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

Area of Science:

• Super-resolution microscopy
• Fluorescence imaging
• Biophysics

Background:

• Stimulated emission depletion (STED) microscopy achieves sub-wavelength resolution using a doughnut-shaped depletion beam.
• 2D-STED utilizes vortex phase modulation for maximal transverse confinement and radial-aberration immunity (RAI).
• RAI in 2D-STED limits axial localization accuracy, causing uncertainty in photon origin.

Purpose of the Study:

• To reduce axial uncertainty in STED microscopy by perturbing the phase mask.
• To develop a method for independent tuning of resolution and contrast in STED.
• To enhance the precision of fluorescence photon origin determination in STED imaging.

Main Methods:

• Modification of the 2D-STED phase mask to alter axial concavity near focus, creating a dilated dip.
• Implementation of coherent-hybrid STED (CH-STED) mode.
• Testing CH-STED in x-y imaging of complex biological structures, including dividing cells.

Main Results:

• The perturbed phase mask, when compressed by laser depletion power, recovers lateral resolution with significantly higher contrast.
• CH-STED demonstrates improved axial localization accuracy compared to standard 2D-STED.
• The method creates an orthogonal direction in STED parametric space for independent control of resolution and contrast.

Conclusions:

• CH-STED offers a novel strategy for enhancing STED microscopy performance.
• Independent tuning of resolution and contrast is achievable with a single depletion beam in conventional STED setups.
• This technique improves the accuracy of axial photon origin determination, advancing biological imaging capabilities.