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.
Highlights
The capacity to label proteins and other macromolecules with highly specific fluorescent reporters makes fluorescence microscopy an essential tool in the life sciences
The same applies for each annular vortex of a CH-Stimulated emission depletion (STED) mask, making existing STED setups adequate for coherent-hybrid STED (CH-STED) microscopy
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
Summary
The capacity to label proteins and other macromolecules with highly specific fluorescent reporters makes fluorescence microscopy an essential tool in the life sciences. The effective point-spread function (PSF) is 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. 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. 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, fundamentally changing the axial response. 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
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