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.

Background reduction in STED-FCS using coherent-hybrid STED

“…The inner radius of the CH-STED mask can be changed to modify the shape of the corresponding depletion pattern. We initially set the value of this parameter to 0.85 (85% of the pupil radius size), as was used in a previous implementation [15]. The influence of the CH-STED radius parameter is discussed in section 4.1.…”

“…The shape of the CH depletion pattern, and thus of the effective observation volume, can be tuned by varying the size of the inner radius ρ of the CH STED phase mask ( Figure 5(a)) [15]. A smaller CH inner radius corresponds to a larger deviation from the vortex pattern, meaning a better axial confinement, at the expense of a lower lateral confinement.…”

Microtubule tyrosination/detyrosination specifies a mitotic error code

“…Persistent merotelic attachments in which a single kinetochore is attached to microtubules from both spindle poles are the main source of anaphase lagging chromosomes in mammalian cells (Cimini et al, 2003). To investigate whether the lagging chromosomes observed after experimental increase in α-tubulin detyrosination result from the formation of merotelic attachments, we used super-resolution CH-STED microscopy (Pereira et al, 2019) in fixed cells after TTL RNAi. Relative to conventional 2D-STED, this novel sub-diffraction technique improves contrast in highly complex environments, such as the mitotic spindle.…”

“…An oil-immersion 60x 1.4 NA Plan-Apo objective (Nikon, Lambda Series) and pinhole size of 0.8 Airy units was used in all confocal acquisitions. Super-resolution images were acquired using a coherent hybrid (CH-) STED beam (Pereira et al, 2019).…”

“…Here we combined powerful gene manipulation tools (including RNAi, small-molecule inhibition, protein overexpression and CRISPR-Cas9 gene editing) with state-of-the-art microscopy, including a novel super-resolution microscopy technique (Pereira et al, 2019), to investigate whether MCAK activity required for mitotic error correction is regulated by microtubule tyrosination/detyrosination. Our findings support a physiological role for microtubule tyrosination/detyrosination in the discrimination between correct and incorrect kinetochore-microtubule attachments, establishing a new paradigm in the control of mitotic fidelity.…”