of 0.8 Airy units and an APD detector gate (800ps-8ns).

z-scans of 40nm-diameter fluorescently-labeled nano-beads (Crimson beads (Abberior), excitation 640nm), show that the CH-STED PSF depletes the ghost spots efficiently as compared to z-STED (Fig. 3(a)) and provides the desired dip around the geometrical focus (absent in 2D-STED). The effect of depletion energy redistribution is therefore a specific suppression of out-of-focus fluorescence signal – an ampoule-shaped PSF (Fig. 3(b), bottom), which should be more suitable than 2D-STED for imaging thick and complex environments.

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.

We tested interphase cell and mitotic spindle imaging (see sample preparations in Appendix D) with CH-STED using the geometrical transformation only: the constant-power mode (Fig. 4). Here, a focus-specific signal rescue (coupled to a loss in lateral resolution) is expected. Fluorescence signal from interpolar microtubules and kinetochore fibers (microtubule bundles attached to chromosomes), which are immersed in a noisy environment, emerges after switching to CH-STED. In brighter spindles (Fig. 4(a)), the gain is attributed to relative background decrease, whilst at lower photon counts (Fig. 4(b)), the signal increase after transition becomes relevant also against detection noise. Microtubules in less dense meshworks clearly show the expected loss in lateral resolution, but even here the SBR is markedly increased (Fig. 4(c), see z-stack acquisition in Visualization 1).

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.

To assess performance we used elementary PSF metrics (defined in Fig. 5(a) inset) in the optical sectioning range, ρ ≈ 0.8 – 1, as well as at a varying STED power. Width was measured at the focal plane (D₀) and at a plane defocused by one Rayleigh range (z_R = 260nm), the standard measure of the Gaussian beam’s (half-) depth of focus. It is observed that, while the PSF undergoes the usual scale transformation at varying STED power (Fig. 5(a)) [37,38], a shape transformation towards D_Z < D₀ is observed when ρ is varied (Fig. 5(b)).

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 (z_R = 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).

Lateral resolution CH-STED data is in agreement with theoretical calculations (Fig. 5(b)), where a parabolic approximation to the depletion beam proves insufficient (see Appendix B). The high-order expansion required to generate the theoretical curve reflects the fact that, in CH-STED, power and resolution are not anymore univocally related (even for a given fluorophore type), making the fluorescent profile sensitive to the detailed structure of the depletion beam whenever high STED power is used for high sectioning (i.e., lateral resolution lower-end, ρ < 0.9).

For a parametric comparison of 2D-STED and CH-STED, D_Z was measured as a function of a common independent variable - lateral resolution, D₀. In these scatter plots (Fig. 5(c)), where each point represent one nano-bead, the instrumental parameters P_STED and ρ are implicit variables that probe the D₀ × D_Z space. The bottom-right half-space is populated by experimental PSFs that get narrower away from the focus (‘ampoule-shaped’), as opposed to the hourglass-shaped PSF typical of confocal and 2D-STED microscopes. In addition to the progressive geometrical confinement, CH-STED more efficiently attenuates the integrated signal emitted from out-of-focus planes (Fig. 5(d)), as measured by the PSF amplitude relative to a background signal measured at a one-wavelength axial distance (defined in Fig. 5(a)).

Thus far, we used either P_STED or ρ modulation to tune the PSF (Figs. 3(b) and 4). Naturally, the two-dimensional parameter space can be explored. Using the simplified parabolic dip approximation and a first-order approximation to the depletion process [37], the combined effect of ρ and P_STED yields a generalized STED equation for lateral resolution,

$$R\cong \frac{\lambda }{2NA\sqrt{1+{(1-2{\rho }^3)}^2\frac{P_{STED}}{P_{SAT}}}},$$ (2)

where P_SAT, a saturation power characteristic of the sample, sets the scale for resolution improvement. From Eq. (2), a constant-resolution mode arises naturally (Fig. 6(a)) through the combination of a decreasing ρ with an increased depletion power. Here, the extra power recovers lateral resolution with increased optical sectioning. As required, Eq. (2) tends to the usual STED equation [37,38] if ρ tends to 1 or 0.

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.

