Vortex Plates for STED Microscopy
Stimulated Emission Depletion (STED) microscopy is a well-known technique for achieving super resolution in microscopy that enables resolving sub-micron details that are smaller than the diffraction limit of the optical system.
Developed to bypass this diffraction limit of light microscopy, which is the main limit to the resolution of traditional light microscopes, STED creates super-resolution images by illumination of the fluorophores in a ring-like (donut) pattern that depletes the fluorescence from the outside area of the donut. This technique minimizes the area of illumination at the sample focal point and enhances the achievable resolution for a given system.
By using the non-linear response of the fluorophores, STED forces the excited fluorophores at the donut profile to emit at a longer wavelength that is then optically filtered out. Only the fluorescence from a small, sub-diffraction limit region is left, enabling super-resolution.
STED Microscopy – Principle of Operation
Typical STED systems include two independent optical channels – one for the long wavelength (red) depletion laser and another for the short wavelength (green) excitation laser. Both channels are combined into the same optical path by dichroic mirrors and are then focused by an objective on a sample. Fluorescent light reflected from the sample goes to a detector. The above figure displays a schematic setup for a STED microscope optical system.
The excitation channel is focused by the objective, while the depletion channel is propagated through a vortex phase plate, otherwise known as a Vortex Lens (VL), before it is focused by the objective. The vortex plate adds a spiral wavefront to the incident Gaussian beam to convert the beam into a donut-shape at the focal plane of the objective. The resulting donut shaped beam at the focal plane shares the same optical properties as a Gauss-Laguerre 01 laser mode.
The vortex laser beam in the STED system, that is the depletion laser, can be easily generated using a single vortex plate Diffractive Optical Element (DOE).
Overview of Diffractive Optical Elements
DOEs are micro-optical, window-like phase elements designed to modify the phase of the light that propagates through them. They create various shaping functions, such as multi-channel beam splitting, spatial beam shaping, and beam focal shaping. Amongst the spatial beam shapes achievable by DOEs are also ring and donut shapes, such as the vortex plate DOE used in STED systems.
The main fabrication process of DOEs consists of several repeating steps, including photoresist wafer coating, followed by direct UV lithography, etching directly into the Fused Silica substrate, and repeating to create a binary pattern microstructure at the surface of the mm-scale-thick optical window. To achieve optimal optical efficiency, it is often recommended to apply up to 4 lithography steps, thus creating 16 levels of microstructures. The last step in the DOE manufacturing process is deploying an anti-reflective coating layer. The next figure shows an actual diffractive structure of a 16-level vortex plate, generating an optical vortex beam that is measured by an optical profilometer.
Diffractive surface of a 16-level Vortex plate DOE measured by optical profilometer
Thanks to this production process, DOEs provide perfect angular accuracy with extremely low manufacturing tolerances. They are flat, thin and easy to integrate into any opto-mechanical design. In addition to these mentioned advantages, Fused Silica-made DOEs have outstanding laser damage threshold, surface deviation, micro-roughness, and mechanical properties. In many cases, DOEs present a much more cost-effective beam shaping method than their refractive counterparts, which frequently demand complex electro-opto-mechanics. Thus DOEs are a more robust solution when considering lifetime value.
STED microscopy, being a technique for measuring submicron structures, requires the system to be highly precise and accurate; otherwise, overall performance may be severely affected. In addition to all the aforementioned advantages, Holo/Or’s vortex plates are polarization insensitive and do not require rotation alignment. For all these reasons, they pose the perfect solution for STED systems.
Holo/Or offers a wide variety of vortex plate DOEs. To calculate the ring and hole diameter of the vortex plate, Holo/Or created this optical vortex calculator to aid our customers in choosing the part fitting their needs.
For more information about this or other Holo/Or products, contact Holo/Or.