A three-acquisition sequence was followed in order to standardize comparative imaging: constant-geometry (of which 2D-STED is one particular case), constant-power and constant-resolution (Fig. 6(a)). To avoid ‘chronological’ artifacts that might over-estimate CH-STED performance (e.g. by photo-bleaching), CH-STED was always acquired after 2D-STED (and z-STED) acquisitions. Different biological contexts (Figs. 6(b)-6(e) and Visualization 2) indicate that entering the CH-STED regime provides background rejection at high focal signal level. This is shown in photo-count profiles (line-plots in Fig. 6(d)) displaying a high CH-STED signal relative to the constant-power counterpart (the high-power 2D-STED) and a low background relative to the constant-resolution counterpart (the low-power 2D-STED), which amounts to a an increased dynamic range. Other samples, such as neuronal structural proteins, nucleoporins and tubulin in other cell stages (Appendix C and Visualization 2) generally deliver increased structural information when observed with CH-STED. An exception is sub-wavelength-thickness objects, where a regime closer to 2D-STED (ρ > 0.95) should be used.

As a practical corollary, if SBR is judged to be too low in a given 2D-STED acquisition, entering CH-STED is proposed as the default path (Fig. 6(e)), provided deleterious photo-physical effects (e.g. photo-bleaching) do not call for a decrease in STED beam power.

4. Discussion and conclusion

We introduced CH-STED as a perturbation to 2D-STED that dilates and contracts the nodal line of the depletion beam as it crosses the focal plane. This simple geometrical modulation, parameterized by the bi-vortex radius ρ, allowed a focus-specific fluorescence signal selection. Lateral resolution was shown to scale inversely with the geometrical and optical factor, (2ρ³ – 1)² P_STED. When required, the resolution decrease caused by setting ρ below 1 can be recovered by increasing P_STED, now accompanied by an improved depth sectioning.

Fundamentally, CH-STED deviates from the conventional search for best lateral resolution, shifting focus towards a tunable compromise between resolution and SBR, with 2D-STED remaining as the limiting case where ρ = 1. Unless sub-diffraction sectioning is provided by the sample itself [39] or by using evanescent excitation fields, such as in TIRF-STED [40], it is unlikely that the exact value ρ = 1 is the best choice in any given context within imaging-based, time-domain [41–44] or lithography [45] STED or RESOLFT variants. Very thin objects will generally benefit from approaching, instead of reaching, ρ = 1. These results indicate that, as a general practical rule for the microscope user, the STED beam power should be increased all the way up to a point in which photo-damaging effects are still considered negligible, even if SBR is already severely compromised. Having defined such set-point, SBR is recovered by decreasing ρ.

CH-STED background suppression is low compared to z-STED, but it displays higher lateral resolution and a more efficient depletion of the secondary excitation lobes (Fig. 3(a)), which is shown to be particularly relevant in x-y imaging (Fig. 4(d)). Alternative strategies for background reduction, such as double-depletion STED (termed STEDD) [46], which directly estimates background signal, can be used along with CH-STED for a cumulative improvement in contrast.

Increased power exacerbates photo-damage, a concern in STED microscopy which led to technical advances [47–51] and conceptual generalizations for the use of an optical doughnut [52,53]. Still, whenever a doughnut is used, the highest intensities at the doughnut crest, which are evidently the most photo-damaging for the sample, are too far away from the center to contribute significantly to resolution improvement [50]. The interesting point here is that the unwelcome intensity overshoot of the classical doughnut gets dispersed orthogonally (along the optical axis) when ρ is decreased below unity, thus decreasing the intensity variance of the STED beam. This decreased overshoot in CH-STED will likely translate into decreased photo-toxicity in a constant-power transition (Fig. 4).

Finally, we note that CH-STED does not require modifications in the polarization state (circular, with the proper handedness) of the STED beam used in typical STED setups. If an SLM is used for beam phase modulation, CH-STED implementation is immediate. Static phase plates are still a simple and very high-performance alternative also for CH-STED. In this case, zoom optics have to be used to persistently image the bi-vortex plate at the back focal plane of the objective at a variable magnification. Independent of implementation details, a CH-STED beam can be incoherently combined to a z-STED beam (Fig. 4(d)) to provide improved sectioning.